SSIEM 2016 Annual Symposium - Abstracts

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 DOI 10.1007/s10545-016-9969-2

ABSTRACTS

SSIEM 2016 Annual Symposium - Abstracts Rome, Italy, September 2016

# SSIEM 2016 01. Inborn errors of metabolism in adults

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Discussion: Complete data from both the SAD and MAD portions of the study, including safety, pharmacokinetic and pharmacodynamic data, will now be presented. Conflict of Interest declared.

Interim data from a randomized, placebo controlled, phase 1 study of ALN-AS1, an investigational RNAi therapeutic for the treatment of acute hepatic porphyria

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Sardh E 1, Harper P 2, Al-Tawil N 3, Penz C 5, Chan A 5, Querbes W 5, Simon A 5, Stein P 4, Rees D 4

A double-blind placebo-controlled trial of triheptanoin in adult polyglucosan body disease

1 Karolinska Univ Hosp, Karolinska Inst, Stockholm, Sweden, 2Porphyria Centre, Karolinska Univ Hosp, Stockholm, Sweden, 3Karolinska Trial Alliance, Karolinska Univ Hosp, Stockholm, Sweden, 4King’s College Hospital, London, United Kingdom, 5Alnylam Pharmaceuticals, Cambridge, United States

Schiffmann R 1, Wallace M 1, Rinaldi D 2, Turner J 1, Hogrel J Y 3, Blankenship D 1, Mochel F 2 4 5

Background: Acute hepatic porphyria (AHP), including acute intermittent porphyria (AIP), is a family of rare metabolic disorders caused by enzyme defects in the heme biosynthesis pathway in the liver. When ALA synthetase (ALAS1), the first and rate limiting step in the pathway, is induced by exposure to certain drugs or fasting, the neurotoxic heme intermediates ALA and PBG can accumulate, leading to acute neurovisceral attacks in AHP patients. RNA interference is a naturally occurring cellular mechanism mediated by small interfering RNA (siRNA) that inhibits protein synthesis through the degradation of a specific mRNA. ALN-AS1 is an investigational RNAi therapeutic that targets ALAS1 in order to decrease ALA and PBG levels and subsequent porphyria attacks. Methods: We are currently conducting a phase 1, multinational, randomized, placebo-controlled, single ascending dose (SAD), multiple ascending dose (MAD) and multiple dose (MD) study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics (changes in ALA, PBG and circulating ALAS1 mRNA) of subcutaneously administered ALN-AS1 in AIP patients who are asymptomatic but biochemically active (SAD and MAD), or who have recurrent porphyria attacks (MD) (ClinicalTrials.gov Identifier: NCT02452372). Results: Interim data presented from the SAD portion of the study demonstrated ALN-AS1 was generally well tolerated, as there were no significant AEs or laboratory abnormalities associated with drug. In addition, a mean (SEM) maximal reduction relative to baseline of 44 % ± 8 % in circulating ALAS1 mRNA (p ≤ 0.01 compared to placebo) and of 77 % ± 7 % and 73 % ± 6 % in ALA and PBG, respectively (p = 0.03 and 0.06 compared to placebo, respectively) at the 0.35 mg/kg dose was demonstrated and sustained beyond 42 days.

1 Baylor Research Inst, Dallas, United States, 2INSERM U 1127, CNRS UMR 7225, Sorbonne Univ, Paris, France, 3Institute of Myology, F-75013, Paris, France, 4Neurometab Res Gr, Univ Pierre and Marie, Paris, France, 5Dept Genetics, Pitie-Salpetriere Hosp, Paris, France

Background: Adult polyglucosan body disease (APBD) is a progressive neurogenetic disorder caused by a deficiency of glycogen branching enzyme. Patients develop in the 5th or 6th decade of life neurogenic bladder, progressive spastic paraparesis, sensorimotor peripheral neuropathy and sometimes dementia. There currently is no effective therapy. We hypothesized that decreased glycogen degradation leads to cellular energy deficit and that anaplerotic therapy via triheptanoin may augment energy production thus preventing or reversing cellular damage. Methods: This was a two-site randomized crossover study over 1 year of 23 patients (age 35–73 years; 63 % male). Vegetable oil served as placebo. Outcome measures included a 6-min walk test (primary), balance testing, and SF-36 health survey questionnaire for all subjects; the Spastic Paraplegia Rating Scale, finger tap and dynamometer testing in French site subjects. Safety monitoring included adverse events, blood chemistries, urine organic acids and acylcarnitine profile in blood. The linear mixed model was used to analyze this study. Results: Nineteen patients were eligible for data analysis. At baseline, patients could walk a mean 389 ± 164 meters (range 95–672). The overall mean difference between subjects on triheptanoin versus placebo was 6 meters; 95 % CI: (−11, 22); p = 0.49. Motion capture gait analysis, gait quality, and stairs climbing did not show a consistent direction of change. SF36 questionnaire showed significant improvement in mental and physical quality of life scores on triheptanoin. There was no significant difference in the number of patients experiencing adverse events in the two treatment groups.

S36 Discussion: Triheptanoin had a good safety profile. However, from this study we cannot conclude that it is effective in the treatment of APBD over a 6month period. Conflict of Interest declared.

02. Novel diagnostic/laboratory methods

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Multi-omics tools for the diagnosis and treatment of rare neurological disease Simmons T L 1, Abela L 1, Spiegel R 2, Steindl K 3, Joset P 3, Klein A 1, Zehavi Y 2, Crowther L 1, Rauch A 3, Plecko B 1 1 Div Neuroped, Univ Child Hosp, Zurich, Switzerland, 2Dept Pediatrics, Rappaport Fac Med, Technion, Israel, 3Inst Med Gen, Univ Zurich, Zurich, Switzerland

Background: Early onset epileptic encephalopathies (EE) are a heterogeneous group of rare genetic disorders that pose major diagnostic and therapeutic challenges. Some EEs are caused by inborn errors of metabolism (IEM) that are amenable to specific treatment, while others remain undiagnosed and untreated. Here we present the comprehensive metabolome and exome analysis in two previously undiagnosed EEs. We were able to characterize metabolic ‘fingerprints’ and novel pathogenic mutations for Snyder-Robinson syndrome and mitochondrial aconitase deficiency. Methods: Plasma was collected from 63 patients (27 male; 36 female) with early onset EE of unclear etiology. Multi-dimensional profiles were generated by LCMS based metabolomics and whole-exome sequencing (WES) of index patients and parents. Comparative data mining and multivariate analysis were employed for gene mutation analysis, metabolic fingerprinting and biomarker discovery. Results: Comprehensive multi-omics analysis of two previously undiagnosed EE cases has revealed the first case as Snyder-Robinson syndrome. Here we have identified significantly elevated N8-acetylspermide in SRS patients harboring defective spermine synthase; we have also identified a new pathogenic R130C mutation in the spermine synthase gene SMS. In the second case we have identified a novel pathogenic mutation in the ACO2 gene encoding mitochondrial aconitate hydratase. We have also characterized a signature metabolic profile (i.e. metabolic fingerprint) for ACO2 deficiency, which includes an array of significantly altered TCA cycle metabolites and others. Discussion: We demonstrate a precision biomedical approach with the ability to reveal diagnostic biomarkers/metabolic fingerprints for rare undiagnosed IEM. This strategy is easily translated to the clinic by providing new tools for rapid disease diagnosis and treatment monitoring, and perhaps novel therapeutic strategies for IEM.

03. Newborn screening

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Investigating applications of next generation sequencing in newborn screening Gladding C M 1, Milano A 1, Dawe J 1, Peck G 1, Winship P 1, Richards S L 1, Grafham D 1, Sharrard M 2, Johnson D 3, Weaver L 4, Bonham J R 5, Goodeve A C 1, Dalton A 1 1 SDGS, Sheff Child NHS Found Trust, Sheffield, United Kingdom, 2Inher Metab Dis, Sheff Child NHS Found Trust, Sheffield, United Kingdom, 3Clin Genet Dept, Sheff Child NHS Found Trust, Sheffield, United Kingdom, 4 Climb (Children Living with Metab Dis), Crewe, United Kingdom, 5Phar, Diag and Gene Div, Sheff Child NHS Found Trust, Sheffield, United Kingdom

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: In newborn screening (NBS) programmes, dried blood spot (DBS) samples are used to identify babies with rare, often fatal, but treatable disorders. Biochemical analysis is performed at low cost with short turnaround times. However, it can be challenging to predict disease severity and appropriate treatment in asymptomatic individuals or for disorders lacking a suitable biochemical analyte or enzyme activity assay. The project team is investigating the use of next generation sequencing (NGS) to (1) improve the diagnostic and prognostic utility of existing UK NBS programmes and (2) assess the technical feasibility of using NGS as a primary or adjunct screening test. Methods: For aim 1, a genotype-phenotype database is being developed to establish correlations between genetic mutations, biochemical changes and phenotypes for NBS disorders. For aim 2, several methods have been tested to optimise DNA extraction for NGS using healthy control DBS samples. Results: For aim 1, database development, genotype, biochemical and phenotypic information is being collected from 130 screen positive newborns, 130 clinically affected individuals and 150 healthy controls. For aim 2, a DBS extraction method has been optimised that produces DNA of sufficient quality and quantity for NGS. The automated process is robust and high-throughput with short turnaround times for multiple NBS samples. Discussion: This project will potentially improve the effectiveness of existing NBS programmes by facilitating personalised treatment and providing prognostic information for patients and families. It will also provide a valuable foundation for further NGS developments, particularly for disorders lacking a suitable biochemical screening assay. Use of NGS technology as a primary or adjunct test will have a significant impact on healthcare systems worldwide and on other areas of medicine. This project was awarded Health Innovation Challenge Funding from the Wellcome Trust and Department of Health.

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A newborn screening method for cerebrotendinous xanthomatosis: data from a pilot validation study Vaz F M 2, Bootsma A H 2, Kulik W 2, DeBarber A E 3, Huidekoper H H 1 1 Cent Lyso Metab Dis, Erasmus Med Cent, Rotterdam, Netherlands, 2Lab Genet Metab Dis, Acad Med Cent, Amsterdam, Netherlands, 3BioAnalyt Shared Resource Facility, OHSU, Portland, United States

Background: Cerebrotendinous xanthomatosis (CTX) is caused by the deficiency of 27-sterol hydroxylase (CYP 27), resulting in reduced production of cholic acid (CA) and chenodeoxycholic acid (CDCA) and accumulation of cholestanol and cholesterol, and is a progressive neurodegenerative disorder. Symptoms can be halted or prevented by supplementation of CDCA, reducing bile acid synthesis through negative feedback. Prognosis is good when CDCA is started at an early age, which makes CTX a good candidate for implementation in newborn screening programs. However, a validated screening method is not yet available. Methods: We developed a screening method for CTX, based on the quantification of bile alcohol glucuronides in dried blood spots (DBS) using both plain loop injection-MS/MS and UPLC-MS/MS after standard methanol extraction without the need for derivatization. DBS material of 5 patients with CTX (3 from original newborn screening cards, 2 from two pediatric patients at diagnosis) were blinded between DBS material of 200 healthy newborns (150 at term, 50 prematures) and 2 Zellweger patients, and analysed using our method. Results: The CTX patients could be clearly discriminated from the healthy newborns and the Zellweger patients with both plain loop injection-MS/MS and UPLC-MS/MS. Ratios between bile alcohol glucuronides and specific bile acids are suggested for a diagnostic algorithm in order to filter out other causes for liver disease and/or cholestasis, and make the method CTX specific. Discussion: Our newborn screening method for CTX based on quantification of bile alcohol glucuronides is specific and can be readily incorporated in existing newborn screening laboratories as no derivatization is required. First, a large scale pilot study is needed in order to determine reproducibility and to accurately estimate the false positive rate. Conflict of Interest declared.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 O-006

A new metabolic disorder in human cationic amino acid transporter-2 mimicking arginase deficiency in newborn screening Yahyaoui R 1 2, Blasco-Alonso J 2 8, Benito C 7, Rodriguez-Garcia E Dayaldasani A 1, Vega A 3 4 5 6, Perez-Cerda C 3 4 5 6, Perez B 3 4 5 6

1 2

,

1

Clin Lab, Malaga Regional Hosp, Malaga, Spain, 2IBIMA, Malaga, Spain, Centro de Diag de Enferm Molec CEDEM, Madrid, Spain, 4IDIPAZ, Madrid, Spain, 5CIBERER, Madrid, Spain, 6Centro de Bio Molec-SO, UAM-CSIC, Madrid, Spain, 7Dept of Genetics, Malaga Regional Hosp, Malaga, Spain, 8 Dept of Pediatrics, Malaga Regional Hosp, Malaga, Spain 3

Background: Arginase deficiency is a rare condition that can be identified by elevated levels of Arg in the current expanded newborn screening performed in many countries. To date, only mutations on the ARG1 gene have been described. Case Report: We present a Spanish child of non-consanguineous parents with moderately increased levels of Arg in DBS newborn screening specimen (67 μmol/L, NV:< 33) with normal Arg/Orn ratio (0.31, NV:< 0.40). A second DBS sample was requested and levels of Arg increased to 112 μmol/L. Plasma concentrations of some amino acids (μmol/L) at 1 month of life were high: Arg 312 (NV:34–88), Orn 177 (NV: 52–116) and Lys 599 (NV: 199–209). The profile of amino acids in urine was similar. The infant was healthy and subsequently started on a protein-restricted diet of 1.2 g/kg/day which included PFD1 metabolic formula. Results: Arginase enzyme activity in red blood cells was normal and no pathogenic mutations were identified in the ARG1 gene. Massive parallel sequencing using the Illumina® clinical-exome TruSight™ One sequencing panel detected two loss-of-function mutations in the SLC7A2 gene: a large genomic rearrangement including at least exon 3 and 4 (c.-22-?_532 + ?del) in the paternal allele and a small deletion c.874delA (p.Ile292Leufs*2) in the maternal allele. Currently, the child is 2 years 3 months old and is on a protein-restricted diet of 2 g/kg/day with PFD1 formula 30 g/day to control amino acid levels. He has been asymptomatic and presents normal psychomotor development. Discussion: A defect in human cationic amino acid transporter-2 has been described for the first time. The characteristic biochemical profile seems to be an elevation of Arg, Orn and Lys in plasma and urine. Natural history of the disorder at long-term follow-up is uncertain although a protein-restricted diet seems to be necessary. Newborn screening centres should know this disorder since it can be incidentally detected while screening for arginase deficiency.

04. Dietetics and nutrition

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Longitudinal study examining nutritional status in children with organic acidaemias on a modular feed using a protein free module especially developed for children with IMD Daly A 1, Chahal S 1, Evans S 1, Ashmore C 1, MacDonald A 1 1

Birmingham Children’s Hospital, Birmingham, United Kingdom

Background: Tube feeding is commonly used as a treatment strategy for children with organic acidaemias (OA), particularly propionic acidaemia (PA) and vitamin B12 non-responsive methylmalonic aciduria (MMA). However, little is known about the ideal composition of tube feeds for this group of children and there is no long-term data reporting the use of protein-free modules (PFM) as a core ingredient in tube feeds for children aged ≥ 1 year with OA. Aim: To report 18-month longitudinal, prospective nutritional data in OA children aged ≥ 1 year, fed exclusively by an enteral tube using a PFM (providing 100 kcal/ 100 ml and a vitamin/mineral profile similar to standard paediatric enteral feeds) as part of a low protein enteral feed. Methods: The PFM replaced either an infant PFM or individual fat, carbohydrate, vitamin, mineral and electrolyte modules. 13 subjects, median age 7.4y

(3–15.5y), m = 6/f = 7, with the following OA (PA n = 6; MMA, n = 4; glutaric aciduria n = 1, and isovaleric acidaemia, n = 2) were recruited. Anthropometry and nutritional intake were analysed. Results: Compared with baseline results, the dietary intake of the following nutrients significantly improved: calcium (p = 0.04), magnesium (p = 0.008), iron (p = 0.007), zinc (p = 0.006), selenium (p = 0.008), sodium (p = 0.01), potassium (p = 0.006), and phosphorous (p = 0.006). During the study, median energy (76 % of estimated average requirements) and natural protein (0.9 g/kg/d) intake remained unchanged. All nutritional biochemical markers improved but the following were significant: plasma zinc (p = 0.01), vitamin B12 (p = 0.04), and MCV (p = 0.002). No significant changes in z scores for weight and height were observed. Discussion: An age appropriate vitamin and mineral supplemented PFM providing an energy source improved long term nutritional intake and biochemical status in patients with OA. It is essential that age appropriate PFM modules are developed for all age groups with OA. Conflict of Interest declared.

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Dietary treatment of 49 MSUD Italian patients Salera S 1, Bernabei S M 2, Dianin A 3, Di Mauro A M 7, Gugelmo G 3, Fasan I 6, Gallo G 2, Musiani C 4, Pozzoli A 5, Pretese R 8, Tarrini G 4, Tursi S 8, Zucchi C 9, Zuvadelli J 10 1 Ped Dept, IRCCS Policlinico, Milan, Italy, 2Bambin Gesu, Children Hospital, Rome, Italy, 3Inherit Metab Dis Unit, Dept of Ped, Verona, Italy, 4S Orsola Malpighi Hosp, Bologna, Italy, 5Piacenza Hosp, Piacenza, Italy, 6Dept Pediatrics Univ Hosp, Padova, Italy, 7Univ Hosp Giovanni XXIII, Bari, Italy, 8Metab Unit, San Gerardo Hosp, Monza, Italy, 9Gaslini Hosp, Genova, Italy, 10Clin Dept of Ped, San Paolo Univ Hosp, Milan, Italy

Background: Only few data are available on maple syrup urine disease (MSUD) dietetic treatment in Italian patients. The aim of this work is to report the data on dietary practices in MSUD Italian patients. Methods: A questionnaire focusing on dietary treatment was sent to all dietitians working in 18 Italian metabolic centers. Results: 49 patients (22 > 18 y) from 10 metabolic centers (30 classic, 15 intermediate, 2 intermittent, 2 thiamine sensitive) were collected. Mean leucine tolerance (mg/day) was: (1) classic form 343 (range 180–500) in the first year of life; 412 (225–600) or 27 mg/kg/day 1–10 y; 429 (300–800) or 10 mg/kg/day 11–18 y; 639 (350–1350) or 11.7 mg/kg/day >18 y; (2) intermediate form: 1000 (950– 1000) or 48.8 mg/kg/day 1–10 y, 1300 (600–2000) or 28 mg/kg/day 11–18 y, 1518 (470–4200) or 24.5 mg/kg/day >18 years. All patients with classic form used branched chain amino acid (BCAA)-free mixtures, in the mean amount (g/kg/day) of: 1.8 < 12 months; 1.4 from 1 to 10 y; 1 from 11 to 18 y; 0.9 > 18 y. 27 classic patients received valine (V) supplementation and 23 isoleucine (I). All patients with intermediate form used BCAA-free mixtures (1.4 g/kg/day from 1 to 10 y; 0.53 from 11 to18 y; 0.69 > 18 years), while only 2 used V and I supplementation. Intermittent and thiamine sensitive forms had only a moderate protein restriction. 3 of 30 patients with the classic form were exclusively treated with enteral nutrition (EN) for more than 1 year as the only way of nutrition in spite of normal psychomotor development. The others used EN only during decompensations. 5 of 10 centres prescribed DHA supplementation. Discussion: Leucine tolerance is variable in MSUD patients. V and I supplementation are used by most but not all patients with the classic form. It would be interesting to study the impact on metabolic balance, growth, puberty and body composition of different dietetic treatments.

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Protein intake and physical activity are associated with body composition in patients with phenylalanine hydroxylase deficiency Jani R 1, Coakley K E 1, Douglas T D 1, Singh R H 1 1

Department of Human Genetics, Emory Univ, Decatur, United States

S38 Background/Objective: This cross-sectional study examined associations between protein intake, physical activity (PA), genotype, and body composition in patients with phenylalanine (PAH) deficiency. Methods: Total protein including intact and medical food (MF) were analyzed using 3-day food records in Nutrition Data System for Research from 59 children and 27 adults (n = 86). PAH deficiency severity was classified utilizing genotype assigned value method. PA was assessed using a studydeveloped question (light vs intense active) as a proxy indicator. Body composition was measured by DEXA, including android:gynoid ratio (A:G: abdominal obesity), fat free mass index (FFMI: lean body mass), fat mass index (FMI) and FMI:FFMI ratio (fat body mass). Spearman’s partial correlation coefficients and generalized linear model examined associations adjusted for covariates. Results: Higher intact protein was associated with higher lean mass and lower fat mass in adults (FFMI: p = 0.008; FMI:FFMI: p = 0.04). MF protein (p = 0.04) and total protein (p = 0.04) were directly proportional with lean mass (FFMI) in children. In adults and children, light PA was associated with higher fat mass indices compared to intense PA (FMI:FFMI p = 0.001, p = 0.007; children only: FMI p = 0.02; A:G p = 0.04). All associations remained significant after covariate adjustment. Genotype was not associated with body composition. Discussion/Conclusion: Source of protein intake impacts body composition differently in adults and children. In adults, intact protein given based on individual tolerance promotes lean versus fat mass. However, in children, total protein maximized with MF promotes lean mass and better metabolic control. Nutrition counselling could be complimented with PA recommendations for optimal clinical outcomes.

05. Phenylketonuria: general

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Secondary pterins alteration in patients with phenylalanine hydroxylase deficit Nardecchia F 1 2 5, Valentini G 1, Chiarotti F 4, Santagata S 3, Carducci C 3, Angeloni A 3, Leuzzi V 1 1 Dept of Pediatr and Child Neuropsychiatry, Sapienza Rome, Italy, 2Dept of Physiol and Pharmacol, Sapienza Rome, Italy, 3Dept of Exp Med, Sapienza Rome, Italy, 4ISS, Dept of Cell Biol and Neurosci, Rome, Italy, 5Dept of Psychology, Sapienza Rome, Italy

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 show that the interindividual variability accounted for the most of the variability seen in pterins excretion in PKU patients, suggesting that individual factors, still unknown, determine an individual response to chronically elevated Phe levels.

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Phenylalanine hydroxylase N-terminal domain is an allosteric binding site and can be target for pharmacological chaperone design Patel D 1, Kopec J 1, Fitzpatrick F 1, McCorvie T J 1, Yue W W 1 1

Struct Genom Consortium, Univ of Oxford, Oxford, United Kingdom

Background: The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary phenylalanine (Phe) to tyrosine. Inherited PAH deficiency due to PAH gene mutations causes the autosomal recessive disorder phenylketonuria (PKU). Phe is not only the substrate for the PAH active site, but also an allosteric activator of the enzyme via an unclear mechanism. Recently, the reported full-length structure of rat PAH in the inactivated state reveals an auto-inhibitory conformation of the N-terminal regulatory domain (PAH-RD) towards the enzyme catalytic domain. Based on this, in addition to the catalytic domain, Phe has been proposed to bind to an allosteric site at the PAH-RD in order to activate the enzyme. Methods: To decipher the molecular mechanism of Phe-mediated PAH activation via its regulatory domain, we have expressed recombinant human PAHRD and determined its structural properties by crystallography, differential scanning fluorimetry and small angle x-ray scattering. Results: The 1.8 Å resolution structure of human PAH-RD bound with Phe reveals a homodimer with Phe bound at the dimer interface. This provides the long-sought structural evidence to support solution studies that a binding site for Phe exists in the PAH-RD, and that Phe binding results in dimerization of PAH-RD. Consistent with this, a disease-associated PAH mutant impaired in Phe binding, namely p.E76A (c.227A > C), disrupts the monomer:dimer equilibrium of PAH-RD. Discussion: Our data support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. This study also yields novel insight into mutation-induced destabilization and aggregation of PAH, and directs efforts in developing pharmacological chaperones for PKU by targeting the PAH regulatory domain.

06. Phenylketonuria: treatment, BH4 Background: Tetrahydrobiopterin (BH4) cofactor is essential for various enzyme activities, included phenylalanine hydroxylase. The controlling point of cofactor biosynthesis is GTP cyclohydrolase I (GTPCH) whose activity is stimulated by phenylalanine (Phe) through the GTPCH feedback regulatory protein. In phenylketonuria (PKU) patients, who are chronically exposed to high Phe level, a higher urinary excretion of pterins is found. However, the persistence of this homeostatic mechanism related to the duration of the exposure to the high Phe levels and individual variability is not known. The aim of the study was the longitudinal investigation of urinary pterins excretion in PKU patients in relationship with their blood Phe levels and age. Methods: 274 urinary pterins analyses of 47 PKU patients at different ages were collected together with concomitant blood Phe and Tyr. Results: Multiple regression analysis evidenced that urinary biopterin and neopterin levels were influenced by age and Phe levels, the percentage of variance explained by the model was 4 % (R2 = 0.0386) and 25 % (R2 = 0.2531), respectively. We found an inverse correlation between biopterin and neopterin levels and age (p = 0.002 and p < 0.001, respectively), and a direct correlation between biopterin and neopterin and Phe levels (p = 0.026 and p = 0.009, respectively). ANCOVA analysis, taking into account age and Phe levels, showed for both biopterin (F (46,134) = 1.92, p = 0.002) and neopterin (F (46,134) = 1.78, p = 0.006) that variability among subjects was significantly higher than variability among repeated measures within subjects. Discussion: The association between pterins excretion, age and Phe levels was already known. However, the longitudinal design of the study allowed us to

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The first structure of full-length phenylalanine hydroxylase has finally been determined Jaffe E K 1, Arturo E C 1 2, Gupta K 3, Heroux A 4, Stith L 1, Cross P J 5, Parker E J 5, Loll P J 2 1

Fox Chase Cancer Center—Temple Health, Philadelphia, United States, Drexel Univ College of Medicine, Philadelphia, United States, 3Perelman School of Medicine, Univ Penn, Philadelphia, United States, 4Brookhaven National Laboratory, Upton, NY, United States, 5University of Canterbury, Christchurch, New Zealand 2

Background: Improved understanding of the dynamic structure of phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria. The conformation and multimerization properties of PAH respond to allosteric activation by phenylalanine (Phe); which is necessary to maintain Phe below neurotoxic levels. PAH exists as a mixture of both a resting-state tetramer and an activated tetramer. The amount of activated PAH normally increases after a protein-containing meal. This causes Phe to return to safe

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 levels; then PAH returns to its resting-state. Our 2013 model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers. Methods: Ion exchange chromatography allowed isolation of a single conformation of resting-state PAH, which facilitated determination of the first X-ray crystal structure of full-length resting-state PAH. Small angle X-ray scattering (SAXS) was used to visualize both resting-state and activated PAH. Results: We present the first X-ray crystal structure for a full-length mammalian (rat) PAH in the resting state (PDB id 5DEN; 2.9 Å). The tetramer structure corrects and supersedes a composite homology model that has been used to rationalize phenylketonuria genotype-phenotype relationships. SAXS confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The SAXS data are consistent with our hypothesis that the domains of activated PAH are dramatically reorganized to form an inter-subunit interface where allosteric Phe likely binds. Discussion: The use of SAXS and X-ray crystallography together provides the first complete view of the structures of tetrameric PAH and a deeper understanding of PAH allostery. The lack of structural detail for activated PAH remains a barrier to the rational design of new structure-based PKU therapeutics. Conflict of Interest declared.

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Evaluation of long-term safety and efficacy of pegvaliase treatment for adults with phenylketonuria: updated year 4 results Longo N 2, Thomas J 4, Wasserstein M 5, Burton B 3, Vockley J 6, Grange D 7, Decker C 1, Weng H H 1, Li M 1, Schweighardt B 1, Zori R 8 1

BioMarin Pharmaceutical Inc, Novato, United States, 2Univ of Utah, Salt Lake City, United States, 3Lurie Child Hosp, Chicago, United States, 4Univ of Colorado, Aurora, United States, 5Mount Sinai Hosp, New York, United States, 6Child Hosp Pittsburgh, Pittsburgh, United States, 7St Louis Child Hosp, St Louis, United States, 8Univ Florida, Gainesville, United States Background: Phenylketonuria (PKU) is caused by phenylalanine hydroxylase deficiency resulting in phenylalanine (Phe) accumulation. Pegvaliase (PGVL), PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase, is a potential enzyme substitution therapy for PKU. Methods: The ongoing PAL-003 phase 2 extension study evaluated long-term efficacy, safety, and tolerability of subcutaneous PGVL in adults with PKU. Subjects entered PAL-003 on the last dose received in the parent study (PAL002, PAL-004 or 165–205), with dose adjustment as needed for safety or to maintain blood Phe of 60–600 μmol/L. Results: 68 subjects entered PAL-003 with 11–24 (mean: 17) weeks of prior PGVL exposure and mean baseline blood Phe of 1041 ± 545 μmol/ L (n = 61). In PAL-003, mean PGVL dose was 190 ± 161 (range: 1–925) mg/wk administered in 1–7 doses/wk and mean treatment duration was 3 ± 1.7 (range: 0.08–6.1) years. At PAL-003 year 1, mean blood Phe decreased 63 % from pre-treatment baseline of 1332 ± 327 to 459 ± 476 μmol/L, 49 % of subjects had blood Phe ≤360 μmol/L and 67 % had blood Phe ≤600 μmol/L (n = 57). This decrease persisted through PAL-003 year 4. Long-term PGVL treatment was well tolerated; adverse events (AEs) were mild to moderate in 61 (90 %) subjects and led to drug withdrawal in 4 (6 %) subjects. The most common AEs were headache (59 % of subjects), nasopharyngitis (56 %), rash (50 %), arthralgia (49 %), and injection site reaction (49 %). Four (6 %) subjects had anaphylaxis AE per National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network criteria that resolved without sequelae (2 continued treatment and 2 withdrew). All subjects developed a sustained antibody response against PAL and most developed a transient antibody response against PEG. Discussion: Subjects treated with PGVL had a substantial and persistent decrease in mean blood Phe. Long-term treatment was well tolerated, with most subjects experiencing mild to moderate AEs. Conflict of Interest declared.

O-014

Large neutral amino acid supplementation as a possible alternative treatment for adult PKU patients: evidence in PKU mice Van Vliet D 1 2, Bruinenberg V M 2, Mazzola P N 1 2, Anjema K 1, Van Faassen H J R 3, De Blaauw P 3, Kema I P 3, Heiner-Fokkema M R 3, Van der Zee E A 2, Van Spronsen F J 1 UMCG, Beatrix Child Hosp, Div Metab Dis, Groningen, Netherlands, 2Univ Groningen, GELIFES, Dept Mol Neurob, Groningen, Netherlands, 3UMCG, Dept Lab Med, Groningen, Netherlands 1

Background: While a phenylalanine (Phe)-restricted diet can improve some behavioral issues in late-diagnosed adult phenylketonuria (PKU) patients, in most cases, such dietary treatment is a severe challenge. Therefore, an alternative less-demanding treatment strategy that could improve the neuropsychological and behavioral outcome in these cases is highly awaited. Clinical experience with large neutral amino acid (LNAA) supplementation has shown promising results. However, the therapeutic mechanism has not been investigated at adult age. Methods: The effect of LNAA treatment on brain monoaminergic neurotransmitter synthesis in relation to brain and plasma amino acid biochemistry was investigated in late-adult C57Bl/6 Pah-enu2 PKU mice, and compared to normal chow and an isocaloric/isonitrogenic high-protein control diet in both PKU and WT mice. Results: LNAA supplementation in late-adult PKU mice increased brain serotonin from 43 to 90 % (p < 0.01), norepinephrine from 51 to 102 % (p < 0.01), and dopamine from 78 to 95 % of WT levels (not significant; p = 0.078). LNAA supplementation partly resolved the brain deficiency of tyrosine and tryptophan in PKU mice, whereas brain Phe concentrations remained unchanged compared to PKU mice on normal chow (p = 0.203). Discussion: The fact that LNAA supplementation significantly increased brain serotonin and norepinephrine concentrations in late-adult PKU mice, even when not influencing brain Phe concentrations, makes it a promising alternative treatment strategy to improve neuropsychological outcome in late-diagnosed adult PKU patients. This may also apply to early- and continuously-treated adult PKU patients, as the current results show that brain tyrosine and tryptophan deficiency might be of special importance for brain neurotransmitter synthesis in adult PKU patients. Therefore, LNAA treatment may not only improve adherence but, even more importantly, may also be more effective to improve brain biochemistry—and thereby outcome—in adult PKU patients. Conflict of Interest declared.

O-015

New generation of chemical scaffolds able to bind to human phenylalanine hydroxylase Leandro J 1, Amaro M P 1, Paterna R 1, Lopes R 1, Gomes K 1, Tavares de Almeida I 1, Gois P M P 1, Leandro P 1 1

iMed.ULisboa, Fac Pharmacy, Univ Lisboa, Lisboa, Portugal

Background: Phenylketonuria (PKU) results from a deficiency of human phenylalanine hydroxylase (hPAH), and due to decreased stability and folding efficiency of the variant PAH proteins, it has been classified as a conformational disorder with loss of function. Recent cofactor supplementation therapy as a pharmacological chaperone offered a new alternative treatment to PKU and proof of principle for the discovery of small molecules to correct the PKU phenotype. Based on a building blocks chemistry approach our group recently synthesized and tested new iminoboronates modulators of the hPAH protein using the substrate and regulator L-phenylalanine (L-Phe) as the key structural motif. In this work a new generation of small molecules based on a quinoline moiety was design in order to test for their capacity to modulate the activity/ stability of the hPAH protein. Methods: The wild-type hPAH was expressed in E. coli as a 6xHis fusion protein and purified by affinity chromatography and size-exclusion

S40 chromatography. The hPAH activity and L-Phe activation was measured according to standard protocols. The hPAH thermal stability was evaluated by differential scanning fluorimetry. Results: From the 18 quinoline derivative compounds tested, one was a strong inhibitor of hPAH activity (compound 3), whereas compounds 4 and 6 were able to activate hPAH (1.7- and 1.6-fold, respectively). Compounds 10 and 11 bound to the hPAH, leading to a protein with lower stability. Compound 13 lead to a 8 °C increase in the Tm of the hPAH regulatory domain while not changing the Tm of the catalytic domain. Discussion: The building blocks chemistry approach utilized here is a powerful tool to design small molecules able to modulate the activity and stability of hPAH. Further modification of compounds 4, 6 and 13 scaffolds could generate useful structures in order to develop pharmacological chaperones able to treat severe forms of PKU. Funded, in part, by iMed.ULisboa (FCT; UID/DTP/04138/2013).

07. Sulphur amino acid disorders

O-016

Effect of enzyme replacement therapy on osteoporosis in several CBSdeficient homocystinuric mouse models Majtan T 1, Park I 1, Carrillo R S 1, Kraus J P 1 1

Dept Pediatr, Univ Colorado, School Med, Aurora, United States

Background: Cystathionine beta-synthase (CBS)-deficient homocystinuria (CBSDH) is an autosomal recessively inherited metabolic disorder characterized by high plasma level of the transsulfuration toxic intermediate, homocysteine. CBSDH patients often suffer from osteoporosis among other clinical signs. We developed an enzyme replacement therapy (ERT) for CBSDH and assessed its efficacy against osteoporosis in several mouse models of CBSDH. Methods: Three CBSDH mouse models, designated as KO, HO and I278T, were examined for the presence of the osteoporotic phenotype by measuring body composition using a dual-energy X-ray absorptiometry (DEXA) scanner. Bone mineral content (BMC), bone mineral density (BMD), total mass and fat content were compared among healthy +/−untreated mice, affected −/−untreated mice and the ERT-treated −/−mice. Plasma metabolites of mice prior to the DEXA scan were determined by GC-MS in order to correlate the observed phenotypes with metabolic profiles as well as to assess the efficacy of the ERT treatment. Results: Both untreated −/−KO and I278T mice showed a profound osteoporosis characterized by low BMC, BMD, total weight as well as fat content, which correlated well with the plasma metabolite profiles showing severe homocysteinemia, decreased levels of cysteine and slightly elevated methionine compared to healthy +/−controls. We have not observed any of these features in −/−HO mice despite similar metabolic imbalance. The ERT essentially normalized cysteine levels and reduced homocysteine by 70–80 %. Treatment of −/−KO and I278T mice resulted in a complete reversal of the osteoporotic phenotype, with BMD and BMC being normalized to the levels found in healthy +/−controls, and significantly improved their total weight and fat content. Discussion: Unlike the HO model, the KO and I278T homocystinuric mice showed a profound osteoporosis, which can be entirely rescued by ERT administration. Conflict of Interest declared.

O-017

S-Adenosylhomocysteine alters methylation of cellular RNA Barroso M 1 2 3, Thuring K 3, Tserovski L 3, Gupta S 4, Kruger W D 4, Blom H J 5, Tavares de Almeida I 2, Loscalzo J 1, Castro R 2 6, Handy D E 1, Helm M 3

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 1

Brigham and Women’s Hosp, HMS, Boston, United States, 2iMed.ULisboa, Lisbon, Portugal, 3Johannes Gutenberg Univ, Mainz, Germany, 4Fox Chase Cancer Center, Philadelphia, United States, 5Center Pediatrics and Adolescent Med Univ, Freiburg, Germany, 6Dept Biochem Human Bio, Univ Lisbon, Lisbon, Portugal Background: S-Adenosylhomocysteine (SAH), which is elevated in hyperhomocysteinemia (HHcy), is, simultaneously, a product and a competitive inhibitor of most cellular methylation reactions. Our previous results have shown that SAH-induced DNA and protein hypomethylation can contribute to homocysteine-associated endothelial dysfunction. Our aim in this study was to investigate the effects of SAH accumulation on RNA methylation, namely, on 12 well-characterized RNA methylation modifications (Am, Cm, Gm, Um, m1A, m5C, m7G, m1G, m2G, m6A, m26A, m5U). Methods: LC-MS/MS was used to analyze methylation modifications in total and size-fractionated RNA samples from human endothelial cells and mouse tissue samples with intracellular excess SAH, induced pharmacologically or due to genetically determined HHcy, respectively. Results: We found that endothelial accumulation of SAH reduces the content of Cm, m1G, Gm, Am, and m6A modifications in total RNA by 16 ± 2.6 %, 18 ± 7.0 %, 11 ± 6.1 %, 7 ± 1.5 %, and 9 ± 1.2 %, respectively (p < 0.05). Additionally, in endothelial cells, methylation of the tRNA fraction was significantly affected by excess SAH with reductions in Cm and Gm modifications by 30 ± 9.6 % and 25 ± 7.6 %, respectively. A comparison of the modified nucleoside content from hyperhomocysteinemic and wild-type mice revealed significant hypo- and hyper-methylation of specific moieties. Specifically, Cm, Gm, and Um content was decreased in RNA from different fractions of liver and kidney (p < 0.05). However, in brain, different methylation modifications were increased during HHcy; namely, in the tRNA fraction, m1A, m5C, m1G, and m5U were each up-regulated by over 11 % (p < 0.05). Discussion: Our results reveal that HHcy can promote both hyper- and hypomethylation of RNA in vivo, depending on the specific RNA modification, RNA fraction, and tissue. These findings show that alterations in intracellular SAH affect RNA methylation and provide insights into new pathways that may be altered in HHcy.

08. Other amino acid disorders

O-018

Minimal NTBC concentrations necessary to prevent formation of succinylacetone in tyrosinemia type 1 patients Van Reemst H E 1, Kienstra N S 1, Van Ginkel W G 1, Van Dam E 1, De Blaauw P 1, Daly A 2, MacDonald A 2, Heiner-Fokkema M R 1, Van Spronsen F J 1 1 Beatrix Child Hosp UMCG, Groningen, Netherlands, 2Birmingham Child Hosp, Birmingham, United Kingdom

Background: In hereditary tyrosinemia type 1 (HT1) the dose and frequency of giving NTBC (nitisinone [OrphadinR]) is still disputable. Unfortunately, raised succinylacetone (SA) concentrations are sometimes found in clinic, suggesting suboptimal NTBC treatment. Therefore, this project aimed to determine the minimal concentrations of NTBC resulting in the absence of toxic metabolites. Methods: 18 patients were analysed. All patients took NTBC (13 once and 5 twice daily). Blood spots for analysis of NTBC and SA were collected four times daily for three consecutive days. SA concentrations were considered to be raised from a level of 0.6 μM onward. Mean NTBC levels and the number of SA concentrations ≥0.6 μM were calculated. Statistical analyses were performed by using Mann–Whitney U tests, Spearman correlational analyses and Friedman test. Results: Mean NTBC intake was 1.1 (0.66–2.0) mg/kg/day. NTBC concentrations did not vary significantly during the day (p = 0.457). Mean NTBC levels were negatively correlated to the number of SA ≥0.6 μM per patient (ρ = −0.514, p = 0.029). When NTBC was given twice a day, no SA ≥0.6 μM were seen, while in the group receiving NTBC once a day SA ≥0.6 μM were seen in 7 patients (54 out of 156 samples). No significant

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 difference in mean NTBC levels between the different treatment regimens was seen (p = 0.289). However, in patients with high SA concentrations, the mean NTBC level was 26.5 μM (range: 19.4–34.0 μM), significantly lower compared to patients who did not have SA concentrations ≥0.6 μM (mean NTBC 35.6 μM, range: 16.8–47.3 μM) (p = 0.027). Discussion: Mean NTBC concentrations should be at least 35 μM to prevent SA formation. Although NTBC concentrations did not significantly differ between the two different treatment regimes, giving NTBC twice a day could possibly prevent the formation of SA more effectively giving the same dose. Conflict of Interest declared.

O-019

Genetic cause and prevalence of hydroxyprolinemia Staufner C 1, Haack T B 2 3, Feyh P 1, Gramer G 1, Ediga Raga D 1, Terrile C 3, Sauer S 1, Okun J G 1, Fang-Hoffmann J 1, Mayatepek E 4, Prokisch H 2 3, Hoffmann G F 1, Koelker S 1 1

Div Neuroped and Met Med, Univ Child Hosp, Heidelberg, Germany, Institute of Human Genetics, TU Munich, Munich, Germany, 3Inst of Hum Gen, Helmholtz Zentrum Munich, Neuherberg, Germany, 4Dept Gen Ped, Univ Child Hosp, Duesseldorf, Germany 2

Background: The genetic cause and prevalence of hydroxyprolinemia, an inborn error of amino acid metabolism considered to be a non-disease, have remained unclear. As in MS/MS newborn screening the mass spectrum of hydroxyproline cannot be differentiated from isoleucine and leucine, cases of hydroxyprolinemia cause false-positive newborn screening test results for maple syrup urine disease (MSUD). Methods: 2 siblings with hydroxyprolinemia were studied via exome sequencing and the candidate gene found was confirmed in 5 further affected individuals, who were all characterized biochemically and clinically. Based on the number of individuals with hydroxyprolinemia detected via MS/MS newborn screening at our centre from 2003 to 2014, the prevalence of hydroxyprolinemia was calculated. Results: In 6 cases, homozygous or compound heterozygous mutations in PRODH2 were identified as the underlying genetic cause of hydroxyprolinemia. One subject was heterozygous for a deletion in PRODH2 and had an intermittent biochemical phenotype with partial normalization of hydroxyproline concentrations. In all individuals with biallelic mutations in PRODH2, plasma hydroxyproline concentrations were clearly elevated. All studied individuals remained asymptomatic, arguing in favour that hydroxyprolinemia is a non-disease. The prevalence of hydroxyprolinemia in Germany is about 1 in 47,300 newborns. Discussion: Mutations in PRODH2 cause human hydroxyprolinemia, an autosomal-recessively inherited benign condition, via impaired dehydrogenisation of hydroxyproline to delta1-pyroline-3-hydroxy-5-carboxylic acid. We suggest PRODH2 be renamed HYPDH. Hydroxyprolinemia is a frequent cause of false positive screening results for MSUD, the prevalence being about 2.5 times higher than that of MSUD.

O-020

Gain of function mutation in GLS1 causes infantile onset cataract and profound cognitive impairment Rumping L 1, Tessadori F 2, Vringer E 1, Pouwels P 5, Savelberg S M C 3, Bakkers M 6, Ramos R J J 1, Schellekens P A W 7, Duran K J 3, van der Knaap M S 5, Prinsen H C M 1, Houwen R H J 4, van Haaften G W 3, Verhoeven-Duif N M 1, van Hasselt P M 1, Jans J J M 1 1 Div Metab Dis, Univ Child Hosp, Utrecht, Netherlands, 2Hubrecht Inst, Utrecht, Netherlands, 3 Div Gen, Univ Med Cen Utrecht, Utrecht, Netherlands, 4Div Ped, UMCU, Utrecht, Netherlands, 5Div Neu, VU, Amsterdam, Netherlands, 6Div Vet Med, Univ Utrecht, Utrecht, Netherlands, 7 Ophthal, Univ Med Cen Utrecht, Utrecht, Netherlands

Background: Lens transparency, essential for optimal vision, is constantly threatened by various cellular stressors. A highly specialized lens metabolism counteracts the effect of these stressors, thereby preventing cataract formation. Uncovering genetic defects leading to cataract may shed light on the molecular mechanism underlying cataract formation and permits the design of previously unimaginable gene-targeted treatments by interfering in the pathophysiological pathway. Methods: We identified a new inborn error of metabolism caused by a de novo mutation in GLS1 encoding glutaminase in a patient with infantile cataract and profound cognitive impairment. Metabolic consequences of the mutation were examined in patient materials and with an in vitro GLS1 transfection model. Furthermore, we developed a genetically modified zebrafish model expressing the mutant GLS1 and investigated the capacity of glutaminase inhibition to prevent cataract formation. Results: The glutamate:glutamine ratio increased in urine, fibroblasts and brain of the patient as well as in our cell transfection model, confirming a gain of function. The reduced:oxidized glutathione ratio decreased and the level of reactive oxygen species increased, both indicating oxidative stress. Expressing mutant GLS1 in zebrafish induced cataract. Glutaminase inhibition normalized the amino acid profile and oxidative state in vitro and reduced cataract severity in vivo. Discussion: Our study describes a new inborn error of metabolism and identifies GLS1 as a causal gene and glutamate as a key molecule for cataract formation, pointing to glutaminase inhibition as a novel strategy for cataract treatment.

09. Urea cycle disorders

O-021

Ammonia activates hepatic autophagy in vivo and its enhancement protects against acute and chronic hyperammonemia Soria L 1, Pastore N 2, Annunziata P 1, Polishchuk E 1, Montefusco S 1, Medina D 1, Polishchuk R 1, Ballabio A 1 2 3, Brunetti-Pierri N 1 3 1 TIGEM, Pozzuoli (NA), Italy, 2Dept Mol and Hum Genet, Baylor Coll Med, Houston, United States, 3DISMET Federico II University, Naples, Italy

Background: Systemic ammonia is elevated in patients with inherited urea cycle disorders, liver injury, or chronic liver disease. Hyperammonemia may result in irreversible brain damage if not treated early and thoroughly, and current treatments are often inadequate. In cell cultures ammonia has been found to stimulate autophagy, a catabolic process that degrades cytoplasmic components and organelles in the lysosome. Here, we investigated whether autophagy is also induced in vivo and whether modulation of autophagy plays a role in the severity of hyperammonemia. Methods: Liver autophagy was determined in wild-type C57BL/6 mice during acute hyperammonemia induced by intraperitoneal (i.p.) injection of ammonium chloride. Ammonia handling was investigated under conditions of either autophagy induction or deficiency, induced either chemically or genetically. Enhancement of autophagy was also tested in chronic hyperammonemia induced by a standard diet supplemented with ammonium acetate (20 % w/w) and in a thioacetamide-induced model of acute liver failure. Results: Hepatic autophagy was activated in livers during acute hyperammonemia and suppression of autophagy impaired clearance of ammonia. Enhancement of autophagy by liver-directed gene transfer of TFEB, a master regulator of autophagy and lysosomal function, protected against acute hyperammonemia. Ammonia detoxification was also ameliorated by autophagy-inducing small molecules. The induction of autophagy was associated with increased ureagenesis and higher levels of N-acetylglutamate precursors, ATP, and aspartate that activate and fuel the urea cycle. Pharmacological enhancement of autophagy reduced blood ammonia in chronic and acute liver failure-induced hyperammonemia. Discussion: Taken together, these data show that hepatic autophagy is an important mechanism for ammonia detoxification. Enhancement of autophagy has potential for therapy of urea cycle disorders and secondary causes of hyperammonemia.

S42 10. Organic acidurias: branched-chain

O-022

A fish model for propionic acidemia: increased survival and improvement of neurological phenotype by anaplerotic diet Ginocchio V 1, De Felice E 1, Barrows F T 2, Sepe R M 3, Sordino P 3, Salierno F G 1, Conte I 1, Brunetti-Pierri N 1 4 1 TIGEM, Pozzuoli (NA), Italy, 2Dept Mol and Hum Genet, Baylor Coll Med, Houston, United States, 3Stazione Zoologica Anton Dohrn, Naples, Italy, 4 DISMET Federico II University, Naples, Italy

Background: Propionic acidemia (PA) is an inborn error of metabolism caused by deficiency of mitochondrial propionyl-CoA carboxylase (PCC). The disease presents with acute and life-threatening crisis of metabolic decompensation starting from the newborn period. Patients suffer from multi-organ complications, neurological dysfunction, and cardiomyopathy. Despite treatment mortality and morbidity remain elevated and investigation of novel and more effective therapies is highly needed. Methods: We used the medaka fish (Oryzias latipes) to generate a model of PA using a pair of transcription activator-like effector nucleases targeting the medaka pccb gene encoding one of the subunits of PCC. The PA medaka model was phenotyped and used for treatment investigations. Results: pccb−/− medaka showed defective hatching, developmental delay, reduced locomotor activity, and early lethality. Importantly, pccb−/− larvae showed a significant increase in the levels of C3 (propionylcarnitine), the biochemical hallmark of the disease. Administration of non-protein energy sources and carnitine improved survival. Because PCC deficiency induces deficiency of Krebs cycle intermediates, we hypothesized that an anaplerotic therapy supplying these intermediates would improve the phenotype of pccb−/ − medaka. To test this hypothesis, sodium citrate, ornithine alphaketoglutarate, and glutamine were administered to pccb−/− larvae and they resulted in significant improvements of both survival and locomotor activity. Discussion: We generated a fish model of PA recapitulating the major clinical and biochemical features observed in human patients. Anaplerotic therapy supplying intermediates of the Krebs cycle significantly improved the fish phenotype and has high potential for clinical translation. The PA medaka model might also be a useful tool to investigate novel drugs given its small size, easy handling and large number of progeny that make it suitable for largescale drug screening.

O-023

Medium term outcome of liver transplantation for children with propionic acidaemia Vara R 1, Mundy H 1, Grammatikopoulos T 3, Heaton N 2, Rela M 2, Dhawan A 3, Hadzic N 3 1 Dept Met Dis, Evelina Children’s Hosp, London, United Kingdom, 2Inst of Liver Studies, King’s Coll Hosp, London, United Kingdom, 3Paediatric Liver Center, King’s College Hosp, London, United Kingdom

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Indications were frequent metabolic decompensations in 9, family history in 2 and elective in 2. Median waiting time for LT was 213 days (49–1064). Seven children received left lateral segment grafts, 2 left lateral segment and 2 right lobe auxiliary grafts, 1 whole graft and 1 live related donor. Two children required re-transplantation (hepatic artery thrombosis and chronic cholangiopathy). Median hospital stay was 25 days (13–482). Patient and graft survival was 75 % and 85 %, respectively. Three children died, 2 following postLT complications (pulmonary haemorrhage and biliary sepsis) at 29 and 42 days respectively. One died 14 years post-LT with lymphoproliferative disorder. There have been no metabolic decompensations or evidence of cardiomyopathy post-LT in 9 of the surviving patients. All are on protein unrestricted diets and standard immunosuppression. Median follow up post-LT is 4 years (0.6– 20.5 years) and median age at follow up is 8.4 years (2.3–22.3 years). Discussion: LT in PA reduces number of metabolic crises and continues to be a valuable treatment option for selected children.

O-024

Propionate anions, accumulated in propionic acidemia, affect cardiac excitation-contraction coupling, gene regulation and cellular growth, which may contribute to heart dysfunction Ford K 1, Hulikova A 1, Swietach P 1 1

Oxford University, Oxford, United Kingdom

Background: Propionic acidemia (PA) is a rare metabolic disorder caused by a deficiency in propionyl-CoA carboxylase. The resulting accumulation of propionic acid and derivatives in blood produces acidosis, but the mechanistic link to organ toxicity is unclear. Since cardiac myopathies are a common cause of mortality, this study explored the effects of propionic acid on cardiac myocytes. Methods: Cardiac ventricular myocytes were isolated from adult or neonatal rats euthanized humanely. Excitation-contraction (EC) coupling was imaged fluorescently in adult myocytes loaded with the Ca2+-reporter dye Fluo3 and paced electrically. In cultured neonatal myocytes, acetylation of histone H3, measured by immunoblot, was used as one readout of epigenetic changes in gene expression. Cellular hypertrophy was measured in neonatal myocytes in terms of cell size. Control solutions contained 5 % CO2 and 24 mM HCO3−. The median base excess reported in PA patients is 6 mM, therefore test solutions contained 5 % CO2, 6 mM propionate, 0.3 mM NH4Cl and 18 mM HCO3− to mimic a mild uncompensated acidosis with hyperammonemia. Results: Propionic acid affected Ca2+ transients by raising systolic Ca2+ and slowing recovery, consistent with an inhibitory effect on the SERCA Ca2+ pump of the sarcoplasmic reticulum (SR). Propionic acid reduced the variability of spontaneous Ca2+ leak from an unstimulated SR, indicating a stabilizing effect on RyR Ca2+ release channels. 24-h incubation with propionic acid increased H3 acetylation to a level comparable to the effect of butyrate, a histone deacetylase inhibitor. This treatment also increased cell size by 30 %, which may relate to altered gene expression. Discussion: Propionic acid affects EC coupling acutely, as well as gene expression chronically, resulting in cellular hypertrophy. These changes are associated with many different types of cardiac myopathy. A better understanding of propionate targets would offer more refined PA management.

O-025 Background: Children with propionic acidaemia (PA) continue to have poor neurological outcome despite maximal medical therapy. Liver transplant (LT) has shown favourable short term outcomes. Methods: Retrospective review of children with PA transplanted in a tertiary liver transplant centre between 1995 and 2015. Results: Thirteen children identified (5 published 2011). Seven male; median age at presentation of 3 days (0–84). Nine had classical neonatal presentation, 2 at 1 and 3 months of age, respectively, with chronic encephalopathy and 2 following prenatal diagnosis. At presentation 6 had seizures and median ammonia of 175 umol/L (47–1978 umol/L). All children were treated with standard medical therapy and 8 required gastrostomy feeding prior to LT. Developmental delay was present in 10 children at LT. Median age at LT was 2 years (1–7).

Axonal peripheral neuropathy in propionic acidaemia: a severe side effect of long-term metronidazole treatment Diodato D 1 2, Maiorani D 1, Deodato F 1, Taurisano R 1, D’Amico A 2, Di Capua M 5, Boldrini R 3, Della Bella J 4, Bertini E 2, Dionisi-Vici C 1 Div Metab Dis, Child Hosp Bambino Gesu, Rome, Italy, 2Musc Neurodeg Dis, Child Hosp Bamb Gesu, Rome, Italy, 3Pathology Div, Child Hosp Bambino Gesu, Rome, Italy, 4Ped Rehab Div, Child Hosp Bambino Gesu, Rome, Italy, 5Neurology Div, Child Hosp Bambino Gesu, Rome, Italy 1

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Standard treatment of propionic acidaemia (PA) is based on a low-protein diet, carnitine supplementation and, although there is very limited evidence of its efficacy, chronic cyclic metronidazole (MTZ) administration, which is often used to reduce the intestinal production of propionate by bacterial gut flora. Here we report on three teenage PA patients, all receiving chronic intermittent MTZ administration, who presented with axonal peripheral neuropathy secondary to MTZ therapy. Case Report: P1 and P2, on good metabolic control and receiving cyclic MTZ courses (P1, 500 mg/day, 3 weeks/month; P2 500 mg/day, 1 week/month), presented with lower limbs hyporeflexia, paresthesias/hyperaesthesias and subacute loss of deambulation. Nerve conduction studies (NCS) displayed a sensory-motor axonal neuropathy of lower limbs. Skin biopsy showed reduced intraepidermal small nerve fibers, consistent with the diagnosis of axonal neuropathy. CSF studies, autoimmune and virological profile, and vitamins serum levels were normal. Suspecting MTZ-induced neuropathy, the drug was discontinued and both patients showed a progressive recovery of deambulation and improvement of NCS parameters. A second skin biopsy (P1), showed the normalization of previous changes with reappearance of subepidermal nerve fibres. Retrospectively, an additional patient on cyclic MTZ presented with identical clinical signs. However, further investigations were not performed because she died suddenly from metabolic decompensation a few days later. Results and Discussion: Axonal neuropathy is a known side effect of MTZ therapy. Our patients were extensively investigated, and the causal and temporal relationship with MTZ therapy was demonstrated. We aim to alert physicians on the potential risk of this severe iatrogenic complication, suggesting physicians reconsider the use of MTZ in BCOAs, whose efficacy is yet to proven. Neurological evaluation and NCS must be periodically performed in BCOA patients receiving MTZ therapy.

O-026

Stable isotope breath testing to assess in vivo metabolite flux in methylmalonic acidemia. From mouse models to patients Manoli I 1, Harrington E A 1, Ktena Y P 1, Gagne J 1, Smyth S 2, Hattenbach J 2 , Senac J 1, Sloan J L 1, Chen K Y 2, Venditti C P 1 1 Organic Acid Res Section, NHGRI, Bethesda, United States, 2Clin Endocrin Branch, NIDDK, NIH, Bethesda, United States

Background: Isolated methylmalonic acidemia (MMA), caused by a defect in the methylmalonyl-CoA mutase (MUT) enzyme, is characterized by increased morbidity and mortality. Elective liver (LT), kidney (KT) or combined liver and kidney transplantation (LKT) are used to stabilize severely affected patients. Methods: Mice expressing the Mut gene under the control of a muscle-specific creatine kinase promoter (Mut−/−;TgINS-MCK-Mut) were used to probe metabolic flux. 1-13C-propionate oxidation was used to assess in vivo Mut activity, while 1-13C-methionine, glycine, α-ketoisocaproic acid and octanoate oxidation studies were used to probe secondary mitochondrial dysfunction. In patients, CO2 production was measured by indirect calorimetry, and serial breath samples were collected following an oral bolus of 1-13C-propionate. 31 MMA patients (mut0 n = 19, mut− n = 6, cblA n = 5, cblB n = 1, age range 4–41 y), including 3 KT, 1 LT and 5 LKT recipients, and 15 controls were studied. Results: Mut −/− ;Tg INS-MCK-Mut mice displayed decreased propionate (p = 0.003), methionine (p = 0.03) and glycine (p = 0.008) oxidation compared to heterozygote littermates. Decreased propionate oxidation was observed in MMA patients versus controls (p < 0.0001), with intermediate results in cblA and mut− patients. Patients carrying two nonsense mutations (n = 3), showed almost no movement of label, despite isolated KT in one individual. LT and LKT recipients demonstrated restoration of oxidation rates to control levels (p = NS). Propionate oxidation was stable on repeat testing despite markedly different plasma methylmalonic acid concentrations. Discussion: 13C-breath testing can be used to track whole body MUT activity and secondary metabolic perturbations, even in the presence of an expanded trace pool. They can serve as outcome measures to interventions, such as transplantation or gene or small molecule therapy, in MMA and other disorders where branched-chain amino acid oxidation and mitochondrial function are disturbed.

O-027

Insights into disease mechanisms of cblA-type methylmalonic aciduria from 67 new patients and functional MMAA missense mutation characterization Plessl T 1 3, Buerer C 1, Lutz S 1, Baumgartner M 1 2 3, Froese D S 1 2 1 Univ Child Hosp, Division of Metabolism, Zurich, Switzerland, 2radiz—Rare Disease Initiative Zurich, Zurich, Switzerland, 3Zurich Cent for Integr Human Physiology, Zurich, Switzerland

Background: In humans, vitamin B12 (cobalamin) is converted to adenosylcobalamin (AdoCbl) to function as cofactor for the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). In this multi-step pathway, the functions of MMAA, a G3E family GTPase, and MUT are inextricably linked. MUT stimulates MMAA GTPase activity and MMAA mediates AdoCbl incorporation into MUT. A disturbed function of this process causes the cblA-type of methylmalonic aciduria, but knowledge of the mechanism of dysfunction in MMAA is still lacking. Methods: We collected data from 67 unpublished patients with cblA-type MMA, referred for diagnostic work-up at Zurich. We analysed all known (22) MMAA missense mutations for their protein stability, nucleotide binding ability, GTPase activity, and complexation with and GTPase stimulation by MUT. Results: We found that most patients from this cohort presented with early onset of disease ( A; p.Lys329Glu). In contrast to other fatty acid oxidation disorders that affect liver, skeletal muscle and in part the brain, MCAD deficiency primarily presents with liver pathology. Previous studies of the cellular and tissue-specific consequences using ex vivo studies of dermal fibroblasts from patients and a knock-out mouse model produced only limited insights into the mechanisms of the liver pathophysiology. Methods: We produced induced pluripotent stem cells (iPSCs) from fibroblasts derived from a patient homozygous for the prevalent mutation, using a lentivirus-based reprogramming method, which delivers a polycistronic cassette of four transcription factors (OCT4, KLF4, SOX2, C-MYC). We characterized the pluripotency and differentiation capacity of the MCADD iPSCs, and investigated relevant parameters related to fatty acid oxidation in both iPSCs and iPSC-derived hepatocytes. Results: Unlike the fibroblasts they were derived from, iPSCs homozygous for the MCAD mutation display strongly reduced growth. Growth inhibition could be rescued by addition of carnitine to the growth medium, allowing the cells to export accumulating C10 and C8 acylcarnitine

14. Mitochondrial disorders: nuclear encoded, disorders of pyruvate metabolism and the Krebs cycle

O-038

Decanoic acid treatment of fibroblasts from patients with nuclearencoded complex I deficient Leigh syndrome: a step towards personalised medicine? Kanabus M 1, Khabbush A 1, Fassone E 1, Hughes S D 1, Bilooei S F 1, Rutherford T 3, O’Donnell M 3, Heales S J R 1 2 4, Rahman S 1 2 1 UCL Institute of Child Health, London, United Kingdom, 2Great Ormond Street Hospital, London, United Kingdom, 3Vitaflo International, Liverpool, United Kingdom, 4National Hospital for Neurology, London, United Kingdom

Background: Decanoic acid (C10) is a ligand for the nuclear receptor PPAR-γ, which is known to be involved in mitochondrial biogenesis. Our previous

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 studies showed that C10 induces mitochondrial biogenesis and increases complex I and catalase activity in neuronal cells. No effective treatments exist for the vast majority of mitochondrial diseases. In this study we now aimed to examine the effects of C10 treatment on mitochondrial function in primary fibroblasts from a cohort of patients with nuclear-encoded complex I deficient Leigh syndrome, the most severe clinical manifestation of mitochondrial disease. Methods: The effects of C10 were investigated in primary fibroblasts, using mitochondrial respiratory chain enzyme assays, gene expression microarray, quantitative polymerase chain reaction (qPCR) and flow cytometry analysis of mitochondrial volume and reactive oxygen species (ROS). Results: Treatment with C10 increased citrate synthase activity in 50 % of studied fibroblasts in a PPAR-γ–mediated manner. Affymetrix GeneChip 1.0 ST array and qPCR studies suggested that treating cells with C10 supports fatty acid metabolism, through increasing ACADVL and CPT1 expression, whilst genes involved in glucose metabolism (PDK3, PDK4) are downregulated. PCK2, involved in blocking glucose metabolism, was upregulated. CAT, encoding catalase, was also upregulated. Discussion: We hypothesize that C10 results in a mild increase of mitochondrial ROS, which is counterbalanced by an increase in catalase, but may be sufficient to trigger adaptive responses including increased mitochondrial biogenesis, which could be of benefit for patients with mitochondrial diseases. Furthermore the assay methods used here could be used to identify suitable patients for further investigation of the efficacy of C10 in a clinical trial, in a ‘personalised medicine’ approach. Conflict of Interest declared.

O-039

A novel causative gene of mitochondrial respiratory chain disorders in an apparent life-threatening event Matsunaga A 1, Murayama K 1, Nyuzuki H 2, Kishita Y 2, Tokuzawa Y 2, Kohda M 2, Okazaki Y 2, Ohtake A 3 1 Dept Metab, Chiba Children’s Hosp, Chiba, Japan, 2Res Cen Genomic Med, Saitama Med Univ, Saitama, Japan, 3Dept Ped, Saitama Med Univ, Saitama, Japan

Background: It is well known that inborn errors of metabolism cause sudden unexpected death (SUD) or apparent life-threatening events (ALTE). Mitochondrial respiratory chain disorder (MRCD) refers to a common group of inborn errors of metabolism, affecting at least 1 in 5,000 individuals. It is also known to be a cause of SUD or ALTE. Case Report: This patient was a boy, born at 39 weeks and 6 days, and his birth weight was 4090 g, a big baby in comparison to the gestational age of Japanese. Cardiopulmonary arrest occurred unexpectedly at 1 month old when he was asleep in the prone position. He was resuscitated, but he died at the age of 1 year due to hypoxic ischemic encephalopathy. Results: The enzyme assay of his dermal fibroblasts revealed reduction of complex IV activity. Whole exome sequencing prioritised mutations in PNPLA4 as a candidate gene. The PNPLA4 gene is located on chromosome X and he inherited a hemizygous nonsense variant c.559C > T in PNPLA4 from his mother. PNPLA4 gene expression and protein expression were reduced in his fibroblasts. The colocalization of PNPLA4 and mitochondrial markers was identified by immunofluorescence microscopic observation. We found reduced assembly of complexes I, III and IV in his fibroblasts. The expression of wildtype PNPLA4-V5 cDNA in his fibroblasts recovered the amount of assembled complexes III and IV. Discussion: PNPLA4 encodes a calcium-independent phospholipase A2η (iPLA2η) that acts as an acylglycerol and retinol transacylase, triglyceride hydrolase. PNPLA4 has not previously been reported to associate with the mitochondria. We assume that PNPLA4 is also required for mitochondrial phospholipid metabolism and respiratory chain function. Conclusion: We report that PNPLA4 is a novel causative gene for MRCD presenting with ALTE. We need further investigation to elucidate PNPLA4 function in mitochondria, which might help to clarify the pathogenic mechanisms of SUD or ALTE and to help prevent them in the future.

O-040

Clinical exome sequencing in 900 index cases: diagnostic rate and new disease genes Haack T B 1 2, Alhaddad B 1, Kovacs-Nagy R 1, Berutti R 2, Strom T M 1 2, Zschocke J 3, Konstantopoulou V 7, Makowski C 8, Mayr J A 4, Sperl W 4, Karall D 3, Hoffmann G F 5, Freisinger P 6, Prokisch H 1 2, Meitinger T 1 2 Technische Univ Muenchen, Munich, Germany, 2Helmholtz Zentrum Muenchen, Neuherberg, Germany, 3Univ Innsbruck, Innsbruck, Austria, 4 Paracelsus Medical University, Salzburg, Austria, 5Univ Heidelberg, Heidelberg, Germany, 6Klinikum Reutlingen, Reutlingen, Germany, 7Univ Wien, Wien, Austria, 8Kinderklinik Schwabing, Munich, Germany 1

Background: Exome sequencing has become a standard tool to investigate patients with rare presumed genetically-based disorders. In a prospective study over the course of 2 years, we evaluated the efficacy of exome sequencing across a broad spectrum of clinical conditions, mainly pediatric cases with neurological and metabolic diseases. Methods: We investigated 900 cases with suspected genetic disorders using clinical exome sequencing. We developed standardized algorithms for the diagnostic interpretation of exome datasets as well as the prioritization of novel disease genes in single individuals or cohorts with overlapping phenotypes. Results: We established firm molecular diagnoses in about 45 % of patients, with mutations being identified in >220 different genes. We identified clinically relevant variants in both known disease genes (≈40 %) and genes not previously associated with human disease (≈5 %) including recently published genes such as UNC80 and TANGO2. Extended analyses of the unresolved cases led to the identification of >20 novel candidate disease genes involved in a variety of cellular processes such as mitophagy, mitochondrial RNA metabolism, myelination, and ribosylation. Discussion: The implementation of standardized interpretation algorithms substantially added to the diagnostic yield. Unbiased statistical approaches contribute to the prioritization of genetic defects in novel disease genes underlying inborn errors of metabolism.

O-041

Cytosolic phosphoenolpyruvate carboxykinase deficiency presenting with acute liver failure following gastroenteritis Santra S 4, Cameron J 5, Shyr C 2, Wassermann W 2, Wevers R 3, Rodenburgh R 3, Gupte G 4, Preece M A 4, Van Karnebeek C 1 1 Dept of Pediatr, Univ British Columbia, Vancouver, Canada, 2Dept of Med Genet, Univ British Columbia, Vancouver, Canada, 3Dept Lab Med, Radboud Univ Med Centre, Nijmegen, Netherlands, 4Birmingham Children’s Hospital, Birmingham, United Kingdom, 5Peter Gilgan Centre Research Learning, Toronto, Canada

Background: We report a patient from a consanguineous family who presented with transient acute liver failure and biochemical patterns suggestive of disturbed urea cycle and mitochondrial function, for whom conventional genetic/metabolic investigations for acute liver failure failed to yield a diagnosis. Methods: Trio whole exome sequencing was performed (42X) with bioinformatics analysis via our TIDEX customized pipeline and subsequent Sanger sequencing of candidates. PEPCK activity was measured in transfected mutant versus wildtype COS-1 cells in the direction of oxaloacetate formation (published methods). Results: Of the 14 genes harbouring recessive/hemizygous (n = 9) and de novo (n = 5) mutations, the homozygous 12-bp deletion (p.GVPLV123V) in PCK1 encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) was the most compelling based on function and (likely) pathogenic nature of the variants. Compared to wildtype transfected COS-1 cells, mutant PEPCK activity was significantly reduced, similar to the empty vector, confirming the deleterious nature of the homozygous PCK1 mutant.

S48 Discussion: We propose that PEPCK deficiency should be considered in a young child with unexplained liver failure, especially with TCA cycle metabolite accumulation on urine organic acid/amino acid profiles with hyperammonemia suggestive of a proximal urea cycle defect during the acute episode. If suspected, intravenous administration of dextrose should be initiated. Long-term management comprising avoidance of fasting with the provision of a glucose polymer emergency regimen for illness management may be sufficient to prevent future episodes of liver failure. This case report provides further insights into the (patho-) physiology of energy metabolism, confirming the power of genomic analysis of unexplained biochemical phenotypes.

15. Mitochondrial disorders: mtDNA

O-042

Urinary organic acids in paediatric single mitochondrial DNA deletion disorders Boenzi S 1, Diodato D 2, Carrozzo R 2, Verrigni D 2, Goffredo B M 1, Semeraro M 1, Bertini E S 2, Taurisano R 1, Dionisi-Vici C 1, Rizzo C 1 1 Div Metab Res Unit Metab Bioch, OPBG, Rome, Italy, 2Unit Neuromusc Neurodeg Dis, OPBG, Rome, Italy

Background: Single mitochondrial DNA (mtDNA) deletions are classified into three main phenotypes: Pearson marrow-pancreas syndrome (PMS), Kearns-Sayre syndrome (KSS) and chronic progressive external ophthalmoplegia. However, clinical overlap is often present. Thus far, few studies have investigated the urinary organic acids profile of these diseases. Methods: We have retrospectively evaluated by GC/MS the organic acids profile in several urine samples collected from a series of 9 pediatric patients with single mtDNA deletion, 4 with PMS and 5 with KSS. Results: All PMS patients (4/4) presented with very large urinary excretion of lactate, pyruvate, 3-hydroxy-butyrate, 3-hydroxy-isobutyrate and fumarate. In 3/4 patients, 2-ethyl-3-hydroxy-propionate and 2-methyl-2,3-dihydroxy-butyrate were detectable. Overall, KSS patients showed less pronounced changes of urinary organic acids excretion when compared to PMS. The following metabolites were detectable: lactate 4/5, pyruvate 3/5, 3-hydroxy-butyrate 2/5, 3-hydroxy-isobutyrate 2/5, fumarate 2/5, 2-ethyl-3-hydroxy-propionate 2/5, and 2-methyl-2,3-dihydroxy-butyrate 2/5. Discussion: Our results showed that PMS patients show urinary organic acids profile sensibly altered compared to KSS patients, in all samples analyzed. Organic acids profile was almost normal in most of samples from KSS, or showed poor organic acids excretion. Both syndromes showed detectable 3hydroxy-isobutyrate and 2-methyl-2,3-dihydroxy-butyrate, which have recently been proposed as the characteristic biomarkers of HIBCH and ECHS1 deficiencies, associated with Leigh-like disease. Differently from previous reports, we did not detect 3-methylglutaconic aciduria. Although the urinary organic acids abnormalities are not specific for single mtDNA deletion disorders, PMS patients show more pronounced changes than KSS. Careful differential diagnosis with newly identified branched-chain amino acids defects is indicated.

18. Lysosomal disorders: mucopolysaccharidoses, oligosaccharidoses

O-043

A reliable multiplex mass spectrometry analysis of glycosaminoglycans for mucopolysaccharidoses Auray-Blais C 1, Lavoie P 1, Tomatsu S 2, Valayannopoulos V 3, Mitchell J 4, Raiman J 5, Beaudoin M 1, Maranda B 1, Clarke J T R 5

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 1

Dept Ped, Universite de Sherbrooke, Sherbrooke, Canada, 2Nemours, Hosp for Children, Wilmington, United States, 3Hosp Univ Necker Enfants Malades, Paris, France, 4McGill Univ Health Center, Montreal, Canada, 5 Dept Ped, Hosp for Sick Children, Toronto, Canada Background: Mucopolysaccharide (MPS) disorders are the result of primary defects in lysosomal enzymes leading to glycosaminoglycan (GAG) accumulation. Objectives of the study were (1) to analyze a wider spectrum of urinary GAGs to improve the detection, diagnosis and monitoring of patients affected with various types of MPSs (MPS I, II, III, IV, and VI); (2) to develop and validate a quantitative multiplex mass spectrometry method for the urine analysis of dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS) disaccharides; and (3) to compare this method with the dimethylene blue (DMB) spectrophotometric method used in most biochemical laboratories. Methods: A methanolysis was performed by adding methanol-HCl·3 N to a dried urine sample, followed by heating at 65 °C for 60 min, evaporation under nitrogen, and dissolution of the residue in 200 mL of a solution containing synthesized in-house deuterated internal standards. 2 mL were injected onto an Acquity I-Class UPLC/Xevo TQ-S MS/MS (Waters Corp). Results: The method is rapid (7 min) and offers high resolution and specificity to differentiate various types of MPS patients and controls. Intraday precision assays were good (RSDs ≤7.6 %), as well as accuracy results with biases ranging from −12.0 to 18.4 %. Regarding interday precision and accuracy assays, RSDs were ≤8.7 % and biases ranged from −12.8 to 14.2 %. The linearity was acceptable (r2 > 0.995) for DS, HS, CS, and KS. Normal reference values were established for 5 age groups. The comparison of the MS/MS method with the DMB method showed that the latter test would have missed 30 % of MPS cases and provided 13 % borderline results, confirming its unreliability. Discussion: This rapid and efficient UPLC-MS/MS method allows the absolute quantification of each GAG separately, offering a profile which is useful for diagnostic purposes, follow up of patients, and evaluation of treatment efficacy. Conflict of Interest declared.

O-044

Long-term outcomes with rhGUS in a phase I/II clinical trial in MPS VII Coker M 2, Gonzales-Meneses A 4, Song W 3, Taylor J 3, Agarwal S 3, Haller C 3, Kakkis E 3, Jones A S 1 1

Ctr for Gen Med, Univ Manchester, Manchester, United Kingdom, 2Ege University, Izmir, Turkey, 3Ultragenyx Pharmaceutical, Novato, United States, 4Hosp Univ Virgen del Rocio, Sevilla, Spain Background: Recombinant human beta-glucuronidase (rhGUS, UX003) is an investigational therapy for mucopolysaccharidosis type VII (MPS VII, Sly syndrome), an ultra-rare autosomal recessive disease with a highly heterogeneous clinical presentation. Methods: Three patients ages 5, 9 and 25 years completed a 36-week dosetitration period in a phase I/II study of rhGUS administered intravenously every other week. The highest dose of 4 mg/kg was well tolerated and resulted in maximal reduction in urinary glycosaminoglycans (uGAG); all subjects continued in a long-term extension at this dose. Assessments included safety parameters, urine and serum GAG, spirometry and tests of endurance, motor function, visual acuity and subjective assessments. Results: Long-term dosing with UX003 resulted in sustained uGAG reduction, with change from baseline in dermatan sulfate of 60.0 % at week 109, 61.5 % at week 108 and 74.3 % at week 97 for subjects 201, 202 and 203, respectively. Although some assessments could not be done due to physical and cognitive impairments, all 3 subjects showed some clinical improvement. One subject showed marked improvement in spirometry from baseline to week 96 in FVC (2.9 L, 68 % predicted to 3.5 L, 83 % predicted) and maximum voluntary ventilation (77 L/min to 103 L/min), respectively. Variable improvements were seen in tests of fine motor precision and manual dexterity, shoulder range-ofmotion and subjective scores of appetite, fatigue/malaise and breathing

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 difficulty. Two of 3 subjects developed anti-drug antibodies (ADA) but no neutralizing activity was detected. No drug-related hypersensitivity reactions were reported and serious adverse events (SAEs) involved surgeries to correct MPS complications. Discussion: Stable reduction in uGAG was observed up to 108 weeks of treatment with no related SAEs or hypersensitivity reactions despite ADA formation in 2 subjects. Some improvement in various clinical parameters was observed including pulmonary function, joint mobility and fine motor function. Conflict of Interest declared.

O-045

Increased collagen glycosylated hydroxylysine in the urine of MPS I, II and VI patients Heywood W E 1, Banushi B 1, Doykov I 1, Patel N 1, Mills P 1, Footitt E 2, Cleary M 2, Clayton P T 1, Grunewald S 2, Chakrapani A 2, Hindmarsh P 1, Heales S J R 2, Burke D G 2, Ruggero T 3, Mills K 1, Gissen P 1 1 UCL Institute of Child Health, London, United Kingdom, 2Great Ormond Street Hospital, London, United Kingdom, 3University of Pavia, Pavia, Italy

Background: Early markers of disease severity for the mucopolysaccharidoses are needed to optimise and monitor treatment. Glycosaminoglycans provide limited prognostic information. Markers of pre-symptomatic affected processes may have potential to address this need. Extracellular matrix (ECM) disruption is an early feature of MPS. A major constituent of ECM is collagen, which undergoes post translational modification (PTM). Of interest are PTMs of collagen lysine, which include hydroxylation of lysine (Lys-OH), galactosylation of hydroxylysine (Lys-O-Glc) and glucosylation of galactosylhydroxylysine (Lys-O-GlcGlc) Methods: 34 patients included 2 groups with mild Sheie or moderate to severe Hurler + Hurler-Sheie (H + HS) MPS I disease (n = 16), MPS II with (severe) or without (mild) neurological involvement (13) and MPS VI (5) were analysed. An LC-MS/MS method was used. Results: Unmodified lysine residues were increased in the H + HS MPS I and MPS VI groups compared to controls (p < 0.03). Lys-O-GlcGlc and Lys-OGlc levels were significantly elevated between control, the H + HS MPSI and both MPSII mild and severe (neurological) groups and MPS VI (p < 0.02). Lys-O-Glc levels were significantly different between Sheie and H + HS MPSI (p < 0.04) Discussion: Lysine hydroxylation occurs in the ER by LH1 and LH2, whereas glycosylation of Lys-OH is performed by LH3 both intra- and extracellularly. Lys-OH levels were not significantly affected, however, there is a significant increase of unmodified lysine in the severe MPS I and II groups, indicating affected collagen turnover. Glycosylated collagen lysines, however, were greater and able to distinguish mild MPS II from controls and stratify the MPS I groups. Increased levels may indicate inefficient degradation of ECM, which may contribute to pathogenesis of MPS disorders. No observed linear relationship with age in the MPS I severe group indicating these markers have potential for use in early disease.

O-046

Initial, 24 week results of heparan sulfate levels in cerebrospinal fluid, brain structural MRI and neurocognitive evaluations in an open label, phase I/II, first-in-human clinical trial of intravenous SBC-103 in mucopolysaccharidosis IIIB Whitley C B 2, Escolar M L 3, Vijay S 4, Parker G 5, Roberts C 5, Zhang X 1, Cinar A 1, Bubb G 1, Patki K C 1, Rojas-Caro S 1 1

Alexion Pharmaceuticals, Inc., New Haven, United States, 2University of Minnesota, Minneapolis, United States, 3Children’s Hospital of Pittsburgh, Pittsburgh, United States, 4Birmingham Children’s Hospital, Birmingham, United States, 5Bioxydyn Limited, Manchester, United Kingdom

Background: Mucopolysaccharidosis IIIB is caused by a marked decrease in alpha-N-acetyl-glucosaminidase (NAGLU) enzyme activity, which leads to the accumulation of heparan sulfate (HS) in the key organs, progressive brain atrophy, and neurocognitive decline. Methods: This first-in-human open label study with 3 parallel dose groups administered SBC-103 [0.3 mg/kg (n = 3), 1 mg/kg (n = 4), and 3 mg/kg (n = 4)] as an intravenous (IV) infusion every other week in patients aged 2–12 years (developmental age ≥1 year). Primary objective: assessment of safety and tolerability of IV SBC-103. Secondary objectives: effect of SBC-103 on total HS levels in cerebrospinal fluid (CSF), brain structures (MRI), neurocognitive status, and pharmacokinetic (PK) profile of SBC-103. Results: Eleven patients were enrolled (median age 4 years). During the 24 weeks, there were 3 treatment-emergent serious adverse events (SAEs) in one patient and 6 infusion-associated reactions in 3 patients. No SBC-103 related SAEs occurred. At week 24, total HS percent change from baseline (mean [SD]) in the CSF was 10.9 [14.7], −0.43 [11.9] and −26.2 [20.9] for 0.3 mg/kg, 1 mg/kg, and 3 mg/kg groups, respectively. HS reduction in CSF was linearly correlated with SBC-103 serum PK exposures. Preliminary volumetric MRI (cortical gray matter volume) and neurocognitive outcomes (age equivalent/developmental quotient) suggested potential for disease stabilization in subjects receiving the 3 mg/kg QOW for 24 weeks compared to their baseline. Overall, response profiles among the 3 mg/kg treatment group suggests a potential dose effect as compared to 0.3 mg/kg and 1 mg/kg groups. Discussion: These initial observations suggest that IV SBC-103 was biologically active and well tolerated in MPS IIIB patients. Potential blood brain barrier penetration of IV SBC-103 was illustrated by PK/PD correlation for HS reduction in CSF. Preliminary evidence of disease stabilization was suggested by changes in brain structures and neurocognitive assessments in some patients. Conflict of Interest declared.

O-047

ZFN-mediated correction of murine MPS I and MPS II models by expression of the human alpha-L-iduronidase and iduronate-2-sulfatase cDNAs from the albumin locus Chester C B 1, DeKelver R 2, Laoharawee K 1, OU L 1, Tom S 2, Radeke R 2, Rohde M 2, Sproul S 2, Przybilla M J 1, Koniar B L 1, Podetz-Pedersen K 1, Cooksley R D 1, Meyer K 2, Holmes M 2, McIvor R C 1, Wechsler T 2 1

Univ of Minnesota, Dept of Pediatrics, Minneapolis, United States, Sangamo Biosciences Inc, Richmond, United States

2

Background: Mucopolysaccharidosis type I (MPS I/Hurler syndrome) and type II (MPS II/Hunter Syndrome) are caused by deficiencies of alpha-Liduronidase (IDUA) and iduronate 2-sulfatase (IDS), respectively, and subsequent systemic accumulation of glycosaminoglycans (GAGs), leading to widespread complications and a shortened lifespan. Current treatments are costly, can be inadequate and/or have significant risks. Methods: A ZFN-mediated genome editing strategy is proposed to permanently modify patient cells by targeted integration of a wt hIDUA or hIDS transgene into the albumin locus. Both MPS I and MPS II mice were treated with a single intravenous infusion of ZFN-AAV and transgene donor-AAV. Results: Following ZFN + donor AAV administration, supraphysiological protein activities were detected in the liver (60–200X of wt). In the plasma, stable, supraphysiological activity levels of IDUA or IDS were detected throughout the study (4 months). Significant IDS/IDUA activity was found in peripheral tissues: spleen, kidney, lung, heart and muscle (6 %–200 % of wt). Up to 2 % of wt IDUA/IDS activity was detected in the brain of treated animals. Urine and tissue GAGs were reduced in all treatment groups in both models. Animals from both studies showed cognitive benefits at 4 months after treatment in the Barnes maze and had behavior similar to wt mice. Histological observations suggested reduced cellular vacuolation, indicating reduced GAG accumulation in several tissues. Biochemical characterization of enzymes showed glycosylation and proteolytic processing similar to recombinant proteins. The proteins exhibited 6-mannose-phosphate depended uptake in primary hepatocytes. Discussion: Our data provides proof of concept for ZFN-mediated targeting of albumin in hepatocytes as an in vivo protein replacement platform to express

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284

therapeutic amounts of hIDUA or hIDS, leading to GAG biomarker correction and neurological benefits in relevant disease mouse models. Conflict of Interest declared.

Cell Research Center and Hospital, Tehran, Iran, Islamic Republic of, Children’s Hospital of Cordoba, Cordoba, Argentina, 15South Stockholm General Hospital, Stockholm, Sweden, 16University Medical Center Mainz, Mainz, Germany, 17Inst Fed de Biologie, CHU de Toulouse, Toulouse, France

19. Lysosomal disorders: sphingolipidoses

Background: Farber disease is caused by mutations in the ASAH1 gene, resulting in acid ceramidase deficiency and accumulation of the proinflammatory and pro-apoptotic lipid, ceramide. Patients usually present in infancy or late childhood with one of three characteristic symptoms: earlyonset polyarticular arthritis, subcutaneous nodules or dysphonia. Acid ceramidase deficiency can also cause spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME). The prevalence of both Farber disease and SMA-PME is currently unknown, and awareness is extremely limited due to their rarity and lack of specific treatments; both are likely underdiagnosed. Methods: Data including genotypes and clinical, biochemical and immunologic phenotypes is being collected on a growing cohort of 40 Farber patients from around the world. This is the largest Farber cohort yet reported. Results: Our findings to date reinforce the validity of the characteristic symptoms of Farber disease. However, they also reveal that there are patients who present with only one or two of these symptoms, and that the spectrum of disease includes remarkably attenuated forms with relatively little associated disability. Such patients are likely to be misdiagnosed or experience a significant delay in diagnosis. Indicative of this fact is the recent case of three siblings who were diagnosed with Farber disease at over 40 years of age. Discussion: These insights provide an indication for diligent screening of certain pediatric and adult patients for acid ceramidase deficiency, and such programs are being initiated. Acid ceramidase enzyme therapy is also under development, and a natural history study is being initiated to better understand disease progression and factors influencing the different phenotypes, as well as what benefits can be derived from currently available symptomatic treatment with anti-inflammatory drugs. Conflict of Interest declared.

O-048

Identification of a new biomarker in Fabry disease by plasma proteomic analysis Lee B H 1, Heo S H 1, Kang E 1, Kim Y M 1, Kim G H 1, Yoo H W 1 1

Univ Ulsan Coll Med, Seoul, Republic of Korea

Background: Fabry disease is caused by α-galactosidase A deficiency. Enzyme replacement therapy (ERT) exerts a protective effect against renal, cardiac, and cerebral disease progression. Although globotriaosylceramide (Gb3) and its deacylated derivative lyso-Gb3 are known as the most useful diagnostic biomarkers, wide variation in their plasma levels has been noted, showing poor correlation with the long-term disease course. This study was performed to identify a new biomarker reflecting the long-term disease course and ERT efficacy. Methods: Plasma proteome profiles were analyzed before and after ERT in 8 patients with classical Fabry disease by 2DE and MALDI-TOF/TOF. The functional role of each protein showing differential expression was assessed with the validation by Western blotting. Results: After 8 months of ERT, the expression levels of 15 proteins involved in inflammation, oxidative stress, and ischemic injury were significantly decreased. Differential expression levels of beta-actin and its binding partners, including profilin-1, especially reflected the dysregulated endothelial nitric oxide synthase activity in Fabry vasculopathy. Of note, altered complement activity was indicated by the differential expressions of the multiple components including C1Q, C3 and C4 and their regulators. Western blotting was done to assess the expression of ACTB, C1Q, C3, and C4 throughout the course of long-term ERT (65 months), but only C3 and its derivative, C3b, showed the gradual reduction along the ERT, which was comparable to that of Gb3 levels. Discussion: The plasma proteomic analysis revealed the diverse inflammatory processes underlying Fabry vasculopathy. Importantly, the altered complement activation may be a major contributor to Fabry vasculopathy and ERT helps its stabilization. Furthermore, activation of C3 and its derivative C3b may be the new candidate markers to monitor ERT efficacy and disease course.

O-049

Farber disease: acid ceramidase deficiency is more common than previously thought and slowly progressive disease may only be diagnosed in adulthood Solyom A 1, Mitchell J 3, Huegle B 4, Makay B 8, Arslan N 8, Ozen S 7, Batu E D 7, Kariminejad A 10, Bonafe L 9, Superti-Furga A 9, Hadipour Z 13, Hadipour F 13, Tanpaiboon P 12, Guelbert N 14, Di Rocco M 6, Magnusson B 11, Grigelioniene G 11, Cuevas Cid A 15, Ehlert K 5, Beck M 16, Simonaro C 2, Levade T 17, Schuchman E H 2 1 Plexcera Therapeutics LLC, New York, United States, 2Icahn School of Medicine at Mt Sinai, New York, United States, 3Montreal Children’s Hospital, Montreal, Canada, 4German Center for Pediatric Rheumatology, Garmisch-Partenkirchen, Germany, 5University Medical Center Greifswald, Greifswald, Germany, 6Istituto Giannina Gaslini, Genoa, Italy, 7Hacettepe University Hospital, Ankara, Turkey, 8Dokuz Eylul University Hospital, Izmir, Turkey, 9 Lausanne University Hospital (CHUV), Lausanne, Switzerland, 10Kariminejad-Najmabadi Genetics Center, Tehran, Islamic Republic of Iran, 11Karolinska University Hospital, Stockholm, Sweden, 12 Children’s National Medical Center, Washington, United States, 13Sarem

14

20. Lysosomal disorders: others

O-050

Low reliability of functional (enzymatic) diagnostics of lysosomal storage disorders in dry blood spots compared to fibroblasts Schoonderwoerd K 1, Weykamp C 2, De Graaf I 2 1 Dept Clin Genet, ErasmusMC, Rotterdam, Netherlands, 2SKML, Beatrix Ziekenhuis, Winterswijk, Netherlands

Background: Enzyme assay is a key component in the diagnostics of the lysosomal storage disorders (LSD). In the case of sphingolipidosis, the diagnosis is totally dependent on enzyme measurement. Therefore, reliable enzyme assay is very important. Since 2010 ERNDIM offers an EQA scheme for lysosomal enzymes. Enzyme assay in fibroblasts is the gold standard. During the last decade enzyme diagnostics in dry blood spots (DBS) became more and more used in several laboratories. To obtain a good picture of the performance of the lysosomal enzyme assays in the different laboratories we compared the reliability of the diagnostics between fibroblasts and DBS among all participating laboratories all over the world in two different years. Methods: Lyophilized samples of fibroblast homogenates were shipped to about 70 participants. DBS made of heparin blood on Whatman filter paper were sent to 2–40 participants. Enzyme activities of different LSDs were measured in different patient samples and two identical control samples to determine the reproducibility in fibroblasts and DBS. Moreover participants were asked to fill in the diagnosis for every sample in a drop down menu on the website. Results: Concerning the reliability of the diagnostics in fibroblasts, only 0–15 % of the participants did not accomplish the correct diagnosis based upon the biochemical enzyme measurements and the diagnosis stated in the drop down menu. In DBS, however, 4–67 % did not accomplish the correct diagnosis. Also the intra-laboratory and inter-laboratory variation were much higher in DBS compared to fibroblasts (intra-laboratory DBS 40–60 % versus fibroblasts 10– 20 %; inter-laboratory DBS 76–127 % versus fibroblasts 40–70 %).

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Discussion: Enzyme diagnostics in fibroblasts is reliable for most LSDs. Enzyme diagnostics for LSDs is much less reliable in DBS, especially for Krabbe, MPS IV, MPS VI, Pompe and Gaucher.

O-051

TAR-DNA binding protein 43 (TDP-43) pathology in Niemann-Pick type C disease Dardis A 1, Zampieri S 1, Canterini S 2, Newell K L 3, Stuani C 4, Murrell J R 5, Ghetti B 5, Fiorenza M T 2, Bembi B 1, Buratti E 4 1 Rare Dis Centre, Univ Hosp Udine, Udine, Italy, 2Dept of Psychol, Univ La Sapienza, Roma, Italy, 3Dept of Pathol, Univ of Kansas, Kansas City, United States, 4 ICGEB, Trieste, Italy, 5Dept of Pathol, Indiana Univ, Indianapolis, United States

Background: Niemann-Pick C disease (NPCD) is a lysosomal storage disorder characterized by the occurrence of visceral and neurological symptoms. The molecular mechanisms that link this disease with the progressive neurodegeneration observed in patients are not fully understood. Recently, TAR-DNA binding protein 43 (TDP-43), a heterogeneous nuclear ribonucleoprotein (hnRNP), has emerged as a new player in the field of neurodegenerative diseases. Indeed, aberrantly phosphorylated and ubiquitinated TDP-43 was identified as a main component of brain inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients. We investigated whether TDP-43 pathology is present in NPCD. Methods: TDP-43 expression and/or intracellular localization were analyzed by western blot and confocal immunofluorescence, respectively, in NPC mice, a human neuronal model of the disease and in a human patient brain sample. The expression of TDP-43 controlled was analyzed by RT-qPCR. Results: The expression and/or intracellular localization of the TDP-43 were altered in both NPC mouse and in a human neuronal model of NPC. Indeed, while in control cells TDP-43 was mostly confined within the cell nuclei, as previously reported, in most NPC derived cells it was mainly localized in the cytoplasm and associated with markers of stress granules. Furthermore, in NPC cells TDP-43 mislocalization was associated with hyperphosphorylation and specific alterations in TDP-43 controlled genes. Treatment with N-acetylcysteine or beta-cyclodextrin partially restored TDP-43 nuclear localization. In parallel, a neuropathologic study of a NPCD patient’s brain showed that TDP43 was completely mislocalized in cerebellar Purkinje cells where it displayed a diffuse cytoplasmic localization. Discussion/Conclusion: The results of these studies extend the role of TDP-43 beyond the ALS/FTLD spectrum and strongly suggest that TDP-43 plays a role in the NPCD neuropathological process.

21. Lysosomal disorders: treatment, enzyme replacement therapy

O-052 Intracerebroventricular cerliponase alfa (BMN 190) in children with CLN2 disease: results from a phase 1/2, open-label, dose-escalation study Schulz A 2, Specchio N 4, Gissen P 5, De los Reyes E 3, Williams R 6, Cahan H 1, Slasor P 1, Jacoby D 1 1 BioMarin Pharmaceutical Inc, Novato, United States, 2University Medical Ctr Hamburg – Eppendorf, Hamburg, Germany, 3Nationwide Childrens Hospital, Columbus, United States, 4Bambino Gesu Childrens Hospital, IRCCS, Rome, Italy, 5Great Ormond St Hospital for Children, London, United Kingdom, 6Guys and St Thomas NHS Foundation Trust, London, United Kingdom

Background: CLN2 disease, a rare, inherited, pediatric-onset, neurodegenerative lysosomal storage disorder caused by TPP1 enzyme deficiency, is characterized by seizures, ataxia, rapid loss of language and motor functions, blindness and early death. Cerliponase alfa (BMN 190) is a recombinant human TPP1 enzyme. This phase 1/2, multi-center, open-label, dose-escalation study evaluated the

safety, tolerability and efficacy of every other week intracerebroventricular (ICV) infusions of cerliponase alfa in children with CLN2 aged 3 to 16 years. Methods: Following a dose escalation period, all patients received 300 mg of cerliponase alfa every 2 weeks by intracerebroventricular (ICV) infusion for 48 weeks. Efficacy was evaluated by monitoring changes in motor and language functions using a CLN2 clinical rating scale. Results: 24 subjects (9 male, 15 female, mean age 4.3 y [median: 4 y; range: 3–8 y]) enrolled in the study. Almost all subjects (96 %) had adverse events assessed as study drug-related, the majority of which were grades 1–2 and included pyrexia (46 %), hypersensitivity (38 %), seizure (38 %), and epilepsy (17 %). Serious adverse events assessed by the investigator as study drug-related were reported in eight (33 %) subjects. There were no anaphylaxis/anaphylactoid reactions, study drug discontinuations or deaths due to adverse events. The mean (SD)/median rate of decline in CLN2 score for subjects treated 48–91 weeks (n = 23) was 0.48 (0.756)/0.00 units/48 weeks, in contrast to 2.09 (0.97)/1.87 units/48 weeks observed in natural history (n = 41). Discussion: Enzyme replacement therapy with ICV-administered cerliponase alfa is well-tolerated and slows the progression of functional decline in children with CLN2. Conflict of Interest declared.

O-053 Novel treatment for Fabry disease—IV administration of plant derived alpha-gal-a enzyme safety and efficacy, 1 year experience Schiffmann R 1, Huges D 2, Giraldo P 3, Gonzalez D 4, Holida M 5, Golper Alpan O 6, Maegawa G 10, Atta M G 7, Nicholls K 8, Tuffaha A 9, Barisoni L 11, Colvin R 12 , Jennete C 13, Alon S 14, Krupko J 14, Szlifer M 14, Almon E 14, Chertkoff R 14 1 Div Metab Dis, Baylor Univ, Dallas, United States, 2LSDU Royal Free Hosp, London, United Kingdom, 3Hosp de Dia Quiron, Zaragoza, Spain, 4Inst Priv Hemato Invest Clinica, Asuncion, Paraguay, 5Iowa Univ Hosp and Clinic, Iowa City, United States, 6O&O Alpan LLC, Fairfax, United States, 7Dept Nephr, Johns Hopkins Univ, Baltimore, United States, 8Dept Nephr, Royal Melbourne Hosp, Melbourne, Australia, 9Kansas Univ Med Center, Kansas City, United States, 10Florida Univ Ped Dept Gen and Metab, Gainesville, United States, 11Path Dept Univ of Miami, Miami, United States, 12Path Dept Mass General Hosp, Boston, United States, 13Path Dept, NC Univ, Chapel Hill, United States, 14Protalix Biotherapeutics, Carmiel, Israel

Background: Fabry disease (FD) is an X-linked disorder caused by a lysosomal α-galactosidase-A insufficiency. PRX-102 (PEGunigalsidase alfa), a novel PEGylated enzyme for the therapy of FD expressed in a plant production system, is comprised of 2 subunits covalently-linked via a 2-kDa PEG moiety resulting in a stable homo-dimer. This interim report is a 1 year clinical experience summarizing ~15 patients’ years treatment. Methods: Dose ranging studies (0.2; 1; 2 mg/kg): PB-102-F01 and PB-102F02 were designed to evaluate the safety, pharmacokinetic (PK) and efficacy parameters in FD patients treated with PRX-102 administered intravenously every 2 weeks in adult symptomatic treatment naive patients. Primary safety outcome consisted of clinical laboratory, physical examination, ECG and antibodies. Secondary outcome included PK and exploratory efficacy parameters such as Gb3 and Lyso-Gb3 in plasma, kidney function: eGFR and UPCR, and questionnaires for GI symptoms and pain, Gb3 inclusions in kidney biopsies (baseline and 6 months), cardiac MRI and MSSI. Results: PK results indicated a plasma half-life of approximately 70 h and a substantially higher AUC. A total reduction in Gb3 inclusion in PTCs of 75.5 % and 86.5 % was achieved by 0.2 and 1 mg/kg, respectively, in kidney biopsies (BLISS). All doses presented reduction in biomarkers, Gb3 and Lyso Gb3, stability in annualized eGFR and in cardiac MRI, and an improvement in MSSI. PRX-102 was well tolerated, with a 95 % incidence of mild and moderate adverse events. Only one of the patients experienced hypersensitivity. 19 % developed treatment induced antibodies. Discussion: PRX-102 has unique PK properties which result in available enzyme throughout 2-week infusion intervals that offer optimal opportunity for attenuating disease progression. The majority of adverse events were mild and moderate with limited formation of antibodies in long-term data. In addition, to the various efficacy parameters indicate a potential positive treatment effect. Conflict of Interest declared.

S52 O-054

The emerging neurocognitive profile of classic infantile Pompe disease Ebbink B J 1, Poelman E 1, Aarsen F K 2, Plug I 1, Lequin M H 3, Van Doorn P A 2, Van der Ploeg A T 1, Van den Hout J M P 1 1 Dept Ped Div Met Dis CLMZ, ErasmusMC, Rotterdam, Netherlands, 2Dept Neur, ErasmusMC, Rotterdam, Netherlands, 3Dept Rad, Univ Med Center, Utrecht, Netherlands

Background: Classic infantile Pompe disease is a rare metabolic myopathy, which untreated leads to death in the first year. In 1999, we pioneered enzyme replacement therapy (ERT), leading to a significant improvement of motor function and life expectancy. However, ERT cannot pass the blood–brain barrier while glycogen storage occurs in the brain as well. Literature on cognitive development is limited with small and young patient populations, reporting a range of cognition between normal and a mild delay and stable restricted periventricular white matter abnormalities. Recently we reported a patient with a cognitive decline and extensive brain involvement. We now present a large cohort of patients up to the age of 15.6 years. Methods: We prospectively assessed brain MRI and neuropsychological development in 10 patients. Results: All patients presented with periventricular white matter abnormalities. From age 8 onwards, these extended to the subcortical areas, sparing the U-fibers, with involvement of the capsula externa and to a lesser extent the capula interna. In the oldest patients, abnormalities were found in the decussation and corticospinal tracts. Consecutive MRIs in 4 patients showed a slow progression. The extension and pace of brain involvement varied per patient, independent of motor function. Intelligence showed a wider range from normal to intellectual disabilities. A specific profile emerged with a risk of a decrease in processing speed, performance intelligence, and problems in attention and working memory. Discussion: This study in our patient population maturing towards adulthood raises signals that Pompe disease also affects the brain. White matter abnormalities are more extensive than was previously considered with a variable slow progression. The emerging neurocognitive profile complies with the brain abnormalities. We want to alert physicians to monitor cognitive development closely and urge for new therapeutic strategies targetting the brain. Conflict of Interest declared.

22. Glycosylation disorders/CDG, protein modification disorders

O-055 A novel group of metabolic disorders due to tissue-specific defects in VATPase assembly Jansen J C 1, Timal S 1, van Scherpenzeel M 1, Ashikov A 1, Jansen E 1, Wevers R 1, Huynen M 1, Foulquier F 2, Veltman J 1, Stevens T 3, Lefeber D J 1 1 Radboud University Medical Center, Nijmegen, Netherlands, 2Institute of Child Health, Athens, Greece, 3University of Lille, Lille, France

Background: Gastrointestinal tract and liver symptoms are frequent in the Congenital disorders of glycosylation (CDGs), commonly as part of a multisystem phenotype. Isolated hepatopathy is a long standing issue as a secondary cause for CDG type II screening profiles. We identified mutations in four assembly factors of the v-type H+-ATPase (V-ATPase) in a group of in total 28 CDG-II patients, presenting from infantile to adult age with a variety of isolated clinical symptoms, including non-syndromal hepatopathy. Methods and Results: Homology detection at the level of sequence profiles indicatedhuman ATP6AP1 as the long-sought human homolog of yeast VATPase assembly factor Voa1.Furthermore, a processed form of ATP6AP1 could restore V-ATPase dependent growth in Voa1mutant yeast. Assembly of this complex, the main regulator of intra-organellar acidification, iswell known in yeast, while limited knowledge is present for human. Previously, we identifiedTMEM199 (O-055) and CCDC115 (P-552) as human orthologs of yeast V-ATPase assemblyfactors Vma12p and Vma22p, respectively. A fourth assembly factor, VMA21, has been linkedto an adult myopathy (XMEA).

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Genetic studies in our cohort of unsolved CDG-II patientsrevealed mutations in all four genes in a total of 28 patients. ATP6AP1-CDG patients displayed a surprising phenotype of mild to severe hepatopathy,immunodeficiency and a spectrum of neurocognitive abnormalities1, while VMA21-CDGpatients showed a mild liver phenotype, different from XMEA patients (unpublished). In-depthbiochemical studies, including pulse-chase labeling in immortalized human hepatocytes andcomplexome profiling, revealed first insights to explain the tissue-restricted clinical symptoms.ATP6AP1 gene expression was very low in human liver, however, considerable levels wereobserved of a 62kDa non-processed protein in the ER. In contrast, we observed the common 40-kDa processed Golgi form in human brain and a novel 50kDa form in human B-cells, indicatingtissue-specific processing of ATP6AP1. For VMA21, we observed a more severe acidificationdefect for the XMEA mutation as compared to the CDG mutations, while XMEA patientsshowed (near)-normal glycosylation. This suggests that VMA21 is at least involved in twodifferent biochemical mechanisms, resulting in either XMEA myopathy or CDG hepatopathy incase of different mutations. Discussion: Our work unmasks v-ATPase assembly defects as a novel group of metabolicdisorders, presenting with tissue-restricted clinical symptoms, in part explained by tissue-specificmechanisms in V-ATPase assembly. 1 Jansen EJ, Timal S, Ryan M, Ashikov A, …. Stevens TH, Lefeber DJ. ATP6AP1 deficiency causes animmunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation. Naturecommunications 2016;7:11600.

O-056

A novel sugar metabolic pathway in humans: ISPD synthesises CDPribitol Riemersma M 1, Froese S D 2, Van Tol W 1, Van Bokhoven H 1, Yue W W 2, Lefeber D J 1 1

Radboud University Medical Center, Nijmegen, Netherlands, 2University of Oxford, Oxford, United Kingdom Background: A unique, unsolved O-mannosyl glycan on the protein αdystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of congenital muscular dystrophies, the dystroglycanopathies. Mutations in isoprenoid synthase domain containing (hISPD) represent the second most common cause of these disorders; however, its molecular function remains uncharacterized. Methods and Results: We crystallized hISPD and observed a canonical Nterminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs in other species. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity towards pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Further studies in CRISPR/cas9 ispd knock-out cells using liquid chromatography-mass spectrometry (LC-MS) revealed CDP-ribitol as the natural product of the ISPD reaction. Discussion: Our combined results indicate that hISPD is involved in a novel sugar metabolic pathway diverging from the pentose-phosphate pathway by production of CDP-ribitol. This previously unnoticed nucleotide-sugar in humans is essential for functional α-dystroglycan O-mannosylation in muscle and brain. ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies with potential opportunities for intervention.

O-057

ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitolphosphate onto α-dystroglycan Gerin I 1, Ury B 1, Breloy I 2, Bouchet-Seraphin C 3, Bolsee J 1, Halbout M 1, Graff J 1, Vertommen D 1, Muccioli G G 4, Seta N 3, Cuisset J M 5, Dabaj I 6, Quijano-Roy S 6, Grahn A 1, Van Schaftingen E 1, Bommer G T 1 1 de Duve Inst + WELBIO, UCL, Bruxelles, Belgium, 2Biochemistry, Cologne University, Cologne, Germany, 3Hopital Bichat-Claude Bernard, Bioch Metabol, Paris, France, 4LDRI, Univ Cath de Louvain, Bruxelles, Belgium,

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 5

Hopital Roger-Salengro, Neuropediatrie, Lille, France, 6Hopital R Poincare, Pediatrie, Garches, France

O-059

Background: Mutations in genes required for the glycosylation of αdystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Likewise, the functions of several genes involved in the assembly of this glycan are still unknown. Methods: Isoprenoid synthase domain-containing protein (ISPD), fukutin (FKTN) and fukutin-related protein (FKRP) were purified. Their enzymatic function was characterized. CDP-ribitol in muscle was detected by a combination of HPLC and mass spectrometry. Fibroblasts from patients with ISPD mutations were treated with ribitol, and functionality of α-dystroglycan glycosylation was assessed by flow cytometry and laminin overlays. Results: We report here that three genes mutated in dystroglycanopathies can collaborate to attach ribitol-phosphate onto α-dystroglycan. Specifically, we demonstrate that ISPD synthesizes CDP-ribitol, present in muscle, and that both recombinant FKTN and FKRP can transfer a ribitol phosphate group from CDPribitol to α-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into α-dystroglycan in HEK293 cells. Glycosylation of α-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium. Discussion: Ribitol-phosphate groups are known components of bacterial capsules. We have found that this modification is also present in mammalian cells and plays an important role in the glycosylation of α-dystroglycan. Supplementation of a metabolic precursor (i.e. ribitol) to ISPD-mutant fibroblast cell lines partially restores α-dystroglycan glycosylation. Hence, dietary supplementation with ribitol needs to be evaluated as a novel therapeutic approach for a subset of dystroglycanopathy patients.

MAGT1-deficiency: new insights into a controversial protein with a key role in N-glycosylation

O-058

Toward a folding therapy for PMM2-CDG Yuste-Checa P 1, Brasil S 1, Gamez A 1, Underhaug J 2, Desviat L R 1, Ugarte M 1, Perez-Cerda C 1, Martinez A 2, Perez B 1 1 CBMSO, CEDEM, UAM, Madrid, Spain, 2Bergen University, Bergen, Norway

Background: Functional characterization of disease-causing mutations allows the development of tailored therapeutic strategies aimed to rescue the specific mutant’s defect. The recent functional characterization of the disease-causing mutations described in patients with PMM2-CDG led to the idea to use small compounds able to rescue mutants’ folding by pharmacological chaperones (PC) or restore protein homeostasis by proteostasis regulators (PR). Methods and Results: A high-throughput screening of a commercial compounds library performed by differential scanning fluorimetry has allowed us to identify a potential PC for PMM2 whose defect causes the most common congenital disorder of glycosylation, PMM2-CDG. Its optimal chemical structure and its lack of apparent inhibitory effect on the pure enzyme make this compound an ideal starting point for optimization and testing in animal models. We also tested the PR MG132, carbenoxolone and celastrol in a cellular disease model. The results showed that celastrol produced the highest activity increase of all tested PMM2 mutants. Deeper studies performed by the inhibition of different molecular chaperones allowed us to unravel at least part of the molecular mechanism responsible of the PMM2 stabilization effect produced by celastrol which is related to Hsp90 and its cochaperone p23. However, this effect appeared to be HSF1 independent, and therefore does not rely on heat shock response activation by the compound. The combination of the selected PC and PR produced a synergistic effect on some PMM2 mutants’ activity. Discussion and conclusion: The present results provide proof-of-concept of a possible PMM2-CDG treatment by small stabilizer molecules and identify a promising chemical structure and a new possible therapeutic target involving molecular chaperones. This paves the way to develop new therapeutic agents for this orphan disease. Work funded by Fundación Gemio and AESCDG.

Peanne R 1, Blommaert E 1, Race V 3, Souche E 1, Keldermans L 1, Rymen D 2 , Jaeken J 2, Matthijs G 1, Biondi A 3 1 Centre for Human Genetics, KU Leuven, Leuven, Belgium, 2Centre for Metab Dis, Univ Hosp Leuven, Leuven, Belgium, 3Ped Dept, Univ MiBicocca, Monza ASST, Monza, Italy

Background: Congenital disorders of glycosylation (CDG) are genetically heterogeneous and the diagnosis of CDG has for long been challenging. In order to obtain a better diagnostic yield, we designed a capture assay for a panel of 60 genes associated with CDG, as well as a small number of candidate genes. Using this NGS-targeted approach, we identified two patients with hemizygous variants in the X-linked gene MAGT1. MAGT1 has been described as an isoform subunit of the STT3B complex of the oligosaccharyltransferase (OST). Still, the cellular role(s) of MAGT1 remain(s) controversial due to conflicting studies rather suggesting that it is a plasma membrane-localized Mg2+ transporter, required for Mg2+ uptake by vertebrate cells. Methods: CRISPR-Cas9 was used to engineer various MAGT1 and/or TUSC3 null cell lines, as well as cell lines harbouring the identified MAGT1 mutations. Pulse-chase analysis was used to assess the glycosylation of STT3B-dependent substrates, and both the steady state and localization of MAGT1 were investigated by immunoblotting and confocal microscopy, respectively. Results: We identified two boys with MAGT1 deficiency. Both patients presented similar phenotypes, including developmental delay, macrocephaly and hepatomegaly without splenomegaly. Cell-surface biotinylation and immunofluorescence experiments confirmed the ER localization of MAGT1 and the pathogenicity of the identified mutations. Furthermore, MAGT1-deficiency appeared to cause a significant reduction of the glycosylation of reporter glycoproteins harbouring STT3Bdependent glycosylation sites. Discussion: We report here the characterization of the recently identified MAGT1-CDG. We believe that MAGT1 is a component of the human OST complex, and thus is involved in the glycosylation of a subset of N-glycoproteins. One of these glycoprotein substrates could be a Mg2+ transporter, to date unknown. An impaired MAGT1 function would then result in a deficiency in magnesium as a secondary phenotype.

O-060

NANS-mediated synthesis of sialic acid is required for brain and skeletal development Van Karnebeek C 1, Bonafe L 2, Wen X Y 3, Lefeber D 4, Wevers R 2, SupertiFurga A 4 Dept of Pediatr, Univ British Columbia, Vancouver, Canada, 2Centre for Molec Dis, Univ Lausanne, Lausanne, Switzerland, 3Dept Med, Univ Toronto, Toronto, Canada, 4Dept Lab Med, Radboud Univ Med Centre, Nijmegen, Netherlands 1

Background: Thousands of monogenetic disorders have been reported in intellectual disability patients, and over 500 with skeletal dysplasia. Integrated analysis of -omics data sets provides the opportunity to identify novel human diseases with these features, as well as their biomarkers and targets for treatment. Methods: We applied combined genomics and metabolomics technologies with experimental studies in 9 patients with intellectual dysability and skeletal dysplasia. Zebrafish knockdown morpholino was used to recapitulate the phenotype and perform further studies. Results: We identified bi-allelic mutations in NANS, the gene coding for Nacetyl-neuraminic acid (NeuNAc; sialic acid) synthase, in 9 individuals with infantile-onset severe developmental disability and skeletal dysplasia. Patient body fluids showed an elevation of N-acetyl-mannosamine, and patientderived fibroblasts had reduced NANS activity and were unable to incorporate

S54 sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development and exogenously added sialic acid partially rescued the skeletal phenotype. Discussion: NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways of sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies for NANS deficiency, and to shed light on sialic acid metabolism and its implications for human nutrition.

O-061

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Methods: We have generated a new murine model of CCSD1 obtained by ubiquitous deletion of 5–7 exons in the Slc6a8 gene and we have generated this model at behavioral, biochemical, morphological and molecular levels. Results: In these mice, we detected a remarkable Cr depletion in the brain tissues, and cognitive and autistic-like defects mimicking the key features of human CCSD1. Furthermore, we discovered alterations of cellular and molecular mechanisms that play a pivotal role in the generation of the CCDS1 neurological phenotype. Mutant mice displayed reduction of hippocampal neurogenesis, marked activation of microglia, and increased oxidative stress, leading to a general cognitive deterioration progressively worsening with age. Discussion: This knowledge will open the important possibility to design intervention strategies aimed at overcoming brain CCDS1 alterations and the CrT−/y murine model will provide an important tool for their preclinical test.

SLC39A8 deficiency is a novel treatable disorder of manganese metabolism and glycosylation

O-063

Park J H 1, Hogrebe M 1, Grueneberg M 1, Reunert J 1, Rust S 1, Marquardt T 1

Aminoadipate semialdehyde synthase (AASS) as a therapeutic target for pyridoxine dependent epilepsy by substrate reduction

1

Klinik f Kinder- u Jugendmed, Univ Klin, Muenster, Germany

Background: SLC39A8 deficiency is a novel disorder of manganese metabolism due to disruptive mutations in SLC39A8. These result in a severe disorder with seizures, cranial and skeletal malformations, and pronounced hypoglycosylation. The underlying pathomechanism is a deficiency in manganese, a trace element, which serves as a cofactor to a variety of enzymes, including galactosyltransferases. While galactose supplementation has been shown to correct glycosylation of serum transferrin, manganese supplementation is emerging as a more comprehensive therapeutic approach. Methods: We administered both galactose and manganese (II) sulfate to a patient with the mutations c.112G > C (p.G38R) and c.1019 T > A (p.I340N) who initially presented with cranial malformation, severe seizures and hypoglycosylation corresponding to a type II congenital disorder of glycosylation. Changes in glycosylation were monitored via high-performance liquid chromatography while the patient was clinically followed up. Results: Galactose supplementation resulted in a complete normalization of serum transferrin glycosylation and a significant clinical improvement. Added manganese supplementation led to further clinical improvement with ceasing of seizures. Discussion: SLC39A8 deficiency is the first known disorder affecting both trace element metabolism and glycosylation resulting in a severe clinical presentation. Both galactose and manganese are promising therapeutic approaches for this novel metabolic disease.

23. Neurotransmitter and creatine related disorders O-062 Progressive cognitive deterioration and pathological hallmarks in murine model creatine transporter deficiency Baroncelli L 1, Alessandri M G 2, Molinaro A 1 8, Cacciante F 3, Napoli D 3, Tola J 1, Putignano E 1, Amendola E 4, Zonfrillo F 4, Gross C 4, Mazzanti M C 5 , Leuzzi V 6, Cioni G 2 7, Pizzorusso T 1 8 1 Neuroscience Institute, CNR, Pisa, Italy, 2IRCCS Stella Maris Scientific Institute, Calambrone (Pisa), Italy, 3Scuola Normale Superiore, Pisa, Italy, 4EMBL, Monterotondo (Roma), Italy, 5Fondazione Pisa per la Scienza ONLUS, Pisa, Italy, 6Dept Paed, Child Neurol, Univ Rome, Roma, Italy, 7Dept Clin Exp Med, Univ Pisa, Pisa, Italy, 8Dept NEUROFARBA, Univ Florence, Firenze, Italy

Background: Mutations in the creatine (Cr) transporter (CrT) gene lead to cerebral creatine deficiency syndrome-1 (CCDS1), an X-linked metabolic disorder characterized by cerebral Cr deficiency causing intellectual disability, seizures, movement and autistic-like behavioral disturbances, language and speech impairment (OMIM #300352). CCSD1 is still an untreatable pathology that can be very debilitating for patients and caregivers.

Pena I 2, Kopec J 1, Rembeza E 1, Arruda P 2, Yue W W 1 1 Struct Genom Consortium, Univ of Oxford, Oxford, United Kingdom, 2Inst of Biology, Univ of Campinas, Campinas, Brazil Background: Mutations in the ALDH7A1 gene of the lysine catabolic enzyme cause pyridoxine-dependent epilepsy (PDE), a metabolic condition characterized by recurring seizures that can result in status epilepticus and death if not treated. The biochemical hallmark of PDE is toxic accumulation of aminoadipate semialdehyde (AASA), substrate for the ALDH7A1-catalyzed reaction. We propose that inhibiting the enzyme upstream of ALDH7A1 in lysine catabolism, aminoadipate semialdehyde synthase (AASS), can reduce cellular levels of AASA and serve as substrate reduction therapy for PDE. This is prompted by genetic evidence that naturally-occurring AASS mutations lead to the benign condition hyperlysinemia. Methods: As a first step towards structure based inhibitor design, we have expressed recombinant human AASS, characterized its ligand binding and catalytic properties by structural and biophysical methods, and carried out small molecule screening using an in vitro activity assay. Results: AASS is a bi-functional enzyme catalysing the first two steps in lysine catabolism, via its lysine ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH) domains. We have expressed, purified and crystallized both LKR and SDH domains of human AASS, allowing the determination of their structures at atomic resolution. We have adopted the SDH activity assay into 384-well format, and screened 2000 compounds from commercially available NIH and LOPAC libraries. 4 candidate hits were identified, exhibiting IC50 values in the 10–200 μM range. Discussion: This work provides the necessary tools and proof of principle to develop small molecular therapy for PDE. The AASS structural data highlight key features in the catalytic mechanism distinct from bacterial homologues, and reveal druggable pockets within the LKR and SDH active sites. The 4 compound hits serve as chemical starting points to further optimize for potency and selectivity by medicinal chemistry, and will be tested in patient derived cells.

O-064

Clinical, biochemical and genetic approaches to improve the diagnosis of neurodevelopmental diseases related to neurotransmitter metabolism Arnoux J B 1, Kuster A 2, Barth M 3, Lamireau D 4, Pedespan J M 4, Goizet C 4, Houcinat N 4, Henrion-Caude A 5, Damaj L 6, Odent S 6, Doray B 7, Clot F 8, Benoist J F 9, Schiff M 9, Cano A 10, Corne C 11, De Pontual L 12, Baneiras C 1, Bahi-Buisson N 1, Brassier A 1, Valayannopoulos V 1, Chaumette B 13, Plaze M 13, Martinez G 13, Gaillard R 13, Krebs M O 13, Desguerre I 1, De Lonlay P 1, Lebre A S 14, Christa L 1 1 Neuro Metab Dept Refer Cent Necker Hosp, Paris, France, 2Neuro Metab Dept, Nantes Hosp, Nantes, France, 3Neuro Metab Dept, Angers Hosp,

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Angers, France, 4Neuro Genet, Pellegrin Hosp, Bordeaux, France, 5INSERM U781 IMAGINE, Necker, Paris, France, 6Pediatric Genet Dept, Rennes Hosp, Rennes, France, 7Genet Dept, Felix Guyon Hosp, Saint Denis de la Reunion, France, 8Genet Dept, Salpetriere Hosp, Paris, France, 9Neuro Metab Dept, Robert Debre Hosp, Paris, France, 10Div Metab Dis, la Timone Hosp, Marseille, France, 11 Bioch Dept, Grenoble Hosp, Grenoble, France, 12 Pediatric Dept, Jean Verdier Hosp, Paris Seine Saint Denis, France, 13 INSERM U894 CNRS GDR3557, Saint Anne Hosp, Paris, France, 14 Genet Dept Maison Blanche Hosp, Reims, France Background: Most patients with abnormal neurotransmitters in cerebrospinal fluid (CSF) lack a genetic confirmation and remain without diagnosis. Methods: Among 1200 patients analyzed for neurotransmitter disorders, 228 (228/1200, 19 %) patients presented abnormal CSF neurotransmitters. Diagnoses were confirmed for 55 (55/228, 24 %) patients, divided in primary and secondary neurotransmitter diseases, 31 (31/228, 13.5 %) and 24 (24/228, 10.5 %) patients, respectively. Results: For primary neurotransmitter and cofactor synthesis deficiency, the most frequently mutated gene was DDC (n = 9). Other mutated genes included PAH (n = 4), PTPS (n = 5), DHPR (n = 3), SR (n = 1), TH (n = 1), and PNPO (n = 1). For dopamine transporter and neuronal development, we identified SL6A3 and FOXG1 (n = 1 for each). For folates metabolism we identified MTHFR (n = 3), FOLR1 and MTHFD1 (n = 1 for each). For secondary deficiencies, regarding the mitochondria, we identified POLG (n = 3), ACFSF3 (n = 2), NFU1 and SDH (n = 1 for each). For one patient each, other mutated genes included: ADAR1, RNASEH2 A and B, SL7A2-INC RNA, and EXOSC3 regarding nuclear and cytoplasmic steps; RanBP2 and CASK regarding post-traductional and scaffolding; SL6A19 regarding amino acid transport; MTM1, KCNQ2, and ATP1A3 regarding nerve cell electrophysiological state; del18p23tris12p23, del6Q21, and dup17p13.3 regarding chromosome abnormalities. Three patients were identified with non-genetic aetiologies. We have matched the patients with their respective diagnosis according to 13 distinctive biochemical profiles and 23 clinical features gathered to motor (n = 7), neurological and neurocognitive (n = 9) and dysautonomic (n = 7) signs. Discussion: The defined clinical presentations and biochemical profiles might help to recognize specific patterns to improve the aetiology of neurological diseases. In respect to these patterns, a gene panel approach might be proposed in the future to improve the diagnosis of complex and rare neurological genetic diseases.

O-065

GNAO1 mutation: a new cause of transmembrane signalling derangement causing early onset movement disorder Danti F R 1 4, Romani M 3, Galosi S 1, Montomoli M 2, Carss K J 6 7, Raymond F L 7 8, Study D D D 9, Valente E M 10, Leuzzi V 1, Guerrini R 2, Kurian M A 4 5 1 Dept Ped, Child Neur Psych, Sapienza Univ, Rome, Italy, 2Ped Neurol Unit and Lab, Meyer Child Hosp, Florence, Italy, 3Neurogenetics Unit, Mendel Lab, San Giovanni Rotondo, Italy, 4Mol Neurosciences, ICH, UCL, London, United Kingdom, 5Dept Neurology, GOSH, London, United Kingdom, 6Dept Hematology, Univ Cambridge, Cambridge, United Kingdom, 7 NIHR Bioresource Rare Dis,Univ Cambridge, Cambridge, United Kingdom, 8Dept Med Gen, CIMR, Univ Cambridge, Cambridge, United Kingdom, 9DDD Study, Wellcome Trust Sanger Inst, Cambridge, United Kingdom, 10Sect Neurosciences, Univ Salerno, Salerno, Italy

Background: GNAO1 gene encodes for a subclass (Gαo) of the Gα subunit of heterotrimeric guanine nucleotide-binding proteins, highly expressed in brain. Gαo mediates the widespread presynaptic auto-inhibitory effect of many neurotransmitters (via α2 adrenoreceptors, M2/M4 muscarinic, μ/δ opioid, GABAB, adenosine A1, or endocannabinoid CB1 receptors) through a reduction of sensitivity to membrane depolarization and a direct inhibitory effect on the vesicle fusion process. De novo GNAO1 mutations have been reported in 21 children with neurodevelopmental disorders ranging from Othahara

syndrome, to less severe epileptic encephalopathies or prominent movement disorders with or without partial seizures. Case Report: In order to better define the clinical phenotype, we report a newly identified cohort of patients with GNAO1 mutations. Results: We report 5 patients with novel GNAO1 mutations (current age 3– 10 years). Developmental delay in infancy/childhood was evident in all patients. Age of motor presentation was 8 months to 9 years. Patients show a variable motor phenotype, from severe choreoathetosis to milder presentations with stereotypies and focal dystonia. 4/5 patients had mild drug-controlled epilepsy. Furthermore, we report several additional features, including isolated stereotypies, mild self-injurious/compulsive behaviours, exacerbation by emotion or intercurrent illness and low CSF 5-MTHF. Discussion: Our findings corroborate the causative role of GNAO1 mutations in multiple types of early onset movement disorders. Recently, impairment in synaptic and postsynaptic signalling cascades has been reported in a number of neurological and psychiatric diseases. In particular, mutations in GNAO1 and other G protein subunits (GNAL), adenylyl cyclase (ADCY5) and cyclic nucleotide phosphodiesterase (PDE10A) have been reported in early onset movement disorders, supporting the key contribution of G-protein-cAMP pathway axis to the pathophysiology of dystonia and chorea.

24. Disorders of vitamins, cofactors and trace elements

O-066

Interaction and characterization of the cblF (LMBD1) and cblJ (ABCD4) membrane proteins Fettelschoss V 1, Burda P 1, Lutz S 1, Suormala T 1, Fowler B 1, Bornhauser B , Froese S D 1 3, Baumgartner M R 1 3 4

2

1 Div Metab, Univ Child Hosp, Zurich, Switzerland, 2Dept Oncology, Univ Child Hosp, Zurich, Switzerland, 3radiz, Univ Zurich, Zurich, Switzerland, 4 Integ Hum Physiol, Univ Zurich, Zurich, Switzerland

Background: Vitamin B12 (cobalamin, Cbl), in its cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of methionine synthase and methylmalonyl-CoA mutase, respectively. Following uptake of Cbl by receptormediated endocytosis, the integral membrane proteins LMBD1 and ABCD4 are required for release of Cbl to the cytosol. The similar clinical and cellular phenotypes stemming from dysfunction of LMBD1 and of ABCD4 suggest that these proteins have a closely related role in intracellular Cbl metabolism. Methods: We co-expressed LMBD1 and ABCD4 tagged C-terminally with the fluorescent markers EGFP and mKate2, respectively, in immortalized fibroblasts, confirmed their colocalization by confocal microscopy and investigated their potential interaction via flow cytometry-based FRET (Förster resonance energy transfer) in living cells. Results: We found an elevated FRET signal from LMBD1 and ABCD4, which was not observed when either protein was co-transfected with LAMP1 or ABCB9, two lysosomal membrane proteins not associated with Cbl metabolism. Further, ABCD4 containing patient mutations or mutations that disrupt ATPase activity resulted in clearly decreased interaction with LMBD1, while not dramatically disturbing their colocalization. Finally, colocalization studies with lysosomal and endosomal markers confirmed that, in fibroblasts, LMBD1 is primarily found within the lysosome, while localization of ABCD4 is inconclusive. Discussion: These studies demonstrate that LMBD1 and ABCD4 participate in a physical interaction, which is dependent on ABCD4 ATPase activity, and is perturbed by patient mutations, but the precise cellular localisation of this interaction remains to be discovered.

O-067

Disturbed regulation of methylenetetrahydrofolate reductase by Sadenosylmethionine

S56 Smith D E 1, Mendes M I 1, Coker M 2, Rennings A J 3, Leandro J 4, Fernandez Ojeda M 1, Pop A 1, Leandro P 4, Salomons G S 1 1

Metabolic Laboratory, VU Univ Med Center, Amsterdam, Netherlands, Dept Pediatrics, Ege Univ, Izmir, Turkey, 3Dept Internal Med, Raboud Univ, Nijmegen, Netherlands, 4Metab and Genet Group, Univ Lisbon, Lisbon, Portugal 2

Background: Methylenetetrahydrofolate reductase (MTHFR) deficiency displays a broad phenotype ranging from asymptomatic to severe neurological decline. It catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5methyltetrahydrofolate, which is used to remethylate homocysteine to methionine. The activity of MTHFR is regulated by S-adenosylmethionine (SAM). Methods: MTHFR activity in both wild type (WT) and MTHFR variant fibroblasts of patients was measured by LC-MS/MS. The percentage of inhibition of this activity by 1.25 mmol/L SAM was determined. MTHFR was cloned into pCMV6. Proteins were overexpressed in HEK293 cells to ensure proper phosphorylation. Following protein purification, differential scanning fluorimetry (DSF) was used to determine the effect of SAM on the thermal stability of WT and variant proteins. Results: Fibroblasts harboring two novel MTHFR homozygous missense variants showed high residual MTHFR activity (4.8 and 6.4 nmol/hour/mg protein) in comparison to other MTHFR deficient fibroblasts of patients homozygous for a null allele (5 mg/dL was higher in patients with the worst neurological prognosis (p = 0.02). The psychiatric outcome was significantly associated with the number of lifetime decompensations, but not with yearly metabolic events. The AUC of Leu >5 mg/dL was higher in patients with psychiatric issues. The psychiatric outcome was associated with the maximum Leu level during decompensations: for every increase of 1 mg/dL of the maximum Leu level during

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 any metabolic event, the odd-ratio [95 % CI] for psychiatric follow-up reqirement was 1.20 [1.00; 1.44] (p = 0.053). Parental involvement in the treatment led to a lesser time with plasma Leu >5 mg/dL (p = 0.016). Discussion: Despite medical progress, the number of decompensations and the apparition of psychiatric morbidity at adult age remain unsatisfactorily high in this cohort, which did, however, have a normal IQ. The adaptation of the family to the disease was linked to plasma Leu levels.

P-008

Cerebrospinal fluid neurotransmitter depletion in adult PKU patients Pilotto A 1 2, Blau N 3, Charyasz E 4, Freisinger P 5, Gramer G 3, Hoffmann G F 3, Scheffler K 4, Berg D 2 6 7, Trefz F K 3 1 Neurol, Dept Clin Exp Scie, Brescia Univ, Brescia, Italy, 2Neurodeg Dept, Tuebingen Univ Hosp, Tuebingen, Germany, 3Dept Ped, Neuroped Met Med, Univ Hosp Heidelberg, Heidelberg, Germany, 4Dept Biom Magn Res, Tuebingen Univ, Tuebingen, Germany, 5Dept Ped, Reutlingen Hosp, Reutlingen, Germany, 6DZNE, Tuebingen Univ, Tuebingen, Germany, 7 Neurol Dept, Schleswig-Holstein Univ Hosp, Kiel, Germany

Background: Neurotransmitter synthesis in phenylketonuria (PKU) patients with high phenylalanine (phe) is impaired by inhibition of tyrosine- and tryptophan hydroxylase activity and by competition through the large neutral amino acid transporter of the brain blood barrier. The neurotransmitter deficit might explain neurological alterations in adult patients with PKU. Methods: In order to elucidate the relation between blood phe concentration, neurotransmitter in cerebrospinal fluid (CSF), brain MRI findings and neurologic status, we performed a cross sectional study in 15 classical PKU patients >30 years of age. Seven of the patients underwent CSF analyses. Neurotransmitters were measured by HPLC and fluoremetric detection. Amino acids concentrations in plasma and CSF were analyzed using Biochrom analyzer and catione exchange chromatography. All patients u n d e r w e n t 3 T- M R I i m a g i n g i n c l u d i n g Wa h l u n d s c o r e f o r leukoencephalopathy, visual rating scale and voxel-based morphometry (VBM) analyses for brain atrophy. Results: Plasma phe levels were 57–1115 μmol/l, CSF phe levels were 47–255 μmol/l, corresponding to the treatment status. CSF 5HIAA (R2 0.83) and homovanellinic acid (R2 -0.91) were negatively correlated with plasma/CSF phe concentration. In 3 patients exceeding a plasma phe concentration of 600 μmol/l, 5HIAA in CSF was below the lower n o r m a l l e v e l . M R I f i n d i n g s sh o w e d a c o r r e l a t i o n b e t w e e n leukoencephalopathy (Wahlund score) and brain atrophy with correspondingly increased plasma phe levels and inverse correlation with CSF neurotransmitter levels. Discussion: Plasma phe levels >600 μmol/l probably lead to neurotransmitter depletion in adult PKU patients and might explain neuropsychiatric features (including depression and parkinsonism) presented by some patients. Replication of these findings in larger samples with CSF analyses are necessary in order to confirm the clinical relevance of neurotransmitter deficits in adult PKU patients.

P-009

A coordinated transition model for patients with cystinosis in Spain: from pediatric to adult care Ariceta G 1, Camacho J A 2, Fernandez-Polo A 1, Gamez J 1, Garcia-Villoria J 7 , Lara E 1, Leyes P 3, Martin-Begue N 1, Perello M 1, Pintos-Morell G 5, Torra R 4, Torregrosa J V 3, Torres-Sierra S 1, Vila-Santandreu A 2, Guell A 6 1 Vall d’Hebron Univ Hosp, Barcelona, Spain, 2Sant Joan de Deu Univ Hosp, Barcelona, Spain, 3Clinic Univ Hosp, Barcelona, Spain, 4Fundacio Puigvert, Barcelona, Spain, 5Germans Trias i Pujol Univ Hosp, Barcelona, Spain,

6

Orphan Europe (Recordati Group), Madrid, Spain, 7Clinic Univ Hosp, IDIBAPS, CIBERER, Barcelona, Spain Background: Improved outcome and longer life expectancy in patients with cystinosis, and disease complexity per se justify planning a guided-transition of affected patients from pediatrics to adult medicine. The aims of the process are to guarantee the continuum of care and patient empowerment, moving from guardian-care to self-care. Methods: A working group for the care and transition of cystinosis in Barcelona (T-CiS.bcn) has assembled a group of experts in the disease to establish recommendations for the comprehensive care of cystinosis and the transition of adolescents to adult-care units in Spain. Sources: article reviews, expert opinions and anonymous surveys of patients, relatives and patient advocacy groups. Results: Developed an ad hoc document to guide and coordinate the transition of patients with cystinosis in clinical practice. The document is divided into: (1) transition framework, (2) distinctive features of cystinosis in adolescents, (3) transition plan and stages: preadolescents (12–15 y—planning and initiation), adolescents (16–18 y—readiness and assessment), young adult (19–25 y—transference), (4) adherence promotion, (5) transition barriers, and (6) patient centered multidisciplinary program adapted to the Spanish healthcare system with specific recommendations. Nephrologists play a key role in transition due to the fact that most cystinotic patients suffer severe chronic kidney disease and need kidney transplantation before adulthood. Many other agents are involved in building ongoing and collaborative partnerships between pediatric and adult healthcare providers: transition leaders, families, patient advocacy groups and primary care teams. Discussion: We present a document providing recommendations and suggesting a chronogram path to guide the process of transition of adolescents and young adults with cystinosis in our area. Conflict of Interest declared.

P-010

Nitisinone in alkaptonuria—quantifying the pigmentary pathway Ranganath L R 1 1

Royal Liverpool University Hospital, Liverpool, United Kingdom

Background: Alkaptonuria (AKU) is characterized by increased homogentisic acid (HGA) due to deficiency of homogentisate dioxygenase. Polymerisation of HGA, termed ochronosis, is an incompletely understood process resulting in black pigment deposition in tissues. A HGA-lowering therapy, nitisinone, is available for AKU, as it inhibits phydroxyphenylpyruvate dioxygenase. The ochronotic pathway remains poorly characterized. The study SONIA 1 (Suitability of Nitisinone for Alkaptonuria) presented an opportunity to quantify the ochronotic pathway for the very first time. Methods: SONIA 1 included a dose of 8 mg of nitisnone as well as no treatment for 4 weeks, each containing 8 AKU patients. Fasting acidified serum and 24-h acidified urine samples were collected and analyzed for phenylalanine (PA), tyrosine (TYR), p-hydroxyphenylpyruvate (HPPA), phydroxyphenyllactate (HPLA), nitisinone (NT) and HGA. 24-h serum tyrosine metabolite profiles were also obtained. Results: At baseline, serum PA, TYR, HPPA, HPLA and HGA were 55 + 11, 54 + 15, 50 % decreased GNL levels while homozygotes had undetectable GNL levels (N = 45.2 ng/ml). Discussion: The loss of function mutations in progranulin are linked to two distinct neurodegenerative disorders, FTLD and NCL, with quite a different age of onset, emphasizing the pleiotropic effect of the mutation in heterozygous or homozygous state.

P-025

Woodhouse-Sakati syndrome: a rare cause of leukodystrophy Louro P 3, Duraes J 1 2, Paiva S 4, Tavares P 5, Macario M C 1 2 CHUC - Centro Hosp Univ Coimbra, Coimbra, Portugal, 2Neurology, Coimbra, Portugal, 3Genetic Department, Pediat Hosp - CHUC, Coimbra, 1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Portugal, 4Endocrinology Cent Hosp Univ Coimbra, Coimbra, Portugal, CGC Genetics, Porto, Portugal

5

Background: Woodhouse-Sakati syndrome (MIM 241080) is a very rare cause of neurodegeneration with brain iron accumulation. Mutations on the DCAF17 gene are associated with this disorder. This is an autosomal recessive multisystemic neuroendocrine disorder characterized by hypogonadism, alopecia, diabetes mellitus, mental retardation, deafness and extrapyramidal syndrome. Usually the cerebral MRI findings suggest abnormal brain iron accumulation with white matter lesions. The treatments are palliative and symptomatic. Case Report: A 30-year-old female patient was referred for neurometabolic consultation with dystonia and tetraparesis. Since adolescence, she had received diagnoses of hypogonadism hypogonadotropic, hypothyroidism, diabetes mellitus with retinopathy, sensorineural hearing loss, alopecia and slight abnormalities of mental development. The neurologic symptoms were progressive and she was now presenting with spastic paraparesis, legs dystonia, dysarthria and dysphagia . MRI showed genera lized leukodystrophy (pons, periventricular bilaterally) without abnormal iron deposits. Common genetic and metabolic leukodystrophy and mitochondrial disorders are excluded. She is the only child of a couple consanguineous and there is no medical disorder in the family. Results: Sequencing the gene DCAF17 shows, in homozygosity, the variation c.1091 + 2 T > C. Discussion: The variant c.1091 + 2 T > C of the gene DCAF17 is not described in the literature; however, as it affects the splicing donor site of intron 10, and the skipping is of exon 10, it will be considered as pathogenic. We must be alert for this syndrome when we study young persons with leukodystrophy or extrapyramidal disorders because there is the possibility of genetic counseling for the family.

P-026

Argininossuccinic aciduria—an atypical presentation Vieira D 1, Silva B 1, Vilarinho L 2, Fonseca C 3, Louro E 4, Macario M C 1 1 Inb Err Metab Ref Cen, Cen Hosp Univ Cbr, Coimbra, Portugal, 2Dr Ricardo Jorge Natl Health Inst, Lisboa, Portugal, 3Nut Dept, Cen Hosp Univ Coimbra, Coimbra, Portugal, 4Int Med Dept, Cen Hosp Univ Coimbra, Coimbra, Portugal

Background: Argininossuccinic aciduria (ASA) is the second most common defect in urea cycle deficiency. It can manifest as either a severe neonatal onset form or as a late onset form. The last can range from episodic hyperammonemia triggered by acute infection, stress or non-compliance with the treatment, to cognitive impairment, behavioral abnormalities or learning disabilities in absence of any documented episodes of hyperammonemia. It has unique clinical features, including neurocognitive deficiencies, hepatic disease and systemic hypertension. Typically plasma citrulline levels are elevated, as is argininossuccinic acid in the plasma or urine. Molecular genetic testing of argininosuccinate lyase (ASL) gene and enzymatic activity testing are helpful. Case Report/Results: A 50-year-old man with asthma and hypertension followed for persistent creatine kinase (CK), aldolase and LDH elevation, on routine blood analyses, was referred to our neurology department. He complained about severe muscle cramps and irritability after protein rich meals. Those symptoms had no association with physical activities or fasting. He had hepatomegaly. The neurologic examination was normal. The electromyography had no abnormalities. The hepatic enzymes were slight abnormal. The acylcarnitine profile and αglucosidase were normal. There was an increase in citrulline plasma levels and in argininossuccinic acid plasma and urine levels. Ammonia levels were normal. These values support the diagnosis of ASA, which was confirmed with a molecular genetic study that identified a homozygotic V178M mutation in the ASL gene. The patient began a protein restricted diet with clinical improvement. Discussion: Late onset metabolic disorders can be associated with attenuated clinical course. Clinical symptoms triggered or worsened by protein intake are common and lead to diagnosis. Despite the metabolic disease, high CK levels are not explained directly by ASA but could be part of an attenuated adult phenotype.

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Discussion: The implementation of NGS has enabled rapid, cost-effective molecular testing and vastly increased the repertoire of genes that we are able to test in-house. We have achieved very high diagnosis rates in patients with known, biochemically diagnosed disorders, indicating that the technology is highly effective. However, where there is no biochemical evidence, our experience has shown that casting the net wider from the beginning is more time- and cost-effective.

02. Novel diagnostic/laboratory methods

P-027

Pompe disease—the proportion of fatty and muscle tissues as an indicator of progression and severity of the disease 1

2

Rozdzynska-Swiatkowska A , Jurkiewicz E , Tylki-Szymanska A

P-029

3

Anthrop Lab, CMHI, Warsaw, Poland, 2Dept of Diag Imaging, CMHI, Warsaw, Poland, 3Dept of Ped, Nutrit and Metab Dis, CMHI, Warsaw, Poland 1

Background: In the present study we report the new possibility to use bioelectrical analysis (BIA) to assess the relative proportions of the fatty and muscle tissues in diseases associated with muscle atrophy as such Pompe disease. Results from BIA analysis were compared with magnetic resonance images. In myopathies, as the disease progresses, the space of atrophic muscle tissue is gradually filled by the fatty tissue. The relative to body mass proportions of these two tissue types allows us to determine the extent of muscle tissue destruction. The study’s objective was to use the BIA analysis method to determine the proportion of the amount of fatty and muscle tissues in patients with Pompe disease. Methods: A study was conducted in a group of 20 patients with Pompe disease aged from 6.7 to 52 years. All patients had a diagnosis of Pompe disease. In all patients the body composition analysis by the electrical bioimpedance method (BIA) was obtained. In 17 patients MRI of the thigh muscles images was obtained. Results: In 12 cases the results of BIA analysis and MRI image were in accordance and were classified to the same grade; in 3 cases the results were not inaccordance and were graded into different classes of severity of the disease. In 2 cases analysis for BIA was not unequivocal. MRI was not performed in 3 patients. Discussion: The accuracy of BIA is comparable with magnetic resonance. BIA is much less expensive, faster and easier to perform. The use of BIA to assess the relative proportions of fatty and muscle tissues in diseases associated with muscle atrophy is an innovative method not previously used in myopathies. The results prove the high sensitivity of the BIA method and suggest great potential for using electric bioimpedance analysis as a cognitive tool enabling the assessment of the proportion of fatty and muscle tissues in progressive myopathies as well as the potential for using the device in diagnostics.

A rapid procedure for the detection of 4-hydroxyglutamate in urine by LC-MS/MS for screening for primary hyperoxaluria type 3 Holwerda U 2, Wamelink M M C 2, Salomons G S 2, Bokenkamp A 1, Struys E A2 1 Dept Ped Nephr, VU Univ Med Center, Amsterdam, Netherlands, 2Metabolic Unit, VU Univ Med Center, Amsterdam, Netherlands

Background: Primary hyperoxaluria type 3 (PH3) is an autosomal recessive disease caused by mutations in the 4-hydroxy-2-oxoglutarate aldolase 1 (HOGA1) gene. HOGA1 is involved in the metabolism of hydroxyproline. Patients with PH3 have, in addition to increased urinary excretion of oxalate, increased excretions of 4-OH-glutamate (PHG). Measuring PHG in urine by LC-MS/MS as a marker for PH3 is shown to be a fast and reliable method. Methods: We developed a LC-MS/MS method for measuring urinary PHG using [15N]α-aminoadipic acid as internal standard. PHG and the internal standard were, after conversion to their fluorenylmethyloxycarbonyl-esters (FMOC), separated on an Atlantis dC18 column and detected by MS/MS (in negative multiple reaction monitoring mode). Urines from non-affected PH3 patients (n = 133 with ages from 0 days to 65 years) were measured as well as urines from two proven PH3 patients. Results: PHG values in control urines showed a decrease in age. PHG in two proven PH3 patients was highly elevated at 29 and 47 mmol/mol creatinine (controls Gen and Mol Path, SA Pathology, Adelaide, Australia Fu X 1, Xiao Y 2, Xu Y 2, Pattengale P 1, Dien Bard J 1, O Gorman M 1

Background: Since the implementation of next generation sequencing (NGS) 2 years ago, our laboratory has tested and reported on approximately 200 patients with known or suspected inborn errors of metabolism. Analysis of a single gene is undertaken where a biochemical diagnosis has been made. Alternatively, small, medium or large gene panels can be requested based on clinical phenotype. The approach taken for the latter may significantly influence the chance of making a diagnosis. Methods: NGS was performed using the 550-gene Inherited Disease and 4813-gene One Illumina TruSightTM platforms, with libraries sequenced on Illumina Miseq or NextSeq sequencing systems. Patients were classified into 4 categories according to clinical indication: (A) Patients affected with a known disorder, with a biochemical or clinical diagnosis. (B) Patients with a small number of specific genes requested based on clinical phenotype without biochemical evidence. (C) Patients with broad, less-defined phenotypes with larger gene panels, multiple panels or clinical exome analysis requested. (D) Sequencing of a single gene of patients where a reproductive partner is a carrier of an autosomal recessive disorder. Results: A clear molecular diagnosis was made in 98 of 104 (94 %) patients from category A; 5 of 41 (12 %) patients from category B; 7 of 30 (23 %) patients for category C; 0 of 15 (0 %) cases in category D.

1 Dept Pathology, Univ Southern California, Los Angeles, United States, 2Dept Pathology, Child Hosp Los Angeles, Los Angeles, United States

Background: Oxidative stress has been demonstrated to be an underlying pathophysiologic process in various inborn errors of metabolism (IEMs). F2IsoPs (8-iso-PGF2α) are a series of prostaglandin F2α-like compounds produced by the free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase. In the Biomarkers of Oxidative Stress Study sponsored by the NIH, the quantification of F2-IsoPs was found to be the most accurate method to assess in vivo oxidant stress. Therefore, early detection of 8-iso-PGF2α is crucial in disease prevention and monitoring treatment efficacy. We report the development and validation of an ultra-sensitive LC-MS/MS test for the measurement of urinary F2-IsoPs. Reference interval for the pediatric population was also established. Methods: Each urine sample was spiked with internal standard and subjected to solid phase extraction with Phenomenex Strata X-AW cartridge. The extracted sample was analyzed with a Thermo UPLC and Quantiva mass spectrometer with a HESI probe. Quantitation was performed with multiple reaction monitoring mode under negative ionization. Urine samples (n = 136) with

S66 normal urine analysis were used to establish reference intervals for children age 2 months to 18 years under RIB approval. Results: The assay was linear from 0.024 nM to 20 nM (R2 = 0.999). Recoveries were above 85 % and matrix effects were below 5 %. The intra-day variability (CVs) ranged from 4.0 to 4.5 %; and the inter-day CVs ranged from 4.3 to 5.7 %. The accuracy was evaluated with a clinical reference laboratory (n = 40), and a correlation coefficient of 0.96 was observed. Reference interval for the pediatric population was established to be A; in P2 and P3 we only identified a heterozygosity for the new c.200C > T (p.Thr67Ile) and c.640-2A > G variants, respectively. The mother of P2, with a history of frequent vomiting and vertigo, resulted homozygous for c.200C > T. Asymptomatic P3’s mother was compound heterozygous for c.640-2A > G and the new c.1399A > T (p.Ile467Phe). In P4 two novel missense variants, c.493C > T (p.Leu165Phe) and c.1381 T > G (p.Phe461Val) in the MCCC2 gene, were found. Discussion: Targeted NGS is a cost-effective and rapid approach to confirm a diagnosis of 3-MCGuria and to distinguish between MCCC1 and MCCC2 forms. Such confirmation is especially important for asymptomatic cases, since they are at risk of developing acute crises.

P-043

Urinary organic acids analysis by LC-TOF high resolution mass spectrometry for the diagnostis of inborn errors of metabolism Korver-Keularts I M L 1, Wang P 1, Waterval H W A 1, Habets D D J 1, Kluijtmans L 2, Bierau J 1 1 Dept Clin Gen, Maastricht Univ Med Center, Maastricht, Netherlands, 2Dept Lab Med, Radboud UMC, Nijmegen, Netherlands

Background: Organic acidurias are among the most frequently occurring inborn errors of metabolism (IEM) that require fast diagnostics. Conventional gas chromatography (GC)-mass spectrometry (MS)-based analyses with derivation are time consuming and labor intensive. We developed a liquid chromatography (LC) high resolution time-of-flight (TOF) MS method with minimal sample preparation for targeted analysis of organic acids. Methods: In a 35-min run cycle with MS full scan in the negative mode, the LC-TOF MS method detected 63 metabolites in urine covering 52/56 organic acidurias. The method was analytically validated. For 24 metabolites from ERNDIM (external quality control scheme) the classical GC-MS method (overall results from all participants) was compared to our LC-TOF MS data by Passing-Bablok regression. Clinical validation on 100 samples (controls and IEMs) was performed. Results: 59/63 metabolites were quantified with standards; the other 4 metabolites were reported semi-quantitatively. For 20/24 compounds from the external QC scheme our LC-TOF MS and overall GC-MS results showed no significant difference (within 25 %); 4/24 metabolites showed >25 % difference (oxoglutaric acid, pyroglutamic acid, 3-OH-isobutyric acid and mevalonic acid). In clinical validation all relevant biomarkers for IEM in 100 samples were successfully identified by two independent laboratory specialists. Discussion: We developed a fast and simple LC-TOF MS targeted analysis for organic acids in urine which can be applied in routine clinical screening on IEM. It is possible to extend the targeted list of metabolites and to include the positive ionization mode to a broader metabolic screening. Finally, full scan MS data can be used for future metabolomics investigation in unraveling new/unknown IEM.

P-044 Background: 3-Methylcrotonylglycinuria (3-MCGuria) is an autosomal recessive disorder of leucine catabolism caused by deficiency of 3-methylcrotonylCoA carboxylase (MCCD), encoded by the MCCC1 and MCCC2 genes. MCCD is the most common organic aciduria detected in expanded newborn screening (ENS) programmes, revealing elevated levels of 3hydroxyisovalerylcarnitine (C5OH). 3-Methylcrotonylglycine (MCG) and 3hydroxyisovaleric acid (HIVA) are the major abnormal metabolites in patients’ urine. Phenotypes vary considerably, ranging from acute neonatal onset with a fatal outcome to asymptomatic adults. Methods: Targeted next-generation sequencing (NGS) of genes linked to 3MCGuria (MCCC1, MCCC2, HLCS, BTD and CA5A), using HaloPlex enrichment and a paired-end 2 × 150 bp sequencing on a MiSeq platform (Illumina). Results: We report 4 asymptomatic cases with high C5OH and low carnitine levels on ENS. Urine organic acid analysis revealed increased excretion of

Monitoring bile acid replacement treatment for 3β-hydroxy-Δ5-C27-steroid dehydrogenase deficiency Mazzacuva F 1, Yang Y 1, Paredes A 1, Wilson M 1, Ball C 3, Samyn M 3, Marquardt T 2, Reunert J 2, Mills P 1, Clayton P T 1 ICH, UCL, London, United Kingdom, 2Munster Univ, Munster, Germany, KCH NHS Foundation Trust, London, United Kingdom

1 3

Background: Deficiency of the enzyme 3β-hydroxy-Δ5-C27-steroid dehydrogenase (HSD), catalysing the second step of the conversion of cholesterol into bile acids (BAs), leads to the accumulation of abnormal BAs causing cholestatic

S70 liver disease and progressive cirrhosis. Administration of cholic acid (CA) results in clinical improvement due to the negative feedback exerted by its metabolites (GCA and TCA) on abnormal BA synthesis. However, because of the hepatotoxicity of excessive accumulation of GCA and TCA, treatment monitoring is crucial. Traditionally, the biochemical follow-up has been assessed by qualitative evaluation of urinary BAs or by GC-MS analysis of plasma BAs. In the present study we propose a new quantitative LC-MS/MS method for dried blood spots (DBS) and we compare the standard administration of CA to a novel treatment induction, using a crossover from ursodeoxycholic acid (UDCA) to CA. Case Report: A 10 year old boy affected with HSD deficiency was treated with CA while two siblings with the same disorder were initially treated with UDC, progressively weaned to CA. LFTs, parameters of fat soluble vitamin malabsorption and BA concentration in urine and DBS were monitored for 1 year. Results: Normalisation of fat soluble vitamin malabsorption and reduction of excretion of abnormal BAs was observed in 6 weeks in the three patients. However, the patient treated with a full dose of CA initially showed a brief but marked increase in TCA (24 μM; control range ≤ 1 μM) and GCA (83 μM; control range ≤ 3 μM). This accumulation can be attributed to the presence, in the liver, at the start of treatment of levels of unsaturated BAs which inhibit the bile salt export pump. Initiation with UDCA can establish bile flow and its accumulation is not hepatotoxic. Discussion: Treatment monitoring by LC-MS/MS of DBS provides a simple way of detecting excessive build-up of CA conjugates. Initiation of therapy with UDCA acid can prevent this.

P-045

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Mammalian mitochondrial complex I (CI) is the largest protein complex in the mitochondrial respiratory chain and forms the major entrypoint for electrons into the OXPHOS system. During this process, electrons can escape from CI and react with ambient oxygen to produce superoxide and derived reactive oxygen species (ROS). Depending on the balance between their production and removal by antioxidant systems, ROS may function as signaling molecules or induce damage to a variety of biomolecules, causing oxidative stress. Defective CI is the most common type of OXPHOS disease. In response to oxidative stress, the transcription factor Nrf2 interacts with the antioxidant responsive element (ARE) sequence modulating the gene expression of the most important antioxidant enzymes, such as those involved in the glutathione synthesis (GCLC and GCLM), detoxification of xenobiotics (heme oxygenase-1) and neutralization of ROS (superoxide dismutase, glutathione peroxidase). Methods: The expression levels of mRNA encoding Nrf2-regulated antioxidant genes have been determined by quantitative RT-PCR in fibroblasts obtained from diagnostic skin biopsies of 4 patients with isolated CI deficiency due to mutations in mtDNA-encoded (ND1, ND5) and nDNA-encoded CI subunits (NDUFB11, NDUFAF4). Protein amounts were measured by western blot analysis. Results: To gain insight into the pathological mechanism underlying ROS production and antioxidant defense in the CI deficiency, we have analyzed the genome expression profiling of Nrf2 signaling pathway and Nrf2downstream antioxidant proteins in cultured skin fibroblasts of patients carrying isolated CI mutations. By this technology, we have defined distinguishable redox profiles for each mutation, highlighting an individual antioxidant pattern. Discussion: This study lays the foundation for a personalized defense profile against ROS, helpful for a targeted therapy.

Knowledgebase of inborn errors of metabolism (IEMBASE): validation and performance P-047 Lee J 2, Wasserman W 2, Hoffmann G F 1, Van Karnebeek C 3, Blau N 1 1 Univ Child Hosp Metabol Center, Heidelberg, Germany, 2Ctr Mol Med Ther, Child Fam Res Inst, Vancouver, Canada, 3Div Biochem Dis, British Columbia Univ, Vancouver, Canada

Background: IEMbase was developed as an online platform, which combines a comprehensive expert knowledgebase and a smart system to support efficient diagnosis and management of inborn errors of metabolism (IEM) for clinicians. The system algorithmically combines semantic and cosine similarity to determine a tiered list of possible IEMs, which match the user-provided disease profile. Clinical profiles were then mapped to HPO (Human Phenotype Ontology) in order to exploit the semantic relationships of symptoms from the profiles. Methods: We extracted disease-characterizing profiles of clinical and biochemical markers (n = 2324) from an expert-generated database of 530 IEMs. IEMbase was tested with 85 cases with clinical and laboratory information and with confirmed diagnosis by different metabolic centers. Results: Using a combination of clinical and biochemical data, exact diagnosis (100 % fit) was reached in 74 % of cases and 97 % were within the top 5 diagnoses. With clinical information alone, only 39 % of cases were with top 5 diagnoses. Diagnosis was missed in 5 cases due to insufficient or missing clinical and/or biochemical information. Discussion: The IEMBASE accepts an array of biochemical markers and clinical information from a user and returns a ranked list of possible IEM disorders. In addition, the system can explain the rationale of its results, provide differential diagnoses, list possible treatment options, and provide access to external resources. IEMbase will be now mapped with the HMDB (Human Metabolome Database) to cover wide terminology of biomarkers.

P-046

Redox profiles in patients with isolated complex I mutations Petrillo S 1, Piermarini E 1, Tozzi G 1, D’Amico J 1, Di Nottia M 1, Carrozzo R 1 , Bertini E S 1, Piemonte F 1 1

Children’s Hospital Bambino Gesu, Rome, Italy

Targeted NGS for 3-methylglutaconic aciduria Ferri L 1 2, Mei D 1, Malesci D 1, Funghini S 1, Pasquini E 3, Procopio E 3, Parrini E 1, La Marca G 1 2, Renzo G 1 2, Morrone A 1 2 1 Paed Neur Unit, Meyer Child Hosp, Firenze, Italy, 2 Dept of NEUROFARBA, Univ of Florence, Firenze, Italy, 3Metab Muscular Unit, Meyer Child Hosp, Firenze, Italy

Background: 3-Methylglutaconic aciduria (3-MGA-uria) is an heterogeneous group of inborn errors of metabolism biochemically characterized by increased urinary excretion of 3-methylglutaconic acid (3-MGA). Type I 3-MGA-uria is caused by mutations in the AUH gene. Types II (Barth syndrome) and III are due to mutations in the TAZ gene and OPA3 gene, respectively. Types IV includes unclassified patients and patients with demonstrated mutations in several genes: TMEM70, ATP5E, ATP12, POLG1, SUCLA2, RYR1. Type V, VI and VII are caused by mutations in the DNAJC19 gene, in the SERAC1 gene and in the CLPB gene, respectively. Methods: Targeted next-generation sequencing (NGS) (exons + splicing junctions) using HaloPlex enrichment method (Agilent Technologies) and PairEnd 2x150bp sequencing on a MiSeq platform (Illumina). Reads were mapped by BWA and variants called accordingly to GATK best practices. Results: An NGS panel including 17 genes linked to 3-MGA-uria was set up. From this NGS panel we identified compound heterozygosity for two nonsense mutations in the AUH gene of a female patient of Chinese origin who was identified by newborn screening, which revealed elevated C5OH levels. Urinary organic acids analyses revealed 3-MGA-uria, 3-methylglutaric aciduria and 3-hydroxy isovaleric aciduria. AUH enzyme activity measured on the patient’s fibroblasts was absent and treatment was immediately started. Recently, by targeted NGS, we uncovered the novel c.253G > T p.(Glu85*) and the known c.589C > T p.(Arg197*) AUH variants. The latter has been previously reported, in an homozygous state, in a patient of Moroccan origin exhibiting speech delay. Sanger sequencing confirmed both variants in our patient and in her parents. At the last follow up, she was 8 years old and asymptomatic. Discussion: Targeted NGS leads to rapid and cost-effective detection of causative mutations in 3-MGA-uria. The parallel sequencing of our gene panel allows us to perform genetic differential diagnosis.

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Development and analytical validation of a next generation sequencing panel to assess lysosomal storage diseases

Discussion: The detection of large deletions is challenging. Heterozygous deletions are now detectable by novel methods such as MPS using CNV tools. Further analyses are necessary to clarify the pathogenic effect of the mutated alleles found here and to determine the allele frequency of these rare deletions.

Sudrie Arnaud B 1, Charbonnier F 2, Dranguet H 1, Coutant S 2, Mezain M 2, Saugier Veber P 2, Bekri S 1

P-050

1 Department of Metabolic Biochemistry, Rouen, France, 2Lab Molecular Genetics, INSERM EMI 9906, Rouen, France

Urease immobilized on magnetic microparticles for urine GC/MS analysis

Background: The lysosomal storage disorders (LSD) diagnosis may be hampered by their significant heterogeneity with phenotypic overlap, variable severity and onset. Early diagnosis, including genetic testing, may enable treatment of some conditions and may prevent clinical complications. Conventional biological diagnostic procedures are based on a series of sequential investigations and require multiple samples and steps. Therefore, we have developed a next generation sequencing (NGS) panel that includes 51 LSD genes. This panel is tested in patients with storage features such as bone abnormalities, organomegaly, central nervous system dysfunction and coarse hair and facies or with a nonspecific “storage-like” presentation or hydrops fetalis presentation. Methods: The design of the lysosomal storage disease panel covered the coding region, promoter region and the flanking intronic sequences for 43 genes. In addition, 3’ untranslated sequences were included for 2 genes (AGA and ARSA) and the entire gene sequences were covered for 6 genes (ARSB, CLN3, CLN8, IDS, SGSH and NAGLU). Library construction was done using SureSelectQXT (Agilent) and sequencing was performed on MiSeq instrument (Illumina®). Eighteen patients with LSD with 30 known pathogenic variations and 70 variations using Sanger method were included for the validation. Results: This method allows the analysis of greater than 72.2 % of the targeted sequence. The current protocol was designed to detect copy number alterations. The data were obtained with an excellent coverage for all targeted exonic and intron-exon junction regions (30X target coverage of 99.6 % and an average coverage of 474X). Expected 100 known variations were found in all patients. Discussion: NGS combined with robust bioinformatics analyses is a very useful tool for identifying the causative variations of LSD

Jacova J 1 2 5, Jorenek M 3, Friedecky D 1 2 5, Najdekr L 2 5, Karlikova R 2 5, Cavar S Z 4, Tarkowski P 4, Zajoncova L 3, Adam T 1 2 5

P-049

Detection of large deletions in the genes FMO3 and PHKB by use of novel analysis techniques Maurer E 1, Schatz U 1, Zschocke J 1, Witsch-Baumgartner M 1 1 Div Human Genetics, Med Univ, Innsbruck, Austria Background: Trimethylaminuria is caused by impairment of hepatic trimethylamine (TMA) oxidation due to mutations in the FMO3 gene. Phosphorylase kinase deficiency of liver and muscle (GSDIXb) is caused by mutations in the PHKB gene. In a number of patients with these disorders the second mutation is not detected. This might be due to unproven deletions in the respective genes. Here we report deletions in a patient with TMA and GSDIXb, respectively. Methods: Targeted amplification by Nextera® Rapid Capture (Illumina), then massive-parallel sequencing (MPS; MiSeq). Data analysis including the CNV tool by NextGENe® (SoftGenetics). Sequencing of the whole CDS of FMO3. Breakpoint detection by long range PCR using specific primers located in the adjacent introns [deletion-specific PCR-products: ~320 bp (FMO3), ~680 bp (PHKB)]. 20 TMA patients lacking at least one mutation have been tested for the FMO3 deletion. Results: The TMA patient, a 5y old male of Turkish origin, carries the FMO3-deletion c.-2082_133-982del (12227 bp) including exon 2 with the start codon homozygously. This deletion was described once in an Australian patient of Greek ancestry (Forrest et al., 2001); it likely removes the FAD binding domain with loss of FMO3 function. The FMO3 deletion was tested in a cohort of TMA patients. The GSDIXb patient, a 34y old male of Austrian origin carries the PHKB-deletion c.2176-255_2257 + 339del (676 bp) including exon 23 heterozygously; this is out of the reading frame and is likely to lead to a truncated nonfunctional protein.

1 Lab Inh Metab Dis, Univ Hosp Olomouc, Olomouc, Czech Republic, 2Lab Metabolomics, IMTM Palacky Univ, Olomouc, Czech Republic, 3Dept Bioch, Faculty of Sci, Palacky Univ, Olomouc, Czech Republic, 4Centre Region Hana Biotech Agricult Res, Olomouc, Czech Republic, 5Dept Clin Bioch, Univ Hosp Olomouc, Olomouc, Czech Republic

Background: Urea is the most abundant polar component of urine complicating analyses of urine metabolome, namely when GC/MS technique is used. Since the 1970s liquid-liquid extraction was predominantly used prior to the analysis. The acidic extraction into ethylacetate significantly distorts urine metabolome (solvent-based selection of structural groups of metabolites). An alternative method using urease was developed, however, it introduces a lot of artifacts and it is very laborious. Methods: The aim of the work was to develop the new method of sample preparation, based on urease immobilized on microparticles, and compare it to other used methods (liquid-liquid extraction, free-urease protocol and no sample treatment). The impact of sample preparation techniques on the quality of analytical data was studied via targeted GC/MS analysis (known levels of organic acids) and targeted LC/MS analysis (non-GC/MS metabolites) of standard material based on human urine (Control Organic acid, MCA Laboratory). Results: The urease immobilized on magnetic cellulose microparticles was prepared by simple two-step procedure (bead oxidation, binding of urease). The specific activity was 141 % (as compared to free urease), operational stability 69 %, functional stability 83 %, and storage stability 82 % (1 month at −80 °C). Immobilization of urease on microparticles decreased both number of artifacts (10 peaks in the sample of free urease vs 1 in immobilized urease) and variability of metabolites (average CVof extraction 27 %, no treatment 11 %, freeurease 8 %, and immobilized urease 4 %). Discussion: The new sample preparation technique was tested using urine from patients with metabolic diseases, where urea comigrates with diagnostically important metabolites (e.g. SCAD, glutaric aciduria). Principal component analysis and Wilcoxon test have shown that immobilized-urease treatment provided similar results to free-urease approach. SUPPORT: IGA_LF_2016_014, NPU I (LO1304), GACR I 1910-N26.

P-051

To study the additive predictability of non-HLA marker amino acid citrulline as a metabolomic signature in children with celiac disease and in their first degree relatives (FDR) Lomash A 1, Kumar S 1, Polipalli S K 1, Kapoor S 1 1

MAM College,Univ of Delhi, New Delhi, India

Background: Citrulline is a non-essential amino acid that is not incorporated into proteins and the small intestine is the main endogenous source. Until now, HLA is the only strongest risk locus for CD. This study was designed to evaluate citrulline levels in patients with CD and in their FDR and to establish a correlation between histopathological findings and the citrulline levels as biomarkers for villous atrophy. Methods: The procedure adopted for measuring citrulline on DBS filter paper was tandem mass spectrometery (LC-MS/MS) and plasma citrulline was analysed by RP-HPLC and the disease state was confirmed by HLA typing.

S72 Results: Plasma citrulline value in 54 serology positive subjects was 9.0 ± SD umol/L, citrulline levels in 124 serology negative subjects (FDR) was 24.3 ± SD umol/L with p-value of 500) and patients with known IEMs were analysed. Samples of spiked urine used in the ERNDIM quality control programme were analysed and results showed a linear correlation to median values obtained from laboratories participating in the quality control programme. Discussion: The method substantially simplifies the laboratory work, reduces diagnostic laboratory time and has a great potential for expanding the range of analytes by addition of ion transitions. Grant support: NPU I (LO1304), GACR I 1910–26, IGA_LF_2016_014.

P-055

Measurement of cellular glycolytic flux by liquid chromatography–tandem mass spectrometry Wang B T 1, Schmidt K 1, Okun J G 1, Sauer S W 1 1

Dietmar-Hopp Metab Cen, Univ Child Hosp, Heidelberg, Germany

S73

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Glucose metabolism is essential in all life. The downstream glycolytic intermediates are responsible for energy generation with the biosynthesis of amino acids, lipids and nucleotides. In additions, the reactive oxygen species are detoxified through pentose phosphate pathway, and the production of fructose-6-phosphate is involved in protein glycosylation post-modification in hexosamine biosynthesis pathway. The disturbance of glycolytic flux has been shown in several acquired and inborn diseases, and indicated to be a hallmark of cancer cells and cells under increased energetic stress. Our study aimed to establish a stable method for measuring the main glycolytic intermediates by a liquid chromatography–tandem mass spectrometry (LC-MS/MS) and to study the metabolic flux in different cell models. Methods: Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose1,6-bisphosphate (FBP), glyceraldehyde-3-phosphate (G3P), 2phosphoglycerate (2PG), phosphoenolpyruvate (PEP), pyruvate (PYR), lactate (Lac), and ribulose-5-phosphate (R5P) were included. Samples were deproteinized and injected into a LC-MS/MS. The liquid chromatographic separation was carried out by a Phenomenex Synergi column with the mobile phase of 10 mM tributylamine and methanol. Results: The standard curves were performed from 1 μM to 10 or 20 μM with the linearity (R2) between 0.98 and 0.99. The recovery rates of G6P, F6P, G3P, 2PG, PEP and PYR were within the range of 82 % to 113 % and the impressions (CV%) were less than 10 %. The glucose metabolites were measured in samples from patients with inborn disease such as phosphoglycerate kinase 1 deficient cells and cancer cells such as Jurkat leukemic T-cell and liver hepatocellular cells. Discussion: We established a reliable LC-MS/MS method for studying glucose metabolites which could be further applied for recording the glucose metabolic profile in cells from patients with metabolic disorders.

P-056

Fibroblast metabolic flux analysis for the work-up of inherited metabolic disorders Habarou F 1 2, Pontoizeau C 1, Tamby C 2, Chadefaux-Vekemans B 1, Mochel F 4 5, Rotig A 2, De Lonlay P 2 3, Ottolenghi C 1 2 1 Metab Biochemistry, Necker Hosp, APHP, Paris, France, 2Imagine Institute INSERM U1163, Paris, France, 3IEM Reference Center, Necker Hosp, APHP, Paris, France, 4Genet Div, Pitie-Salpetriere Hosp, APHP, Paris, France, 5 INSERM U 1127, ICM Institute, Paris, France

Background: Palmitate loading tests in cultured patient fibroblasts have long been part of the diagnostic work-up of suspected mitochondrial fatty acid oxidation disorders. In other circumstances, however, loading tests using stable isotope tracers are not common practice. We found that in several novel inherited metabolic disorders (IMDs), stable isotope labeled tracer analysis provided an interesting alternative to traditional biochemical methods. Methods: Cultured patient fibroblasts were incubated with uniformly 13C labeled glucose, glutamine, leucine and/or other amino acids for up to 18 h. Metabolite extraction was followed by silylation and gas chromatography– mass spectrometry (Scion TQ, Brüker). Results: Compared to loading tests with radioactive substrates, stable isotope labeled tracer analysis was as informative yet much simpler and faster to detect reduced branched chain ketoacid dehydrogenase activity in novel defects of lipoic acid biosynthesis (LIPT1, LIPT2). We recently introduced a stable isotope labeled tracer technique to validate proline biosynthesis defects involving P5CS/ALDH18A1, for which enzymology is particularly cumbersome. In addition, flux analysis outperformed enzymology to detect increased glutaminolysis in a putative gain-of-function mutation involving glutaminase (GLS). In all these cases, standard metabolic screenings were only partly or not informative and flux analysis provided functional validation for mutations identified by exome studies. Furthermore, fibroblasts from patients with mild clinical forms of NARP mitochondriopathy (ATP6 mutations) incorporated reduced amounts of labeled carbons, contrasting with normal oxidative phosphorylation, thus providing a surrogate readout for therapeutic tests. Discussion: Progress in GC-MS technology now allows sensitive tracer analysis in patient fibroblasts. This facilitates and expands the applications of metabolic flux analysis for diagnostic and therapeutical purposes in IMDs.

P-057

Whole exome sequencing, consanguinity and inborn errors of metabolism: when you need more than one genetic disease to explain the phenotype Monti F 2, Piazzon F 1 2, Monteiro F 1, Costa L 1 2, Kitajima J P 1, Bueno C 2, Porta G 3, Wanderley H 4, Schwartz I 5, Kok F 1 2 Mendelics Genomic Analysis, Sao Paulo - SP, Brazil, 2Neurometabolic Unit, HC-USP-SP, Sao Paulo - SP, Brazil, 3Ped Hepat Unit, HC-USP-SP, Sao Paulo - SP, Brazil, 4Genetic Unit, Hosp Est Inf, Vitoria - ES, Brazil, 5Genetic Serv, HC-PA, Porto Alegre - RS, Brazil 1

Background: Whole exome sequencing (WES) became a very efficient tool to investigate challenging cases and the finding of more than one symptomatic genetic disorder in a patient is currently not uncommon, especially in inbred populations. Methods: WES was performed using Nextera Extended Capture Kit and Illumina HiSeq2500 platform, we identified 4 patients, born to consanguineous parents, with a combination of one inborn error of metabolism and another recessive genetic disorder, revealing an unusual phenotypic association. All reported variants were in homozygous state. Results: Case 1: 3-year-old boy with osteogenesis imperfecta (OI) associated with developmental delay, upward gaze palsy, dysphagia and dysarthria. WES showed autosomal recessive OI 15 (WNT1, p.Glu343Serfs49*), with the remaining clinical phenotype attributed to PRICKLE1 (p.Thr275Met). Case 2: 3year-old girl with severe intellectual deficiency (ID) and untreatable seizures. WES disclosed deleterious variants in ATIC (p.Tyr362Cys), leading to AICAribosiduria, and PGAP2 (p.Phe89Ser), causing ID with elevated alkaline phosphatase. Biochemical tests supported both diagnoses. Case 3: 37-year-old man, with marfanoid habitus and lens subluxation, camptodactyly, arthropathy and deafness. Plasma homocysteine was elevated. WES revealed classical homocystinuria (CBS, p.Ile278Thr), AR deafness 8 (TMPRSS3, p.Ala138Glu) and camptodactyly-arthropathy-coxa vara-pericarditis syndrome (PRG4, p.Lys1253*). Case 4: 1-year-old girl, with liver failure, rickets, ptosis, myasthenic facies and hypotonia. Succinylacetone was elevated. WES confirmed tyrosinemia type 1 (FAH, c.555-1G > T) and diagnosed cystinuriahypotonia syndrome (CAMKMT, p.Ser93Arg). Discussion: Association of more than one genetic disorder in a patient should always be considered, especially in inbred families. Recurrence risk of at least one of the disorders can be as high as 37.5 %, but can be lower if the responsible genes are linked, as occurred twice among our patients. Conflict of Interest declared.

P-058

Exact mass multi-stage fragmentation for structural elucidation of biomarkers of inherited metabolic disorders Vaclavik J 1, Friedecky D 1 2, Adam T 1 2, Kluijtmans L A J 3, Wevers R 3 1 Inst Molec Translat Med, Palacky Univ, Olomouc, Czech Republic, 2Lab IMD, Palacky Univ, Olomouc, Czech Republic, 3Translat Metabol Lab, Radboud Univ, Nijmegen, Netherlands

Background: Specific diagnostic markers are key to effective diagnosis of IMDs. Current mass spectrometry provides a powerful tool for structural elucidation of unknown compounds even in complex biological matrices. This approach was used to determine molecular structure of uncharacterized compounds, that were observed in plasma samples from phenylketonuria (PKU) and 3-hydroxy-3-methylgluratyl-CoA lyase deficiency (HMGLD) patients. Methods: We used liquid chromatography coupled to a high-resolution mass spectrometer Orbitrap Elite (Thermo Fisher Scientific, MA, USA). Multi-stage fragmentation mass spectra were acquired via two different fragmentation techniques, collisional-induced dissociation (CID) and high-energy collisional dissociation (HCD). In order to determine the molecular structure, we searched Metlin (Sana TR, J Biomol Tech, 2008), ChemSpider (http://

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P-059

the clinical heterogeneity and diagnostic difficulty in mtDNA mediated mitochondrial disorders. In this study, the specifity and sensitivity of next generation semiconductor DNA sequencing technology was investigated for the detection of a heteroplasmic mtDNA mutation. Methods: mtDNA from a patient with MELAS syndrome harbouring mt.3946G > A mutation was mixed with a control mtDNA at different concentrations (1:99, 5:95, 10:90, 20:80, 30:70, 40:60 and 50:50) and was sequenced with semiconductor DNA sequencing technology at various mean depths from 100 to 1000. The data was analyzed with a special bioinformatic software. Results: Semiconductor DNA sequencing technology had a considerably high sensitivity for detecting the mtDNA mutation even at 1 % concentration at relatively high mean depths. Discussion: Semiconductor DNA sequencing technology has the potential to facilitate the reliable detection of heteroplasmic mtDNA mutations in patients with mitochondrial disorder.

Quantitation of plasma organic acids (OAs) in patients with mitochondrial disorders and other inborn errors of metabolism (IEMs) by gas chromatography–mass spectrometry (GC-MS)

03. Newborn screening

www.chemspider.com/), mzCloud (https://www.mzcloud.org/) and HMDB (Wishart DS, Nucleic Acid Res, 2009) databases together with fragmentation predictive software MassFrontier (HighChem, SK). Exact masses of molecular ions as well as their fragments were determined with resolution 120 000 FWHM within mass accuracy ?ppm < 1.5. Results: The fragmentation experiments led to structural elucidation of 3methylglutaconyl carnitine in HMGLD patients. In samples from PKU patients two novel compounds were characterized: Phe-Glu-Glu conjugate and Phe-hexose conjugate. Discussion: Our results proved that structural elucidation of unknown metabolites in human biofluids can be done by current exact mass multi-stage fragmentation techniques even at relatively low concentrations.

Laxen W J 1, Loken P R 1, Danielson M A 1, Gavrilov D 1, Raymond K 1, Rinaldo P 1, Tortorelli S 1, Matern D 1, Oglesbee D 1 1 Mayo Clinic, Rochester, United States Background: Metabolomic screening promises to uncover new biomarkers, or previously unrecognized metabolite profiles, common to a biochemical genetic disorder. For patients with a primary mitochondrial disorder, or other IEM, the analysis of multiple small metabolites within a single plasma sample may enhance diagnostic yield. Thus, we developed a GC-MS method to simultaneously quantify 17 OAs and related metabolites and characterized plasma from a cohort of individuals with primary mitochondrial disease and other IEMs. Methods: A GC-MS, isotope-dilution method was developed for measuring 17 OAs, including citric acid cycle intermediates, lactate, pyruvate, branch-chain amino acid intermediates, and ketone bodies. Samples from 20 donors with a primary mitochondrial disease, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes, pyruvate dehydrogenase complex deficiency, and Barth syndrome, as well as maple-syrup urine disease (MSUD), 3hydroxyl-3-methylglutaryl-CoA (HMG-CoA) lyase deficiency, and other IEMs were analyzed. Reference values for analyte profiles were obtained 300 donors without an IEM. Results: Pentafluorobenzyl-oximation improved the stability of several target analytes, such as acetoacetic acid, and sensitivity was enhanced with positive ion mode. Plasma from donors with a primary mitochondrial disease demonstrated significantly elevated levels of lactate, pyruvate, 2-hydroxybutyrate, and citric acid cycle intermediates, while MSUD and HMG-CoA lyase deficiency donor samples showed characteristic patterns of elevated branch-chain amino acid intermediates as compared to reference controls. Discussion: Plasma OAs from patients with a primary mitochondrial disorder, and specific IEMs, displayed a distinctive metabolic profile as analyzed by this GC-MS method. The measurement of a single plasma OA profile may enhance the detection of certain mitochondrial disorders and reduce the necessity to obtain a suite of individual tests.

P-061

Supporting parents following positive newborn screening results for an inherited metabolic disorder Bonham J R 1, Moody L 2, Atkinson L 2 1 Sheffield Child Hosp, Sheffield, United Kingdom, 2Coventry Univ, Coventry, United Kingdom

Background: Positive newborn screening results raising the possibility of an inherited metabolic disorder can be upsetting for parents and may have lasting consequences even when confirmatory tests subsequently prove normal. Methods: A recent qualitative synthesis of 18 articles examining the views and experiences of parents revealed a number of ways in which the interaction between healthcare professionals and parents could be improved. Some of these themes were also reflected in the results of semi-structured interviews undertaken with parents of children who had received a positive screening result (n = 10) and healthcare professionals (n = 11) who had been involved with the diagnosis and support of parents. Results: The key findings from the literature search included: the timing of screening results, communication styles and ongoing information and support for families. The key findings from the interviews also emphasised the importance of the way in which the results are communicated to parents, rapid turnaround of results and the consistency of approach from those involved in family contact during this period. Discussion: Based upon this experience the authors have sought to design an app which can be used by parents and health professionals to provide information and support during this anxious time from the news of a positive screen result to the conclusion of confirmatory testing. The content, structure and philosophy of this app together with the basis for this approach is outlined.

P-060 P-062 The specificity and sensitivity of next generation semiconductor DNA sequencing in detecting mitochondrial DNA heteroplasmy

Multiplex screening for lysosomal storage diseases (LSDs)

Ezgu F 1, Topcu B 1, Ciftci B 1, Okur I 1, Tumer L 1

Eyskens F J M 1, Devos S 1

1

1

Background: Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. They can be caused by mutations of genes encoded by either nuclear DNA or mitochondrial DNA (mtDNA). Mitochondrial disorders resulting from mutation of mtDNA may harbor a mixture of mutated and wild-type mtDNA within each cell which is called heteroplasmy. Heteroplasmy is one of the reasons responsible for

Background: The interest in neonatal screening for LSDs has increased substantially because screening has been made possible by recent technical advances. The results of pilot LSD screening studies show that the current clinical prevalence is underestimated. Methods: The LC-MS/MS method has advantages for expanding the assays to include additional products and internal standards for multiplexing all nine currently

Gazi Univ, Labor Inborn Metab Disord, Ankara, Turkey

Div Metab Dis, PCMA, Antwerp, Belgium

S75

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 available lysosomal enzyme assays in dried blood spots (Gaucher/ABG, NiemannPick A/B, Fabry/GLA, Krabbe, Pompe/GAA, MPS-I/IDUA, MPS-II, MPS-IVA and MPS-VI) (Spacil et al., 2012, Clin Chem). Validation of this method to the full set of treatable LSDs has been performed in our laboratory. Results: An anonymous pilot study was performed from 2014 to 2015. ABG, GAA, GLA and IDUA enzyme activities in over 20,000 newborn samples were analyzed. Statistically significant higher GAA and GLA enzyme activities were observed in female newborns compared to male newborns. Newborns with a higher birth weight and gestation age have a statistically significant lower GAA and GLA enzyme activity compared to newborns with lower birth weight and gestation age. This proves that it is of importance to define the reference intervals for lysosomal enzyme activities as well as cut-off limits for newborn babies with regard to birth weight, gestational age and sex for each population. One true positive of non-cardiac type of Pompe was found retrospectively as this child presented in the clinic at the age of 1 month with severe muscle hypotonia and motoric development delay. Discussion: We report for the first time results of a 7-plex enzymatic activity UHPLC-MS/MS assay for the LSDs Gaucher, Fabry, Pompe, MPS I, MPS II, MPS IV and MPS VI in Europe. This method is accurate, fast, inexpensive and easy to implement next to the routine newborn screening and therefore suitable for LSD newborn screening.

Methods: From the summer of 2016, the Heidelberg newborn screening centre will start with a pilot study to evaluate newborn screening for 21 additional metabolic disorders for the German newborn screening panel, including e.g. remethylation disorders, classical homocystinuria, vitamin B12 deficiency, tyrosinemia type I, methylmalonic and propionic aciduria and urea cycle disorders. Results: The designated diagnostic algorithms including second tier strategies will be presented. It will be prospectively evaluated whether the extension of the newborn screening panel fulfills the criteria for a population based screening program, especially concerning technical feasibility, process quality, and medical benefit. As an example, the clinical histories of two patients diagnosed symptomatically with one of the new target disorders (combined cobalamin deficiency and severe maternal vitamin B12 deficiency) are presented and their newborn screening results retrospectively evaluated. Screening results of 10 additional patients with remethylation disorders or vitamin B12 deficiency are presented and evaluated with regard to the proposed strategy of the pilot study. Discussion: We expect a considerable number of children to benefit from screening for the additional target disorders in the course of the pilot study and in case of a future extension of the newborn screening panel for Germany.

P-065 P-063 Serendipity in newborn screening work flow: a case report of a methylenetetrahydrofolate reductase deficiency Neonatal screening for phenylketonuria in Kazakhstan Svyatova G S 1, Salimbayeva D N 1, Berezina G M 1, Khoroshilova I G 1, Kirikbaeva M S 1 1

Scientific Center of Obstetrics, Gynecol, Almaty, Kazakhstan

Background: Phenylketonuria (PKU; OMIM: #261600) is an inborn error of phenylalanine metabolism. The most effective method of early diagnosis and prophylactics of PKU is neonatal screening. The national program of neonatal screening for phenylketonuria was introduced in Kazakhstan in 2007. Methods: In Kazakhstan we use immunofluorescence method with standard kits for neonatal screening of PKU. Results: In 2007–2015, 2043533 newborns were tested for PKU. The middle coverage of neonatal screening in Kazakhstan increased from 46.2 to 75.7 %. Kazakhstan has 16 regions and 2 megalopolis. The coverage of PKU in these regions is different, but in 9 regions is over 90 %. The effective state program of neonatal screening in Kazakhstan allowed us to shorten treatment initiation time for PKU to T (p.A222V) polymorphism (rs1801133). Discussion: The present case report describes an occasional identification of a MTHFR deficiency by NBS, due to lab work flow procedures. More cases should be collected in order to establish whether the MTHFR deficiency should be included in NBS panel.

P-064 P-066 Pilot study for evaluation of 21 additional metabolic disorders for the German newborn screening panel Gramer G 1, Okun J G 1, Hoffmann G F 1 1

Univ Children’s Hospital Heidelberg, Heidelberg, Germany

Background: The German newborn screening panel currently includes fourteen target disorders. Recent improvements in diagnostic and therapeutic options suggest an extension of the newborn screening panel.

MCAD deficiency with severe neonatal onset, fatal outcome, and normal acylcarnitine profile Fingerhut R 1 2 5, Joset P 3, Sluka S 1 2 5, Herget T 3, Azzarrello-Burri S M 3, Rauch A 3, Baumgartner M 2 4 Swiss Newborn Screening Laboratory, Zurich, Switzerland, 2Childrens Research Center, Zurich, Switzerland, 3Inst Medical Genetics, University Zurich, 1

S76 Switzerland, 4Div Metabol, Univ Childrens Hospital, Zurich, Switzerland, 5 Univ Childrens Hospital, Zurich, Switzerland Background: Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessively inherited disorder of fatty acid oxidation with potentially fatal outcome in undiagnosed patients. Introduction of tandem mass spectrometry into newborn screening (NBS) has led to the inclusion of MCADD into NBS in many countries, which has resulted in a significant reduction of morbidity and mortality. Methods: Acylcarnitines were measured within the routine NBS. Whole exome sequencing was performed using the Agilent SureSelectXT Kit with paired-end sequencing on a HiSeq2500 System. Results: We report a child with MCADD presenting neonatally with apnoea and heart arrest. Despite intensive efforts to rescue the child, including reanimation for 90 min, the child died at the 2nd day of life. Autopsy revealed fatty liver and fat storage in heart muscle, which was suggestive of a fatty acid oxidation defect. However, acylcarnitines determined from stored EDTA blood were not suggestive for MCAD deficiency. Nevertheless, a subsequent whole exome sequencing analysis revealed homozygosity for the ACADM gene c.1084A > G / p.Lys362Glu mutation. Discussion: This case report illustrates that (a) a normal acylcarnitine profile cannot exclude MCADD, and (b) NBS for MCADD cannot always prevent a fatal neonatal outcome. However, the most astonishing thing about this case is the coincidence of both.

P-067

Japan-wide gene panel study for target metabolic diseases in newborn mass screening using tandem mass spectrometry Sasai H 1, Otsuka H 1, Fujiki R 2, Ohara O 2, Nakajima Y 3, Ito T 3, Kobayashi M 4, Tajima G 5, Sakamoto O 6, Matsumoto S 7, Nakamura K 7, Hamazaki T 8, Kobayashi H 9, Hasegawa Y 9, Fukao T 1 10 1

Dept of Pediatr, Gifu Univ, Gifu, Japan, 2Kazusa DNA Res Inst, Kisarazu, Japan, 3Dept of Pediatr, Fujita Health Univ, Toyoake, Japan, 4Dept of Pediatr, Jikei Univ Med, Tokyo, Japan, 5Div of Neonatal Screening, NCCHD, Tokyo, Japan, 6Dept of Pediatr, Tohoku Univ, Sendai, Japan, 7Dept of Pediatr, Kumamoto Univ, Kumamoto, Japan, 8Dept of Pediatr, Osaka City Univ, Osaka, Japan, 9Dept of Pediatr, Shimane Univ, Izumo, Japan, 10Div of Clin Genet, Gifu Univ Hosp, Gifu, Japan Background: Newborn mass screening (NBS) using tandem mass spectrometry began in 2014 throughout Japan and the target metabolic diseases (TMDs) increased from 6 to at least 19 diseases. Although supportive laboratories to perform analyses of urinary organic acid and serum acylcarnitine became available, molecular diagnosis for TMDs are not commercially available and until recently such molecular analyses were mainly performed by pediatricians with “volunteer spirits”. Methods: To change the situation, we planned to create a gene panel which consists of 54 genes and allows the molecular diagnosis of the TMDs and the related diseases in our research center as substitute for “volunteer pediatricians”. This research was financially supported by Japan Agency for Medical Research and Development. DNA was purified from bloods in Gifu University and the gene panel was analysed using the MiSeq (Illumina®) in Kazusa DNA Res Institute Sanger sequencing was performed to confirm the mutations. Results: We analysed 62 patients who had been positively detected from April 2014 to March 2016. The number of patients with TMDs detected by NBS were as follows: propionic acidemia (13), methylmalonic acidemia (10), primary systemic carnitine deficiency (7), methylcrotonylglycinuria (6), VLCAD deficiency (5), MCAD deficiency (3), phenylketonuria (3), citrullinemia type 1 (3), citrin deficiency (2), glutaric acidemia type 1 (2), OTC deficiency (2), others (6). In most cases, we could find the gene mutations in their corresponding genes. Discussion: In Japan, pilot tandem mass screening showed that total incidence of TMDs was 1:8000 newborns. Thus our study covered about 25 % total patients with TMDs. Coauthors in this study play a role as experts of each disease in reporting the result of gene analysis. Since the cost of gene panel

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 analysis is decreasing in these years, centralization of mutation analysis using NGS for TMDs is now feasible and promising.

P-068

Second tier test for isovaleric acidemia using LC-MS/MS in Tokyo, Japan Ishige N 1, Watanabe K 1, Hasegawa S 1, Konishi K 1, Mashita M 1, Sera Y 1, Ishige M 2, Owada M 1 1 Div Newborn Scr, Tokyo Health Serv Assoc, Tokyo, Japan, 2Dept Pediatr, Nihon Univ Hosp, Tokyo, Japan

Background: In newborn screening (NBS) using tandem mass spectrometry (TMS), a well-known cause of false-positive testing for isovaleric acidemia (IVA) is the presence of pivaloylcarnitine (PC), an isomer of C5-acylcarnitine, which is formed in the presence of pivalic acid on dried blood spots (DBS). These false-positive cases are caused by the administration of pivalic acid (PVA)-containing drugs (some antibiotics and sivelestat sodium). These are not rare cases in Japan. We showed the false-positive cases of NBS for IVA for all of Japan, and evaluated the effectiveness of a 2nd–tier test using LC-MS/ MS method for NBS for IVA in the Tokyo Metropolitan Area. Methods: The 2nd-tier test we described here quantifies C5 isoforms and isovalerylglycine (IVG) in DBS. For individual quantification we combined LC using multi-mode ODS column with TMS in ESI (+) mode. Results: Four patients with IVA were reported by TMS in Japan from 2013 to 2014 (1/426,800). The recall rate (0.087 %) for C5 isoforms was the highest in Japan during NBS by TMS. We screened 301,171 babies from April 2012 to March 2016 in Tokyo, and no IVA patients were detected. We analyzed 14 medical examinations by the 2nd-tier test. The results were that elevation of PC was detected and IVG was not detected. In analysis of 62 recall cases with elevated C5 (>1.0 nmol/mL) by the 2nd-tier test, PC was detected in 54 out of 62 cases (87.1 %) in 2015. All of these were false-positive caused by PVAcontaining drugs. Discussion: About 90 % of elevated C5 were excluded as false-positive caused by PVA-containing drugs. The analysis of C5 isomers and IVG are useful for confirmation that elevation of C5 is caused by IVA or false-positive. The 1st DBS sample for newborn screening can be utilized for this 2nd-tier test, thus cutting collection and shipping time and costs. The 2nd-tier method described here could reveal false positive C5 elevations in newborn screening for IVA.

P-069

A severe LCHADD identified at newborn screening with an atypical genotype Santos H 1, Fonseca J 1, Teles A 4, Vieira A 3, Sousa C 2, Rocha H 2, Sales Marques J 1, Vilarinho L 2 Div Neurosc, Pediatr Dept, CHVNGE, Vila Nova de Gaia, Portugal, 2Div Newb Scr, National Inst Health, INSA, Porto, Portugal, 3Div Nutrition, Pediatr Dept, CHVNGE, Vila Nova de Gaia, Portugal, 4NICU, Pediatr Dept, CHVNGE, Vila Nova de Gaia, Portugal 1

Background: Long chain fatty acid beta oxidation disorders include isolated LCHAD deficiency and MTP deficiency (LCEH, LCHAD and LCKAT activities). They are autosomal recessive disorders caused by mutations in HADHA gene. They can have protean manifestations, but a number of patients with milder defects can never develop symptoms if they are not exposed to the necessary environmental stress. Case Report: A 9-day old male referred by national neonatal screening program for LCHAD/MTP deficiency. Mother had four previous spontaneous foetal losses. He was the first son of an unrelated couple with advanced parental age and had intrauterine growth restriction and low birth weight. He was discharged at 4th day of life with exclusive breast feeding. Reassessed at day 8th, he had 8 % weight losts, and formula feeding was initiated. In day 9 after referral he was emaciated but with good tonus and vitality, and had

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 blood residues in umbilical scar. Complementary examinations showed elevated BT and BD, liver transaminases, GGT and alkaline phosphatase; coagulation abnormalities (low fibrinogen, elevated APTT and PT). He was started on hypercaloric fluids, very low lipid formula, vitamin K and insulin until normal coagulation, and was discharged after 10 days. He is now 18 months, has normal growth, development, tonus and osteotendinous reflexes. He is on a fat-restricted diet and DHA, and had severe rhabdomyolysis crisis at 15 and 18 months. No evidence of neuropathic or retinal complications. Results: Genetic study of HADHA gene showed a compound heterozygous (c.1528G > C and c.921_923delGGT—not yet described). A functional betaoxidation study in skin fibroblasts confirmed isolated LCHAD deficiency. Discussion: This case illustrates the importance of newborn metabolic screening, allowing timely initiation of the appropriate etiological treatment of liver failure. The patient is a compound heterozygous for HADHA, with severe precocious manifestations resembling a more severe MTP deficiency.

P-070

Mimicking medium chain acyl CoA dehydrogenase deficiency (MCADD) on newborn screening (NBS): an unusual iatrogenic cause of deranged acylcarnitines

Background: Mucopolysaccharidosis type I (MPSI) was recently added to the US Recommended Uniform (Newborn) Screening Panel. Available screening assays for several MPS are sensitive but have high false positive rates. To improve this situation we developed and validated a 2nd tier test to measure dermatan (DS) and heparan sulfate (HS) in dried blood spots (DBS) when low iduronidase activity is found by the primary NBS assay. Methods: DS and HS are extracted from DBS and enzymatically digested to disaccharides prior to LC-MS/MS analysis. Run time is 7 min per sample. Results: 954 newborn DBS, 173 pediatric DBS (age >1 week – 18 years) and 170 adult DBS controls were analyzed for DS (median: 66.3, range: 26.0–216.4; median: 32.8, range: 12.8–129.3 median: 34.6, range: 14.8–151.6 nmol/L, respectively) and HS (median: 42.3, range: 6.2–105.4; median: 32.1, range: 6.1–110.6 median: 31.7, range: 12.6–111.6 nmol/L, respectively). DBS from 20 MPSI patients were analyzed and showed elevated DS (median: 509.3, range: 162.4– 3457.9 nmol/L) and elevated HS (median: 164.2, range: 100.7–671.4 nmol/L). MPSI pseudodeficient patients (N = 9) had normal DS (range: 32.4–62.9 nmol/L) and HS (range: 20.9–49.5 nmol/lL) concentrations. We also analyzed other MPS disorders: eight MPSII, three MPSIII, five MPSIV and three MPSVI. Discussion: Preliminary data show that our test is a rapid and specific method for timely diagnosis and treatment of newborns with MPSI, allowing identification of cases with low iduronidase activity due to pseudodeficiency.

P-072 Hulley S L 2, Dalley J 1, Shakespeare L 1, Downing M 1, Bonham J R 1, Sharrard M 2 1

Dept Clin Chem, Sheffield Childrens Hosp, Sheffield, United Kingdom, 2Div Metab Med, Sheffield Children’s Hosp, Sheffield, United Kingdom Background: All babies born in the UK are screened for MCADD by tandem mass spectrometry, estimation of octanoyl carnitine (C8) in dried blood spots, regardless of clinical condition or gestation. Case Report: A female infant was born at 28 weeks gestation by emergency caesarian section due to abnormal heart rate monitoring and umbilical doppler studies. There was also intrauterine growth restriction and her birth weight was 700 g. Total parenteral nutrition (TPN) was commenced early due to her weight and suspected necrotising enterocolitis. She developed presumed TPN related conjugated hyperbilirubinaemia. She underwent routine NBS by dry blood spot on day 5 of life. Results: Initial screening results showed an increased octanoyl-carnitine C8 at 0.57 μmol/L (ref. < 0.50) and C10 μmol/L = 0.037 (ref. < 0.02), with a grossly abnormal C8/C10 ratio of 15.1 (cut off T]. In a boy with intermittent MSUD (1st tier NBS negative), only one variant was detected by the panel, NM_001918.3 (DBT):c.901C > T,p.Arg301Cys. Sanger sequencing of the missing gaps in IEM panel’s amplicon design revealed another variant: c.1291C > T,p.Arg431*, previously reported disease-causing in ClinVar. His younger, assumed healthy sister also had the two DBT variants, and their parents carried one variant each. Discussion: The tested workflow could shorten genotyping to within the first few days after a positive screening test with a standardized workflow for all diagnoses. Filtering before variant evaluation avoids incidental findings outside the screening mandate and drastically shortens analysis time. A smaller gene panel would lower costs and allow higher coverage of the variants found, and likely remove remaining gaps in the amplicon design.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 P-075

Identification of patients suspected of clinically significant very-long chain acyl CA dehydrogenase deficiency (VLCADD) from newborn screen positive babies. Bhattacharya K 1 2, Carpenter K 1 2, Ho G 1 2, Devanapalli B 1, Wilcken B 1 2, Wiley V 1 2 Sydney Child Hosp Network, Westmead, Sydney, Australia, 2Discipline Peds, Univ of Sydney, Sydney, Australia 1

Background: VLCADD is clinically variable; phenotypes include neonatal cardiomyopathy, hypoketotic hypoglycaemia, recurrent rhabdomyolysis and asymptomatic individuals. In New South Wales, skin fibroblast enzyme assay has been the confirmatory test for VLCADD identified clinically, or, from 1998, by newborn screening (NBS). Identification of true cases from NBS parameters remains difficult Methods: Clinically ascertained cases with diagnostic enzymology retrospectively surveyed were compared with NBS-identified cases of VLCADD born from 1998 to March 2010, and from April 2010 to April 2015, with those predicted to have VLCADD from Region 4 (R4S) post analytical interpretive tool. Biochemical and molecular data were reviewed and R4S scores were recalculated using the 2014 algorithm. Results: Seven cases of VLCADD were identified clinically with typical symptoms and fibroblast enzymology. A further 8 were identified by NBS between April 1998 and March 2010 with NBS C14:1 of 1.5 to 6.1 μmol/L, (incidence 1 in 140,000). Between April 2010 and April 2015, 35 further cases initially screened positive with C14:1 or C14 > 0.8 μmol/L. Eleven, with NBS C14:1 from 1.3 to 8.8 μmol/L, were classified true positive (1 in 48,000) with diagnostic VLCAD enzyme activity or R4S scores of >165, similar to the historic cohort. NBS C14:1 in the false positive cohort was 0.85 to 1.61 with R4S scores from 51 to 156. Homozygous c.1226C > T was, identified in 2 subjects with R4S of 110 and 122 Discussion: Using our NBS methodology in the NSW population with comparison of clear-cut clinically-presenting control cases, R4S score can distinguish a clinically affected VLCADD population from those in the NBS cohort who either do not have VLCADD or are at extremely low risk of having symptoms during childhood. However, because of screening method and population differences the value above which the R4S score defines a case must be determined for each screening program using local population data.

P-076

Six years of neonatal screening of inherited metabolic disorders in the Czech Republic Peskova K 1, Chrastina P 1, Bartl J 1, Friedecky D 2, Hlidkova E 2, Pinkasova R 1 , Dvorakova L 1, Pazdirkova R 3, Prochazkova D 5, Jesina P 1, Hruba Z 4, Adam T 2, Kozich V 1 1

Inst IMD, Gen Univ Hosp, Fac Med I CharlesUniv, Prague, Czech Republic, Lab IMD, Dept Clin Bioch, Univ Hosp, Olomouc, Czech Republic, 3Dept Pediat, 3thFac Med, Charles Univ, Prague, Czech Republic, 4C Mol Biol Gen T, Fac Med Univ Hosp, Masaryk Univ, Brno, Czech Republic, 5Dep Ped, Fac Med Univ Hosp, MasarykUniv, Brno, Czech Republic 2

Background: Early diagnosis of inherited metabolic disorders (IMD) enables early treatment, and improves prognosis, and quality of patients’ lives. We present the results of a 6-year expanded neonatal screening program targeted at 10 IMDs in the Czech Republic. Methods: Amino acids and acylcarnitines were extracted from dried blood spot samples and analyzed by tandem mass spectrometry. Results: Between October 2009 and December 2015 we analyzed samples from 688,243 newborns. We detected 194 patients with subsequently confirmed IMD yielding a detection rate of 1:3,550. The positive predictive value was 27 % and false positive rate 0.07 %; we are aware of one missed patient with intermittent MSUD. For the most frequent diseases (PKU/HPA 1:5,250;

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 MCADD 1:20,900 and LCHADD 1:76,500) frequency of pathological alleles and its geographical distribution in regions were evaluated. Discussion: Although performance of expanded neonatal screening met the criteria recommended by the R4S collaborative project further improvement is desirable. Our laboratory algorithms are still being optimized in order to reduce number of false positive cases. After carrying out a pilot project the national authorities are preparing expansion from 10 to 15 IMDs by adding citrullinemia type I, argininemia, CBS/methylenetetrahydrofolate reductase deficiency and biotinidase deficiency. After expansion the neonatal screening program in the Czech Republic will be able to detect a total of 37 IMDs. Supported by MZ CR—RVO VFN64165 and OPPK CZ.2.16/3.1.00/24012.

P-077

Genotype-phenotype correlation study through protein model of 3methylcrotonyl-CoA carboxylase Tolve M 1, Paiardini A 2, Janson G 3, Artiola C 1, Scarno G 1, Giovanniello T 1, Pasquali A 1, Angeloni A 4, Carducci C L 1, Leuzzi V 5, Carducci C A 1 1 Dept Sper Med University La Sapienza, Rome, Italy, 2Dept Biol and Biotech La Sapienza, Rome, Italy, 3Dept Bioch Sci University La Sapienza, Rome, Italy, 4Dept Mol Med University La Sapienza, Rome, Italy, 5Dept Ped and Child Neurol and Psych, Rome, Italy

Background: 3-Methylcrotonyl-CoA carboxylase deficiency (MCCD), is the more frequent organic aciduria, with an uniform diffusion through different ethnicities. The phenotypic spectrum is very broad (from fatal neurological childhood failures to asymptomatic adults identified through familiar studies). MCCD can be considered a genetic condition with low penetrance and high clinical expression variability. The genotyping in this disease seems to have low phenotypic predictivity. Methods: We performed molecular studies of 21 subjects selected by NBS and 3 subjects of maternal MCCD. We identify 9 new mutations with a high heterogeneity. This molecular framework doesn’t permit easy interpretation of molecular results. For these reasons we have developed a proteic model of the heterododecamer (6 α and 6 β subunits) to assess the variants, impact in amino acid neighborhood and therefore on the enzymatic stability and activity through in silico mutagenesis. Using Pymol and Pymod we have built, through homology modeling, the proteic structure of MCC using as templates propionyl-CoA carboxylase and biotin domain of acetyl-CoA carboxylase of Homo sapiens and methylcrotonyl-CoA carboxylase of Pseudomonas aeuriginosa. Results: The model validation has shown that the structure obtained has an energetic profile that overlaps with those obtained from proteins with a crystallographic structure of same size. The new variants have been successfully validated on this quaternary structure. Discussion: The model obtained was very useful to understand the enzymatic effect of 8 rare, undescribed amino acidic substitutions of patients collected from the NBS. Thanks to the in silico mutagenesis we have been able to appreciate how some substitutions can interfere with the stability of the 6 alpha-6 beta subunits through different mechanisms: destabilizing the interchain interactions; destroying the amino acidic interaction intra-chain due or at steric obstruction not tolerated or for the chemical nature of amino acids.

P-078

Newborn screening for lysosomal storage disorders in Tuscany and Umbria (Italy): current overview and first preliminary results La Marca G 1 2, Forni G 1, Ombrone D 1, Catarzi S 1, Poggiali S 1, Daniotti M 4 , Ferri L 1, Malvagia S 1, Funghini S 1, Chilleri C 1, Zavataro L 4, Pasquini E 4, Morrone A 1 3, Donati M A 4 1 Paed Neur Unit and Labs Meyer Child Hosp, Florence, Italy, 2Dept Experim Clin Biom Sc, Univ Florence, Florence, Italy, 3NEUROFARBA Dept, Univ Florence, Florence, Italy, 4Metab Newb Scr Clin Unit Meyer Child Hosp, Florence, Italy

Background: Lysosomal storage disorders (LSDs) are a group of more than 50 different diseases caused by genetic defects in genes encoding proteins involved lysosomal degradation. Although LSDs are individually rare, their overall incidence has been estimated to be 1 in 2,500–5,000 live births. Early detection in the presymptomatic state and early start of treatment could modify the natural history of the disease. The improvement of treatments and strategies for screening tests on dried blood spot (DBS) encourage the development of pilot newborn screening program for LSDs. Since November 2014, a prospective pilot project for Pompe (PD), Fabry (FD) and mucopolysaccharidosis type I (MPS I) has been introduced into the routine newborn screening program of Tuscany and Umbria. Methods: We modified a previous protocol of a triplex assay for a simultaneous dosage of alpha-glucosidase (GAA), alpha-galactosidase (GLA) and alpha-iduronidase (IDUA) from a single DBS punch. After enzymatic reaction, a trapping and cleanup on-line procedure were achieved by perfusion column connected with an analytical column for the chromatographic separation. For subsequent mass spectrometric analysis internal standards were used. Results were available 4.0 min after injection. Results: Over 30,000 DBS samples were assayed for GAA, GLA and IDUA enzyme activity. On 49 samples with positive results at newborn screening, 24 were confirmed by enzymatic assay on leukocytes (10 GAA, 8 GLA and 6 IDUA). Molecular analysis showed 12 true positives (4 PD and 8 FD), 9 pseudo-deficits (4 PD and 5 MPS I) and 3 heterozygotes (2 PD and 1 MPS I). Discussion: All patients identified by newborn screening are in long-term followup to assess when they need to start treatment. Only one patient (PD) with hypertrophic cardiomyopathy is currently treated with enzyme replacement therapy. Conflict of Interest declared.

P-079

Outcome in short chain acyl-CoA dehydrogenase deficiency (SCADD) detected by newborn screening (NBS) Giovanniello T 1, Nardecchia F 3, Tolve M 1, Artiola C 1, Santagata S 1, Carducci C A 1, Carducci C L 1, Angeloni A 2, Leuzzi V 3 1 Dept Sper Med University La Sapienza, Rome, Italy, 2Dept Mol Med University La Sapienza, Rome, Italy, 3Dept Ped and Child Neurol and Psych, Rome, Italy

Background: SCADD is an autosomal recessive disorder characterized by decreased mitochondrial oxidation of fatty acids associated with mutations in the ACADS gene (OMIM #606885). More than 55 pathogenetic and 2 susceptibility variants have been found. The biochemical hallmark is an increase of blood butyrylcarnitine (C4) and urinary ethylmalonic acid (EMA). SCADD shows a wide phenotypic spectrum, ranging from failure to thrive, hypoglycemia, seizures and behavioral disorders to quite asymptomatic. Patients diagnosed by NBS are largely asymptomatic. Thus, the pathogenicity of this condition and its inclusion in NBS programs becomes uncertain. Methods: Since 2014 our NBS centre has identified 20 newborns suggestive for SCADD. Acylcarnitines were measured in dried blood spot by ESI-MS/ MS and urine organic acids by GC/MS. Sequencing analysis of exons and flanking sequences of ACADS gene was performed. The follow-up ranged from 1 to 30 months. Results: 20 newborn patients underwent confirmatory genetic testing: 6 were heterozygous for the common variant c.625G > A, 4 homozygous for c.625G > A, 4 compound heterozygous for a deleterious variant and c.625G > A, 6 biallelic deleterious mutations. Significant and persistent increase of C4 and EMA was observed during follow up in patients with two pathogenetic variants contrasting to compound heterozygous for a deleterious variant and c.625G > A or homozygous for c.625G > A. During follow up, no significant symptoms or signs were observed, except for slight increase of transaminases in 3 patients with pathogenic variants. Discussion: Although SCADD usually presents as a benign condition, there are still not enough studies to assess the long term outcome of patients diagnosed through NBS and to evaluate the potential toxicity of a persistent excretion of EMA in SCADD. The genotype/phenotype relationship could suggest the candidate patients to follow up, to avoid medicalization of children with no evidence of adverse prognostic factor.

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Prematurity and lysosomal enzymes activities: a caveat for the newborn screening Polo G 1, Viggiano E 1, Zanonato E 1, Zampieri M 1, Cazzorla C 1, Burlina A B 1

1

Screen Cntr, Div Inh Met Dis, Univ Hosp, Padova, Italy

Background: Newborn screening for some lysosomal storage disorders (LSDs) has been proposed in the newborn screening panels in different countries. We have previously reported that neonates and prematures can exhibit unexpectedly elevated levels of α-galactosidase activity (Goi et al. 1998). The objective of this study was to evaluate variations in the activities of four lysosomal storage enzymes determined during newborn screening with respect to prematurity. Methods: A pilot study of newborn screening for four LSDs (Fabry, Pompe, Gaucher and mucopolysaccharidosis type I) is currently being pursued by LCMS/MS method in our region. The enzyme activities of α-galactosidase A (GLA), α-glucosidase (GAA), β-glucocerebrosidase (ABG) and α-Liduronidase (IDUA), are analyzed in dried blood spot (DBS) by stable isotope dilution flow injection analysis MS/MS (FIA-MS/MS) using the Perkin Elmer (PE) Six-Plex LSD reagents kit (NeoLSD). Data collected were stratified into four gestational age categories: < 28 weeks, 28–31 weeks, 32–36 weeks, and 37–42 weeks. Results: Data from 17356 neonates were reviewed. Neonates extremely preterm ( G (p.Asp120Gly) mutation and confirmed the diagnosis of multiple acylCoA DH deficiency. 2nd tier testing and clinical-biochemical follow-up are essential for the diagnosis of rare diseases characterized by a complex and sometimes misleading profile in the NBS 1st tier test.

P-082

Maternal vitamin B12 deficiency detected by expanded newborn screening: incidental finding or additional value? Paci S 1, Gasperini S 2, Alberti L 3, Race V 2, Montanari C 1, Galimberti C 2, Ravazzani V 3, Salvatici E 1, Lucchi S 3, Portella C 3, Biondi A 2, Banderali G 1 , Corbetta C 3, Parini R 2, Riva E 1 1

Ped Dept, Univ Milan, S Paolo Carlo ASST, Milan, Italy, 2Ped Dept, Univ Mi-Bicocca, Monza ASST, Monza, Italy, 3Reg Lab for Neon Scr, Buzzi Child Hosp, Milan, Italy Background: Humans are unable to synthesize vitamin B12 (cobalamin), which is essential for fetus and child development; it must be introduced through the consumption of meat, fish, egg and dairy products. During the neonatal period, B12 deficiency is mostly caused by maternal dietary restriction and/or malabsorption. Methods: 36.765 term newborns were screened thanks to an independent research project, led in Lombardy from August 2014 to September 2015, using a tool (R4S-Region 4 Stork Collaborative Project) combined with second tiertest for methylmalonic acid (MMA) on dried blood spot (DBS). 17 newborns with a suspected secondary maternal B12 deficiency, of unknown origin, were admitted to one of the clinical centres, partners of the project (Ped Dept, University of Milan, San Paolo Hosp, Milan–Ped Dept, University of Milano-Bicocca, San Gerardo Hosp, Monza). Results: 15/17 positive newborns were exclusively breastfed; all of them presented a variable cobalamin deficiency; 12 cases were also associated with hyperhomocysteinemia (Hcy), and urinary MMA above the reference limits. Maternal screening resulted in 14 mothers with a secondary B12 deficiency, of whom: 6 had a restricted B12 intake and APCA+ (anti-parietal cell antibodies); 6 were vegetarians or vegans not adequately supplemented during pregnancy; 1 had amebiasis and a diet low in B12 and folate; 1 had gastric by-pass and familiarity for hyper-Hcy. We supplemented both mothers and newborns with personalized schedules, leading to a rapid normalization of biochemical and urinary parameters and allowing a still-in-progress follow-up. Discussion: Our data show a prevalence of secondary maternal cobalamin deficiency of 1:2163, well above what is currently reported by the Italian Society for the Study of Inborn Metabolic Diseases and Newborn Screening (1:7945-Technical Report 2013) and stress the importance of a specific investigation during pregnancy and breastfeeding to avoid serious consequences in infants.

P-083

The first year of full population newborn screening for lysosomal storage disorders (LSDs): the North-East Italy experience Burlina A B 1, Polo G 1, Viggiano E 1, Zampieri M 1, Zanonato E 1, Cazzorla C 1

1

Screen Cntr, Div Inh Met Dis, Univ Hosp, Padova, Italy

Background: There is an intensive discussion about the inclusion of LSDs on newborn screening panel. Screening of LSDs presents several challenges due

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 to the nature of the conditions and lack of information regarding phenotypic spectrum, timing of treatment interventions, as well as a lack of professionally approved guidelines. We report our preliminary experience of LSDs screening for Fabry, Pompe, Gaucher and Mucopolysaccharidosis Type I (MPS I) diseases in the North East of Italy. Methods: The enzyme activities of α-galactosidase A (GLA), α-glucosidase (GAA), β-glucocerebrosidase (ABG) and α-L-iduronidase (IDUA), were analyzed in dried blood spot (DBS) by stable isotope dilution flow injection analysis MS/MS (FIA-MS/MS) using the NeoLSD kit (Perkin Elmer). Infant with low activity ( T) in the SLC52A2 gene. Discussion: We can hypothesise that the observed alterations may be due to the fact that in case of hRFT3 deficiency, maternal riboflavin does not contribute to biochemical profile normalisation as in the case of hRFT2 deficiency. To our knowledge this is the first case of a hRFT3 deficiency detected after an abnormal NBS result, which raises the question of the possibility of detecting this disorder by NBS.

P-085

Expanded newborn screening in Lombardia. Results of a pilot study Gasperini S 2, Paci S 4, Galimberti C 2, Molinari S 2, Salvatici E 4, Cefalo G 4, Banderali G 4, Corbetta C 3, Riva E 4, Alberti L 3, Calabria M 2, Motta S 2, Brambilla A 2, Biondi A 1, Fedeli T 5, Tagliabue P 5, Ravazzani V 3, Lucchi S 3 , Portella C 3, Parini R 2 Dept of Ped, MBBM Found S Gerardo, Monza, Italy, 2Rare Metab Dis, Ped Dept, San Gerardo Hosp, Monza, Italy, 3Regional Newborn Screening Lab, Milano, Italy, 4Metab Dis Unit, Ped Dept, S Paolo e Carlo, Milano, Italy, 5 Neonat Unit, San Gerardo Hosp, Monza, Italy 1

Background: The Lombardia region in Italy has recently concluded a pilot project on expanded newborn screening (NBS). It became region-wide on March 1st and was concluded on September 31th 2015. Methods: NBS was performed only in at-term newborns with multiplex assay for tandem mass spectrometry (MS/MS) at the regional laboratory for neonatal screening, Paediatric Hospital Buzzi Milano. Positive newborns were evaluated and followed up in two different metabolic centers according to geographic area: S. Gerardo Hospital in Monza and San Paolo Hospital in Milano. Results: Specimens had been tested on 37.828 newborns, with a recall rate of 1,34 %. Metabolic alterations were confirmed in 35 newborns. In particular we identified: 2 methylmalonic acidurias (MMA) with homocystinuria (cblC type), 1 isovaleric acidemia (IVA), 1 argininosuccinic aciduria, 3 3methylcrotonylglycinurias (1 from maternal defect), 4 fatty acids beta-oxidation defects, 1 hypermethioninemia, 3 maternal systemic primary carnitine deficiency, 2 transient neonatal tyrosinemia and 17 newborns with Vitamin B12 deficiency due to maternal deficiency (see abstract Paci et al.). At first clinical evaluation only 2/35 newborns presented mild neurological symptoms: the MMA cblC patient had hypotonia and poor feeding and the IVA patient presented lethargy and tremors due to hyperammonemia. 4/35 patients were hospitalized in neonatal intensive care unit for 4–20 days. All the patients were treated according to dietetic and pharmacological guidelines with good results and continued breastfeeding. One child with MMA cblC showed nystagmus and retinopathy at follow-up. Discussion: NBS during 6 months pilot in Lombardia was confirmed as a valuable and efficient tool to early identification and treatment of acute metabolic diseases at risk for severe acute decompensation and, as expected, allowed to identify maternal nutritional deficiencies potentially severely impacting on the neonatal health.

P-086

A case study from the LCHADD newborn bloodspot screening pilot in England Mozley E 1, Hutton I 1, John K 1, Cregeen D 2, Vara R 3, Olpin S 4, Carling R 1 1

Biochem Sci, Viapath, St Thomas’ Hosp, London, United Kingdom, Biochem Gen, Viapath, Guy's Hosp, London, United Kingdom, 3Centre for IMD, Evelina, St Thomas’ Hosp, London, United Kingdom, 4Clin Chem, Sheffield Children’s Hosp, Sheffield, United Kingdom 2

Background: Long chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is an inherited defect of fatty acid oxidation with an approximate incidence of 1/220,000. Symptoms may include failure to thrive, vomiting, hypoglycaemia and hypotonia, as well as systemic disease such as cardiomyopathy, rhabdomyolysis and neuropathy. A pilot newborn screening programme for LCHADD was performed in England in 2012. Case Report: A day 5 bloodspot sample from a healthy child revealed a 3hydroxyhexadecanoylcarnitine (C16OH) significantly higher (0.14 μmol/L) than the population mean but not above the clinical cut-off (0.15 μmol/L). The decision was made to perform a derivatised acylcarnitine parent scan on the screening sample. The results were considered to be consistent with LCHADD and the baby was referred. However, confirmatory acylcarnitines and analysis of the HADHA gene did not support the diagnosis and dicarboxylic aciduria on the organic acids was attributed to MCT feed. The patient was thriving and well and was discharged.

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At the age of 4 months the patient presented with lethargy, poor feeding and vomiting. There was evidence of liver and renal failure, dilated cardiomyopathy and lactic acidosis; the patient further deteriorated and passed away. Results: During this admission, multiple samples for urine organic acids and bloodspot acylcarnitines were analysed. The results showed biochemical changes consistent with a fatty acid oxidation defect such as LCHADD, mitochondrial trifunctional protein (MTP) deficiency or ACAD9 deficiency. A diagnosis of MTP deficiency was made after extensive fibroblast enzyme analyses on a post-mortem skin biopsy. Discussion: Following review of the evidence from the pilot, it was decided not to continue screening for LCHADD in England. This case illustrates the difficulties in determining an appropriate C16OH cut-off concentration, as well as diagnosis of LCHADD due to its variable biochemical and clinical phenotype.

04. Dietetics and nutrition

P-087

The effect of ketogenic diet on serum selenium levels in patients with intractable epilepsy 1

1

Arslan N , Kose E , Guzel O

treatment, data on energy requirements in this patient group are lacking. Our aim was to determine the optimal method to estimate TEE in MD patients. Methods: After an overnight fast, resting energy expenditure (REE) was measured in adult MD patients (carrying the m3243A > G mutation) using indirect calorimetry (IC) and compared with 2 predictive equations for REE. Physical activity level (PAL) was measured using actometry (Sensewear®) and compared with average measured PAL (set at 1,4) and estimated PAL based on patients selfestimated activity level. Accuracy of predictive equations for REE and estimated PAL were evaluated as percentage of subjects predicted within ±10 % of measured REE/PAL, root mean squared error (RMSE) and mean absolute percentage difference (bias) between predicted and measured REE/PAL. Results: 38 MD patients (age: 48 yr ±13, mean ± SD; male n = 5) signed informed consent. At the individual level, the Henry equation based on weight and height was most accurate: 76 % accurate predictions, bias 7.6 %, and RMSE 164 kcal/day. The widely used Harris & Benedict (1984) scored second best (71 % accurate predictions). The self-estimation methods for PAL were 18–34 % individually accurate. The average measured PAL (1,4) was most accurate (i.e. 53 %). Usual care: Harris & Benedict and estimated PAL (1,3– 1,5) predicted TEE not accurate in 58 % of cases. Discussion: In MD patients the Henry equation based on weight and height is the most reliable alternative for IC to predict REE. As PAL is not reliably estimated by patients, measurement of PAL using actometry is recommended in this population.

P-089

2

1

Div Metab Dis, Dokuz Eylul Univ Hosp, Izmir, Turkey, 2Div Ped Neu, Behcet Uz Child Hosp, Izmir, Izmir, Turkey

Changes of thyroid hormonal status in patients receiving ketogenic diet due to intractable epilepsy Kose E 1, Guzel O 2, Demir K 3, Arslan N 1

Background: Ketogenic diet (KD) is one of the most effective therapies for intractable epilepsy. In the literature, only a few studies evaluated the selenium deficiency in patients with KD treatment. The aim of this study was to evaluate serum selenium levels of children who were receiving olive oil-based KD for intractable seizures at least 1 year. Methods: Out of 320 patients who were initiated on KD, patients who stayed on KD for at least 12 months were included. 16 patients who had selenium deficiency at the beginning of the KD were excluded. Finally, 110 patients (mean age: 7.3 ± 4.2 years, 58 boys, 52.7 %) were enrolled. Serum selenium levels were measured at baseline and 3, 6, and 12 months of treatment by using atomic absorption spectroscopy. Selenium deficiency was defined as serum selenium level G (exon 8) (p.Y236D) and c.1012C > G (exon 9) (p.L338V). Both mutations are not yet published. They are considered pathogenetic as at both sides a well-conserved amino acid is altered. The molecular genetic testing confirmed the diagnosis of GGM. Discussion: We report the first Belgian patient affected by the rare autosomal recessive inherited glucose–galactose malabsorption.

P-091 P-093 Breast milk feeding in inherited metabolic disorders other than phenylketonuria–year single center experience Pichler K 1, Michel M 1, Zlamy M 1, Scholl-Buergi S 1, Ralser E 1, JoergStreller M 1, Karall D 1 1

Medical University of Innsbruck, Innsbruck, Austria

Background: Knowledge about breast milk feeding in infants suffering from inherited metabolic disorders (IMD) other than phenylketonuria (PKU) is limited and described outcome is variable. In this retrospective study we report on breastfeeding and breast milk feeding as well as the neurological outcome of patients with any IMD other than PKU, diagnosed within the last 10 years in a metabolic center. Results/Case Report: 20 patients diagnosed with either organic acidemias, fatty acid oxidation disorders, urea cycle disorders, aminoacidopathies or disorders of galactose metabolism were included in the study. Infants were either breast fed on demand or received expressed breast milk. If necessary, additional modular feeds were given via bottle-feeding. All infants were evaluated clinically and biochemically in 2–4 week intervals, with weight gain as leading parameter to determine metabolic control. Good metabolic control, as well as adequate neurological development were achieved in all patients but one, who experienced the only metabolic crisis observed within the study period. The main reason for stopping breast milk feeding in the studied infants was insufficient supply of breast milk. Discussion: Breast milk feeding is safe and feasible in most IMD. Physicians should be encouraged to allow not only breast milk feeding but also breastfeeding in babies with IMD, as it offers nutritional and immunological benefits for the infant.

Nutritional management of maternal phenylketonuria (MPKU): importance of early dietary phenylalanine (phe) control for the prevention of embryopathy Rossi A 1, Rhoden K J 2, Bettocchi I 3, Musiani C 1, Tarrini G 1, Cassio A 3, Marchesini G 1 1 Metab Dis Clin Diet Unit, Univ Bologna, Bologna, Italy, 2Med Genet Unit, Univ Bologna, Bologna, Italy, 3Paed Endocrinol Unit, Univ Bologna, Bologna, Italy

Background: The number of women with phenylalanine-hydroxylase (PAH) deficiency who become pregnant is increasing. It is known that a low-phe diet and blood phe levels between 120 and 240 (360) μM can prevent teratogenic effects of MPKU, however, neonates with mild to severe anomalies continue to be born due to inadequate dietary compliance. Methods: The aim of the study was to assess the efficacy of nutritional management in preventing MPKU complications, in relation to diet therapy onset, compliance and offspring outcome. Seven pregnancies of six PAH-deficient women (4 classic and 2 mild phenotype) were analyzed retrospectively. We evaluated mothers’ gestational diet, anthropometrics, blood phe/tyr levels, compliance and neonatal outcome. Results: Diet therapy was initiated in 2/7 cases (29 %) before conception and in 5/7 cases (71 %) during pregnancy. Blood phe levels were decreased by diet therapy in all pregnancies: 2/7 cases reached target blood phe levels before conception (Group pre-conc; initial phe 129–210 μM, final 120–174 μM), 2/7 cases before the 8th gestation week (Group < 8th wk; initial phe 56–320 μM, final 144–132 μM), 3/7 cases after the 8th gestation week (Group >8th wk; initial phe 810–914-1236 μM, final 126–132-102 μM). Fetal and neonatal

S84 complications were: Group pre-conc, none; Group < 8th wk, small for gestational age (1 case) and congenital heart disease (1 case); Group >8th wk, small for gestational age (3 cases), intrauterine growth retardation (1 case), premature delivery (2 cases). Group >8th wk neonates had the lowest percentile birth weight, length and cranial circumference. Discussion: MPKU complications were avoided only when metabolic control was maintained before and during pregnancy. Women with poor control of blood phe levels delivered babies with a suboptimal outcome. This confirms the importance of continued dietary compliance and pregnancy planning in PAH-deficient women, in order to prevent embryopathy.

P-094

Selective feeding behavior simulating hereditary fructose intolerance and loss of walking in two children: scurvy, a neglected disease in the 21st century Taurisano R 1, Gallo G 1, Barbuti D 2, Maiorana A 1, Deodato F 1, Dionisi-Vici C1 1

Div Metab, Bambino Gesu Children’s Hosp, Rome, Italy, 2Dept Radiol, Bambino Gesu Children’s Hosp, Rome, Italy Background: Selective feeding behavior with aversion to specific foods like fruits and vegetables is a cornerstone for the diagnosis of hereditary fructose intolerance (HFI). Case Reports and Results: A 3-year-old patient with a medical history of congenital cataract, mild psychomotor and speech delay came to our attention for a complete gait refusal complaining of leg pain. He underwent extensive investigations for hematologic, neurologic, metabolic and rheumatologic diseases, which showed only a mild anemia. Although his parents reported normal feeding, a NPI evaluation revealed selective feeding behavior, simulating HFI, with almost no intake of fruits and vegetables with the diet. Skeletal X-ray and MRI showed specific anomalies of long bones with subperiosteal hematoma and diffuse osteopenia. Plasma vitamin C level was found profoundly deficient (0.1 mg/dl; nv 26, 1–84,6). The second patient who had a highly selective feeding behaviour with refusal of fruit and vegetables since infancy, at 3 years old had loss of walking and failure to thrive. The combination of loss of walking with selective refusal of fruits and vegetable led us to suspect scurvy. The diagnosis was confirmed by plasma vitamin C determination, which showed an almost undetectable level and by the observation of the typical radiological signs. After replacement with vitamin C, both patients recovered and started to walk again with disappearance of leg pain. At long-term follow-up both patients tolerated the introduction of vegetables and fruits. Discussion: These cases were useful to remind that scurvy, a neglected disease in the current clinical practice, should be considered in the differential diagnosis in children with selective feeding behavior, similar to HFI, presenting with leg pain and loss of walking. The importance of early diagnosis of scurvy lies in the fact that it is an easily treatable condition with an excellent outcome.

P-095

Illness management in maple syrup urine disease (MSUD) from a single centre in the United Kingdom Billmore K 1, Slabbert A 1, Champion M P 2, Lemonde H 2, Mundy H 2, Gribben J 1 1 Dietetics Dept, St Thomas’ Hosp, London, United Kingdom, 2Ctr Inh Met Dis, Evelina Children’s Hosp, London, United Kingdom

Background: During illness in classical MSUD toxic metabolites rise markedly and can result in irreversible neurological impairment. We describe our emergency regimens (ER) for prompt management during illness.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Historically, one standard MSUD ER recipe was taught: full maintenance fluid, branched chain amino acid (BCAA) free supplement to total protein requirements and glucose polymer. The full maintenance volume is often difficult to achieve at home due to poor tolerance, therefore lower volume interim recipes were introduced. Methods: From 2013 patients were taught a 3 stage ER. The three ER variations are i) low volume ER which is standardised, ii) interim ER and iii) full ER which are both patient specific. Results: All 8 patients have classical MSUD, median age 7.1 (range 0.7– 15.4 years). Interim ER recipe: Child is beginning to become unwell, ketones present or recovering from illness (60 % energy requirements (EAR), 100 % protein requirements from BCAA free supplement, 60 % fluid requirements, double usual isoleucine/valine (iso/val) dose and approximately half usual exchanges). In the authors’ experience, the reduced fluid and energy prescription improves tolerance and allows patients to consume additional energy from solids. Full ER: Illness deteriorating while on the interim ER (100 % EAR, 100 % protein requirements from BCAA free supplement, 100 % fluid requirements, double usual iso/val dose and zero exchanges). Usually administered via NG at home or in hospital due to the larger volume. Low volume ER: Hospitalised with vomiting or diarrhoea on IV dextrose +/− lipids (for fluid and energy) and unable to tolerate enteral fat/ carbohydrates or volumes. Provides 100 % protein requirements from BCAA free supplement, double usual iso/val dose and zero exchanges. A concentrated BCAA free supplement was used (10 g PE per 100 ml, 12 % dilution). Discussion: Use of an interim ER is an alternative to the high volume ER and may provide a more tolerable home illness regimen.

P-096

Case report: glycogen storage disease IXγ and glucose galactose malabsorption Billmore K 1, Champion M P 2, Mundy H 2, Mutalib M 3, Gribben J 1 1 Dietetics Dept, St Thomas’ Hosp, London, United Kingdom, 2Ctr Inh Met Dis, Evelina Children’s Hosp, London, United Kingdom, 3Dept Paed Gastro, Evelina Hospital, London, United Kingdom

Background: GSD Type IXγ (phosphorylase kinase deficiency) causes impaired glycogen breakdown, hypoglycaemia and ketosis. An emergency regimen (ER) of glucose polymer solution is used in illness. Case Report: We report the case of a 19 month old female with GSD IXγ confirmed by mutation analysis. She presented at 6 months with hepatomegaly, deranged transaminases and high triglyceride levels. She was also noted to have a history of loose stools. Trials of extensively hydrolysed formulas did not improve symptoms, thereby excluding cow’s milk protein and lactose intolerance. Safe fasting time was 3 h and ketones were suppressed with a dietary prescription of continuous overnight feed, regular carbohydrate containing food/feed and a standard ER of glucose polymer. At 13 months, she had a gastrointestinal illness requiring hospital admission. After recovery on IV dextrose repeated attempts to re-introduce glucose polymer failed due to diarrhoea, recurrent hypoglycaemia and ketosis. A putative diagnosis of glucose and galactose malabsorption was made. Results: This was a challenging treatment scenario: a requirement of regular carbohydrate without glucose and galactose, compounded by parental low literacy and numeracy. The ER was changed from glucose polymer to fructose module. She was commenced on a glucose and galactose free infant formula. Solids were stopped as there were limited suitable options available. Ketones remained suppressed and growth was maintained. At 16 months glucose containing solids were successfully reintroduced during a hospital admission with no ketones or hypoglycaemia. At home as solids were increased, symptoms reappeared suggesting a possible tolerance level. Discussion: A GSD IXγ patient was successfully treated on a glucose and galactose feed following gastrointestinal illness.

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Amino acid medical foods provide a high dietary acid load that increases urinary calcium excretion and is associated with decreased lumbar spine Z-scores in phenylketonuria Stroup B M 1, Sawin E A 1, Murali S G 1, Ney D M 1 1

Dept Nutri Sci, Univ WI, Madison, United States

Background: Skeletal fragility is a common, but poorly understood, complication of phenylketonuria (PKU). Glycomacropeptide (GMP) medical foods provide a source of intact dietary protein with a lower dietary acid load compared to amino acid (AA) medical foods. GMP provides a safe and acceptable option for PKU management. Objective: To determine how medical foods differing in protein source (GMP vs. AA) and acid load impact bone-related outcomes in human and murine PKU. Methods: Eight PKU subjects consumed commercially available high-acid AA or low-acid GMP medical foods (Glytactin™, Cambrooke Therapeutics) with equivalent protein intake for 7 days each; each subject provided 24-hr food records and one 24-hr urine collection for each diet. One DXA scan was obtained from each subject. Weanling PKU and wild type (WT) mice were fed isoenergetic high-acid AA, low-acid GMP, or low-acid casein diets for 20 weeks, followed by assessment of bone biomechanical and renal parameters. Results: PKU subjects fed high-acid AA medical foods demonstrated a decrease in urinary pH and an increase in 24-hr urinary excretion of renal net acid, calcium, and magnesium compared to low-acid GMP medical foods. There was a significant negative correlation with increased urinary calcium excretion and decreased lumbar spine Z-scores with AA, but not GMP, medical foods. In WT and PKU mice, the AA diet significantly decreased femoral size and strength. Consistent with greater renal workload, the AA diet increased renal mass, urinary volume, and 24-hr urinary excretion of renal net acid and calcium compared to the GMP diet in WT and PKU mice. Discussion: Current use of AA medical foods provides a high dietary acid load, which may contribute to skeletal fragility in human PKU. GMP/ Glytactin™ medical foods support bone health in PKU. Funding disclosure(s): USDA Hatch grant WIS01790 and R01 FD003711 from the Office of Orphan Products Development Conflict of Interest declared.

P-098

Nutritional characterization of PKU adult outpatient clinic in 2015 Guerra A 1, Nunes P A 1, Oliveira A 1, Gomes D C 1 1

North Lisbon Hosp - Metab Dis Ref Center, Lisboa, Portugal

Background: Phenylalanine (PHE) is an essential amino acid that by action of the enzyme phenylalanine hydroxylase (PAH) is metabolized to tyrosine. This reaction requires a co-factor tetrahydrobiopterin (BH4). The deficiency in PAH or the enzymes necessary for synthesis or BH4 regeneration results in hereditary disease phenylketonuria (PKU). The diagnosis is made at birth, by determining the concentration of PHE in the blood, and dietary treatment started early on. The treatment consists essentially of a low protein and PHE diet. PKU without treatment produces a severe developmental impairments specifically in the cognitive area. The outpatient specific protocol for the adult population was created in April 2003 and included all patients older than 18 years of age. There are 57 patients with PKU, 47.4 % (27) were female and 52.6 % (30) male. Of the 57 patients, 14 (24.6 %) failed the appointment of 2015 and 1 patient was pregnant, so she will not be included in the characterization Methods: A nutritional evaluation was made. Results: Regarding the 2015 assessment, the data are as follows: Average age, 28.7 years. Weight distribution: the average weight is 66.7 kg; he average body mass index (BMI) is 24 kg/m2. Body Composition: the fat mass assessment was

on average is 14.6 kg. 90 % of patients followed in this outpatient clinic consumed a low protein diet, supplemented with synthetic amino acids. The protein contribution made with synthetic amino acids had an average of 57,8 g/day, which corresponds on average to 0.9 g/kg/day, values which are similar for both genders. The average measurement of PHE during 2015 was 13.1 mg/dl. Discussion: In conclusion we can surmise, as seen in other treatment centres, the drop out rate is high. Regarding nutritional assessment, it is important to mention that the BMI is border line on overweight values, without exceeding them. Regarding the metabolic control of their disease, we ascertain the subjects are slightly above the recommended values.

P-099

Impact of the amino acid profile of casein glycomacropeptide on metabolic control in children with PKU Daly A 1, Chahal S 1, Evans S 1, MacDonald A 1 1

Birmingham Childrens Hospital, Birmingham, United Kingdom

Background: Casein glycomacropeptide is supplemented with additional Lamino acids (CGMP-AA) in PKU. The importance of the ratio of added Lamino acids on metabolic control is unclear. In a prospective controlled trial in PKU children, the longitudinal changes in blood phenylalanine (Phe) and tyrosine (Tyr) over 15 months in CGMP-AA was examined when the amino acid component of CGMP-AA was modified in 3 different ways. Results were compared with a parallel control group on traditional L-amino acids supplements (L-AA) only. Methods: 21 children, median age 11 y (6–16 y) were recruited, 12 on CGMPAA, 9 L-AA only as their protein substitute (PS) source. Three CGMP-AA products were used: CGMP–AA1 (amino acid profile matched WHO 2007 amino acid requirements) for 6 months, CGMP–AA2 (extra tyrosine) for 4 months, and CGMP-AA3 (extra large neutral amino acids) for 5 months. Weekly blood spots were collected for Phe and Tyr. Results for study and control groups were compared with results for 12 months prior to study entry. The CGMP-AA contained 30 mg Phe/20 g protein equivalent. Results: The median % of total PS provided by CGMP-AA in CGMP1, 2 and 3 was 50 %, 50 % and 75 %, respectively, provided the following Phe (mg/ day) intake: GMP-AA1, 45; GMP-AA2, 45; and GMP-AA3, 68. The median blood Phe were unchanged with CGMP-AA2 and CGMP-AA3 compared with pre-study but was significantly higher with CGMP-AA1 (p = 0.009) (Phe [μmol] pre-study, 275; CGMP-AA1, 378, CGMP2-AA2, 268; CGMPAA3, 290). Control group Phe [μmol] was pre-study 325 μmol; L-AA1, 280; L-AA2, 405; L-AA3, 340). Median tyrosine results significantly improved with CGMP-AA 3 (p = 0.03) compared with pre-study (Tyr [μmol] pre-study, 50; CGMP-AA1, 45, CGMP2-AA2, 50; CGMP-AA3, 60). Control group median Tyr (40 μmol/L) remained unchanged throughout the study. Discussion: The amino acid profile of added L-amino acids in CGMP-AA has a significant impact on influencing metabolic control in PKU children. Conflict of Interest declared.

P-100

Selenium status in inborn errors of metabolism patients Ormazabal A 1 2, Batllori M 1, Meavilla S Cazorla A 1 2, Campistol J 1 2, Artuch R 1 2

1 2

, Garcia-Volpe C 1, Garcia-

Hospital Sant Joan de Deu, Barcelona, Spain, 2CIBERER, Barcelona, Spain

1

Background: Patients with inborn error of metabolism (IEM) are at risk to display nutritional deficiencies. Our aim was to measure the status of three trace elements (Cooper [Cu], Zinc [Zn] and Selenium [Se]) in a large cohort of paediatric patients under different restrictive diet treatments. Patients: We studied 149 patients age range 11 months–17 years (average 7.5 years). Patients were classified into 3 categories: Group 1: IEM under protein restricted diet (n = 87; phenylketonuria, urea cycle disorders,

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aminoacidopathies and organic acidemias), group 2: IEM with no protein restricted diet (n = 38; galactosemia, beta oxidation defects, lysosomal storage diseases and others) and group 3: Cystic fibrosis (n = 24). Results were compared with our reference values. Trace element status was followed up for a period between 2 and 6 years in the different IEM. Baseline results were compared with trace elements results after supplementation. Methods: Cu, Zn and Se were measured in plasma samples by inductively coupled plasma mass spectrometry (ICP-MS). Results: In baseline conditions plasma selenium values were below our reference intervals (0–5 years 54–99 μgr/L; 6–18 year 67–104 μgr/L) in 42 out of 87 patients from group 1, in 10 out of 38 from group 2 and in 9 out of 24 from group 3. When compared baseline conditions and results after Se supplementation, Se values significantly increased in groups 1 and 3 (student t test for paired data p = 0.001, p = 0.006 respectively). In group 2 (no supplemented) no differences were observed over the evolution of the disease. No differences were detected in plasma Cu and Zn concentrations among the 3 groups of patients and our reference values. Discussion: Se deficiency is a relatively common finding in IEM and other paediatric diseases. Supplementation with Se seems advisable since normal values are achieved in these patients after this intervention.

P-101

Background: Patients with phenylketonuria (PKU) in the UK have similar rates of overweight and obesity compared to the general population, but little is known if the prevalence is increasing. The aim is to find out if the prevalence of overweight in PKU adults is increasing compared to the general population. Methods: BMI data on adult PKU patients (>16 years) on a low phenylalanine diet from a single centre was collated from 2013 to 2014 (n = 80) and analysed against data from 2008 to 2009 (n = 53). Maternal PKU patients were excluded. Data was then compared to the Health Survey for England. Results: Prevalence of overweight PKU patients (BMI >25 kg/m2) increased from 58.8 % (2008–2009) to 62 % (2013–2014). PKU males now have a higher prevalence compared to PKU females (m 55.5 % vs f 61 % 2008– 2009) and (m 63 % vs f 60.5 % 2013–2014). The prevalence of overweight PKU males has increased by 7.5 % to 63 %, in comparison to a 3 % increase in the male general population to 67 %. PKU female overweight prevalence is stable, with a small drop of 0.5 % to 60.5 % compared to an increase by 1 % in the general population to 57 %. No statistical difference was seen between the overweight 2013–2014 PKU males and females and the overweight general population. Discussion: The prevalence of overweight PKU male patients in one centre has increased over 5 years, with females remaining stable, following the same trend as the general population. This highlights the need to continue to educate patients, especially males on a healthy, low phenylalanine diet to prevent further increases in BMI and the development of weight associated comorbidities.

Managing feeding difficulties in a phenylketonuria (PKU) patient with poor metabolic control P-103 Cross S 1, Webb M 1, Newby C 1, Smith K 1, Costelloe S 2, Hart J 2, Pierre G 1 1 Dept Met Med, Univ Hosp, Bristol UK, Bristol, United Kingdom, 2Paed Dept, Wonford Hosp, Exeter UK, Exeter, United Kingdom

Background: Management of phenylketonuria (PKU) involves lifelong restriction of natural protein, the consumption and even distribution of protein substitute throughout the day and dietary supplementation with protein free foods. The rigidity of the diet can have an adverse effect on feeding behaviour in children and on quality of life for parents or carers. Case Report: We present a 3 year old girl diagnosed with classical PKU from newborn screening managed jointly by the specialist and local centre. She struggled to maintain phenylalanine levels within recommended target ranges. Only 17 % of levels were within target range (120–360 μmol/l) and there was huge variability in levels up to over 1000 μmol/l. She was a very fussy eater often having less natural protein and calories than advised and sometimes struggling to take her protein substitute. There was a family history of food aversion in the father. Results: After no improvement with input from dietetic and psychology teams locally she was admitted over a 4 week period for consideration of gastrostomy placement at the end. Discussion and observation in the first week showed significant restriction of normal family functioning with few outings, considerable time input, a negative attitude towards the PKU diet and poor coping and support from the extended family. Following an intense programme of re-education to parents and the wider family, parenting techniques, play therapy and dietetic input, there was significant improvement in control with over 50 % of levels within target and less variability of levels overall. Discussion: Food aversion is not uncommon in early childhood. In addition negative food behaviour can be seen in PKU in childhood. Admission and a whole family approach with an emphasis on normal functioning led to a significant improvement in compliance.

Dietary assessment of an adult cohort with McArdle disease Carruthers R 1, Ellerton C 1, Freedman F 1, Hansen K K 1, Midolo T 1, Booth S 1 , Chatfield S 1, Coskeran H 1, Godfrey R 1, Pattni J 1, Siciliani Scalco R 1, Turner P 1, Quinlivan R 1 1 National Hosp Neuro, London, United Kingdom Background: There is limited scientific evidence for the use of specific dietary modification in McArdle disease and no data has been published on the usual dietary intake. We offered individual assessment and tailored dietary advice to suggest specific dietary changes, where appropriate. The information was collated to identify dietary patterns amongst people with McArdle disease. Methods: 30 adults attending the nationally commissioned McArdle disease service completed 3 day food diaries. These were analysed with DietPlan6 software. Clinic notes provided clinical outcomes and bodyweight. Results: Mean percentage energy from: carbohydrate 43.4 % (24.2–57.5 %), protein 17.9 % (9.6–29.4 %), fat 32.9 % (23.4–49.4 %), saturated fat 11.7 % (6.2–22.8 %), alcohol 4.9 % (0–28.1 %). Mean fibre intake 15.1 g/day (7.8– 28.6 g/day). Mean intake of fruit or vegetables was 3.7 portions daily. Mean BMI 27.1 kg/m2 (18.7–36.7 kg/m2). 67 % had BMI > 24.9 kg/m2, 27 % had BMI > 30 kg/m2. 47 % of patients have been reviewed since being provided with their dietary assessment report and 70 % of those had made at least one positive dietary change when suggested. Discussion: Current diet composition varies considerably without obvious dietary patterns. Dietary advice focused around heart health including optimal fruit, vegetables, fibre, alcohol and omega 3 with a regular meal pattern based on slow release carbohydrates and limited saturated fat.

P-104 P-102 Updated, web-based nutrition management guideline for PKU: An evidence and consensus based approach Trends in body mass index in adult patients with phenylketonuria Tomlinson L , Robertson L , Howe S , Bolton S , Dawson C , Geberhiwot T1

Singh R H 1, Cunningham A C 2, Mofidi S 3, Douglas T D 1, Frazier D M 4, Hook D G 5, Jeffers L 6, McCune H 7, Moseley K D 8, Ogata B 9, Pendyal S 4, Skrabal J 10, Splett P L 11, Stembridge A 1, Wessel A 12, Rohr F 12

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IMD, Queen Elizabeth Hospital, Birmingham, United Kingdom

Department of Human Genetics, Emory Univ, Decatur, United States,

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 2

Hayward Genetics Center, Tulane Univ, United States, 3Metab Dis Center, New York Med College, Valhalla, United States, 4Univ of North Carolina, Sch of Med, Chapel Hill, United States, 5University of California, Davis, United States, 6Cleveland Clinic, Center for Human Nutri, Cleveland, United States, 7 Ped Genetics and Metab, Univ of Florida, Gainesville, United States, 8 Genetics Div, USC Keck Sch of Medicine, Los Angeles, United States, 9 Univ of Washington, Dept of Pediatrics, Seattle, United States, 10Dept of Med Genetics, Univ of Nebraska, Omaha, United States, 11Eval Consultant Splett and Associates, Stanchfield, United States, 12Div Genetics and Geno, Boston Child Hosp, Boston, United States Background: In 2014, recommendations for the nutrition management of phenylalanine hydroxylase deficiency were published as a companion to the American College of Medical Genetics and Genomics guideline for the medical treatment of phenylketonuria (PKU). These were developed primarily from a systematic literature review. Since then, the Genetic Metabolic Dietitians International and the Southeast Regional Newborn Screening and Genetics Collaborative have partnered to create a web-based technology platform for the update and development of nutrition management guideline utilizing evidence and consensus based methodology. The purpose of this PKU guideline is to establish harmonization in treatment, to guide the integration of nutrition therapy in the medical management of PKU, and improve outcomes for individuals with PKU in all life stages while reducing associated medical, educational, and social costs. Methods: Six research questions critical to PKU management were formed to support guideline development: Review, critical appraisal, and abstraction of peer-reviewed studies and unpublished practice literature, along with expert Delphi survey, nominal group process, and external review from metabolic physicians and dietitians were used for development of recommendations relevant to each question. Recommendations address nutrient intake, updated protein requirements, optimal blood phenylalanine concentrations, nutrition interventions, monitoring parameters specific to life stages, adjunct therapies, pregnancy and lactation. Recommendations were graded using a rigorous system derived from the Academy of Nutrition and Dietetics. Results and Discussion: These guidelines, updated using a thorough and systematic approach to literature analysis and national consensus process, are now accessible to the global community via the newly developed platform. For additional details on specific topics, readers are encouraged to review materials on the online portal: https://GMDI.org/.

P-105

Classical galactosaemia in infants: the Russian experience of dietary treatment Bushueva T 1, Borovik T 1, Skvortsova V 1, Yatsyk G 1, Roslavtseva E 1

lactose free diet in all patients the blood level of TG had decreased to 2,39 ± 0,45 mg/dl (p = 0,000) and did not depend on the kind of lactose free formulas: I group: 2,47 ± 0,74; II group: 2,39 ± 0,37; III group: 2,37 ± 0,57. The blood serum levels of TB had decreased to 12,1 ± 3,17 mmol/l (p = 0,000), of ALT16,3 ± 3,17 U/L (p = 0,000), of AST-15,1 ± 3,5 U/L (p = 0,000). Discussion: Lactose free soy-based and hydrolysed casein-based infant formulas are the main products of choice for the treatment galactosaemic newborns as well as the whey-based formula fortified by MCT can successfully be used in diet treatment of classical galactosaemia especially in infants with severe hepatic and renal insufficiency.

P-106

Rapidly increasing phenylalanine intake in phenylketonuria (PKU) whilst on growth hormone (GH)—maternal concerns Cochrane B 1, Robinson P 1, Shaikh G 1 1

Royal Hosp for Child, Glasgow, United Kingdom

Background: PKU is an inherited metabolic disorder of protein metabolism caused by the absence of phenylalanine hydroxylase leading to a rise in plasma phenylalanine, which if not addressed, will result in severe neurological damage. Treatment consists of a semi artificial diet comprising of a phenylalanine free amino acid supplement, restricted amounts of phenylalanine containing foods, prescribed low protein staple foods and low protein natural foods. Guidelines advise that children with PKU are regularly monitored for growth, biochemical and haematology markers. Case Report: A boy with well controlled classical PKU (c.1222c > T and c.896 > G) was stabilised on 300 mg phenylalanine. At 18 months of age, an accidental fall resulted in a left cerebellar haematoma and hydrocephalus. From the age of 5 years his growth was noted to be slowing. Subsequent MRI showed no structural abnormality, however, GH stimulation tests demonstrated suboptimal GH levels. Daily GH injections were started. Results: In the 3 months prior to starting GH growth velocity was 7.65 cm/year and increased to 11.48 cm/year in the subsequent 3 months. A rapid increase in phenylalanine tolerance was noted (300 mg to 600 mg with a peak of 750 mg). Twice weekly blood spots, including some at various points during the day, ensured the phenylalanine levels were kept within the target range of 120–480 umol/l for age. Discussion: Maternal anxiety centred on the uncertainty the effect GH has on phenylalanine requirement. In addition there was difficulty in persuading the child to eat unfamiliar foods to complete his phenylalanine allowance. Taking note of parents’ concerns and regular anthropometry is key to identifying abnormal growth patterns. Parental concerns around dietary changes are understandable given the uncertainty of how long the anabolic effect of GH treatment will continue and the effect increased phenylalanine requirement has on phenylalanine levels. Conflict of Interest declared.

1

Scientific Centre of Children Health, Moscow, Russian Federation

Background: Type I galactosaemia is an inherited disorder of galactose metabolism. Galactosaemic newborns feeding breast milk or infant formula demonstrated severe symptoms of multiple organ failure in the first weeks of life. The treatment of classical galactosaemia is strong diet without lactose and galactose. Methods: We examined 39 full-term infants with classic galactosaemia before the appointment and on the different lactose-free formulas. The blood level of total galactose (TG) was determined by fluorimetric method, amd total bilirubin (TB), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were estimated on the automatic biochemical analyzer. Results: The average blood level of TG while breast-feeding milk or formula was 63,7 ± 16,2 mg/dl (norm in Russia is not more than 7 mg/dl). The blood serum levels of TB was 163 ± 126 mmol/l, ALT-182 ± 35,5 U/L, AST-191,3 ± 35,9 U/L. Manifestation of acute severe clinical symptoms observed from 5 to 21 days of life (at average age 6,7 ± 1,7 days). Soy-based infant formulas was used in 41 % infants (I group), hydrolysed casein-based formulas – in 33 % babes (II group) and hydrolysed whey-based formula fortified by MCT was used in 26 % infants with severe hepatic and renal insufficiency (III group). After a months of

P-107

Use of parenteral nutrition in children with organic acidaemias (OA), single-centre experience Cawtherley S 1, Skeath R 1, McSweeney M 1, Davison J 1, Grunewald S 1, Cleary M 1, Chakrapani A 1, Dixon M 1 1

Great Ormond Street Hosp for Children, London, United Kingdom

Background: Children with OA can experience frequent episodes of vomiting and metabolic acidosis requiring hospital admission. Treatment includes IV 10 % glucose and emergency regimen (ER) based on glucose polymer. Prolonged and repeated use of glucose does occur when weaning back to usual enteral nutrition (EN) is not tolerated. We manage these episodes with parenteral nutrition (PN) as inadequate nutrition leads to catabolism with high risk of metabolic decompensation. We describe our experiences of PN.

S88 Methods: A review of patients who received PN from January 2013 to May 2016: MMA B12 non-responsive n = 11, MMA B12 responsive n = 1, PA n = 4, IVA n = 1. PN and EN data was collected from case notes including indication for use, days on IV glucose and ER, days on PN, composition of PN, type of access, days to wean back to usual EN, bodyweight. Results: 17 patients had PN: overall 40 separate episodes (range 1–6 per patient), 5 episodes at diagnosis. Commonest indications for PN were: vomiting, metabolic acidosis and possible or confirmed pancreatitis (n = 17). All had ER, IV 10 % glucose and at least one trial of reintroducing usual EN via NG n = 6, gastrostomy n = 6 or PEG-J n = 5, before commencing PN. Days off EN pre PN ranged 1–11 (median 4). Number of days on PN ranged from 2 to 71d (median 8d). PN was administered via Portacath n = 21, PICC line n = 16, or femoral line n = 3. Standard PN solutions were given amino acids limited to provide usual protein intake, lipid and glucose to normal energy. To wean off, full strength protein feeds (ER or ½ strength, rarely) were titrated against PN, to maintain usual protein and energy intake. Time taken to wean ranged from 1 to 26d (median 4d). Weight was maintained or increased. Discussion: PN was successfully used to prevent catabolism and metabolic decompensation when usual EN was not tolerated. PN was essential to provide adequate nutrition during episodes of pancreatitis, a known complication of OA.

P-108

Very long-chain acyl-coenzyme A dehydrogenase deficiency dietary management pitfalls Mexia S 2, Costa C 1, Janeiro P 1, Nunes P A 2, Gaspar A 1 1

Metab Dis Unit, Lisboa, Portugal, 2Diet Nut Serv, Lisboa, Portugal

Background: Very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCADD) dietary management consists of fasting avoidance, total fat restriction and medium-chain triglycerides (MCT) supplementation, especially before exercise, in order to prevent myopathic symptoms. Case Report: Sixteen years-old female, single daughter of nonconsanguineous parents, admitted at 14 years-old due to severe rhabdomyolysis after exercise. Laboratorial investigation was suggestive of VLCADD, confirmed by molecular analysis (p.P65Tfs*7(c.187_192insA and p.R366H(c.1097G > A). Nutritional counselling was provided based on lowfat diet supplemented with 10 % MCT caloric intake and uncooked cornstarch. Throughout follow-up she had 4 episodes of severe rhabdomyolysis after physical activity, requiring hospitalization. Results: Currently, she weighs 60.2 Kg (p50-75), height 163 cm (p50–75), BMI 22.2 Kg/m2 (p50–75). Nutritional intake: 2135 kcal/day, 47.6 g/day from fat, supplemented with MCT (20 % of the calories). She stopped regular exercise, remaining asymptomatic for the last 6 months. Discussion: There is no consensus for dietary management of VLCADD with myophatic phenotype. Some authors (Spiekerkoetter et al. and Georgianne et al.) argue that total fat intake restriction with MCT/cornstarch supplementation can prevent mobilization of long-chain fatty acids from adipose tissue during exercise, although others (Primassin et al.) debate that these recommendations may result in significant muscular acylcarnitine increased after exercise. In this patient, clinical and laboratory improvement was linked to MCT and uncooked cornstarch supplementation, at a sedentary lifestyle. Additional studies are needed to better correlate genotype/phenotype and adjust dietary recommendations in order to improve outcome, regarding life quality and expectancy.

P-109

Obtaining, prescribing and dispensing of low protein manufactured foods (LPMF): what are the issues? Cochrane B 1, Adam S 1, Lang K 2, Ross K 3, Wire F 4, Dawson S 4 1 Royal Hosp for Child, Glasgow, United Kingdom, 2Ninewells Hosp, Dundee, United Kingdom, 3Child Hosp, Aberdeen, United Kingdom, 4Royal Hosp Sick Child, Edinburgh, United Kingdom

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Phenylketonuria (PKU) is an inherited inborn error of metabolism treated by a semi-artificial diet low in phenylalanine. When untreated, raised phenylalanine levels result in severe neurological damage. The diet includes the use of LPMF available on prescription in the UK which will contribute towards a small, but significant part of the general practitioner (GP) and pharmacist’s workload. Obtaining sufficient product in a timely manner can cause difficulty for patients. To help overcome this, an on-line formulary was developed for use by GPs. Methods: In order to determine the usefulness of the formulary, a questionnaire with fixed and open ended questions was sent to all patients on active diet and their GPs. Patients and carers were asked to hand a questionnaire to their pharmacist. A stamped addressed envelope was included for return. Reuslts were analysed on Minitab 17.0. Results: 220 questionnaires were sent to each group: patients, GPs and pharmacists with a response rate of 28 % (n = 62) from patients; 31 % (n = 68) from Gs and 10.9 % (n = 24) from pharmacists. Of patients, 42 % (n = 26) had a comment from a health professional about their prescriptions. 34 % (n = 23) of GPs and 79 % (n = 19) of pharmacists were not given information on the diet. 73 % (n = 50) of GPs were not aware of the formulary; 87 % (n = 59) of Gs and 83 % (n = 20) of pharmacists were not aware of the NSPKU recommendations on LPMF usage. Discussion: The surveys highlight the need for improved communication between primary and tertiary care staff. Some G.P’s do not feel that it should be their responsibility to prescribe the products: “Prescribing low protein food and snacks should be nothing to do with me. The current system of prescribing food via GP is bureaucratic, inefficient and wasteful”. The use of a similar system for access to LPMF such as that used for gluten free foods in Scotland may ensure patients receive the amount of LPMF they require to manage their diet effectively. Conflict of Interest declared.

P-110

Stricter and differing interpretation of protein food labelling by parents/ adults with PKU than by IMD dietitians MacDonald A 1 10, Adam S 4, Ash J 24, Ashmore C 1, Caine G 28, Chan H 17, Clark A 3, Coates E 29, Cochrane B 4, Daly A 1, Dines K 15, Donald S 9, Dunlop C 4, Ellerton C 22, Ford S 11, French M 18, Gingell C 25, Gribben J 2, Grimsley A 15, Gubb L 16, Hallam P 6, Hendroff N 26, Hill M 14, Hoban R 1, Howe S 23, Hunjan I 7, Judd S 27, Kitchen S 1, Lang K 13, Lowry S 12, Males J 21 , McStravick N 15, Micchiche A 17, Newby C 5, Pereira R 20, Rennie C 4, Ripley S 19, Roberston L 23, Simpson E 8, Singleton K 24, Skeath R 6, Thom R 15 , Tomlinson L 23, Thomas P 5, Tooke A 25, White F 8, White L 12, Wildgoose 7 J , Winstone R 2 1 Birmingham Childrens Hospital, Birmingham, United Kingdom, 2Evelina London Childrens Hospital, London, United Kingdom, 3National Centre for IMD, Dublin, Ireland, 4Royal Hospital for Sick Children, Glasgow, United Kingdom, 5Bristol Royal Hospital for Children, Bristol, United Kingdom, 6 Great Ormond Street Hospital for Children, London, United Kingdom, 7 Bradford Teaching Hospital, Bradford, United Kingdom, 8 Royal Manchester Childrens Hospital, Manchester, United Kingdom, 9 Addenbrookes Hospital, Cambridge, United Kingdom, 10 University College London Hospitals, London, United Kingdom, 11North Bristol NHS Trust, Bristol, United Kingdom, 12Sheffield Childrens Hospital, Sheffield, United Kingdom, 13 Ninewells Hospital, Dundee, United Kingdom, 14 Northern General Hospital, Sheffield, United Kingdom, 15Belfast Hospital for Sick Children, Belfast, United Kingdom, 16Royal Berkshire Hospital, Reading, United Kingdom, 17 St Thomas’s Hospital, London, United Kingdom, 18University Hospitals of Leicester, Leicester, United Kingdom, 19 Salford Royal Hospital, Manchester, United Kingdom, 20Norfolk and Norwich University Hospital, Norwich, United Kingdom, 21Royal Gwent Hospital, Gwent, United Kingdom, 22National Hospital Neurology and Neurosurgery, London, United Kingdom, 2 3 University Hospital Birmingham, Birmingham, United Kingdom, 24University Hospital Wales, Cardiff, United Kingdom, 25Nottingham University Hospital, Nottingham, United Kingdom, 26National Centre for IMD, Mater Hospital, Dublin,

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Ireland, 27Royal Liverpool University Hospital, Liverpool, United Kingdom, 28 Dewsbury and District Hospitals, Dewsbury, United Kingdom, 29NHD, Warrington, United Kingdom Background: Interpreting the protein content of manufactured foods from food labels is challenging for health professionals and phenylketonuria (PKU) patients/caregivers. The European Commission and Consumers Regulation (EU) No 1169/2011, on the provision of food information to consumers’ states that manufacturers need not declare protein content if manufactured foods contain ≤0.5 g protein/100 g, and so foods may be listed as containing 0 g protein even if they contain protein containing ingredients. The protein content of some vegetables appears higher than calculated phenylalanine (Phe) amount. Phe content is only declared on specialist low protein foods. Aim: A comparison of the interpretation of protein analysis by PKU patients/ caregivers and dietitians working in IMD. Methods: A series of 12 questions on protein food labelling on different manufactured foods were presented by PowerPoint presentation to PKU patients/caregivers at a national meeting and were sent separately to IMD dietitians. Respondents were given a choice of 5 answers for each food label: allow without measurement; allow in moderation, count as exchange, disallow, or other. Maternal PKU was excluded. Results: Completed questionnaires were received from 49 of 67 IMD dietitians (73 % response) and 45 PKU patients/caregivers. Dietitians were more liberal in their interpretation of protein labelling than patients/caregivers. PKU patients/caregivers were more likely than dietitians to use exact protein analysis from labelling (38 % vs 6 %), calculate phenylalanine content of special low protein foods (64 % vs 30 %), and exclude foods if their label stated zero protein but contained protein ingredients such as beef gelatine (73 % vs 21 %). Discussion: Uncertainty about food protein content interpretation has led to an overly cautious approach by caregivers/patients with PKU. It is important that IMD dietitians institute standard national guidelines on protein food labelling interpretation.

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Dietary management in pediatric mitochondrial cytopathies—retrospective study of 9 patients Vieira A I 1, Santos F 3, Santos H 2, Marques J S 2 1 Nutr Diet, Div Met Dis Centro Hospitalar, Vila Nova de Gaia, Portugal, 2Div Ped Metab Dis, Centro Hospitalar, Vila Nova de Gaia, Portugal, 3Div Neuroped, Centro Hospitalar, Vila Nova de Gaia, Portugal

Background: Mitochondrial cytopathies (CM) are a heterogeneous group of diseases that may cause mild to severe clinical outcomes. Mutations in the mitochondrial or nuclear DNA can result in impaired respiratory chain or oxidative phosphorylation function and compromise the ATP production. Metabolism of carbohydrates will be affected with increase in lactate/pyruvate ratios, making dietary management (DM) one of the therapeutic measures to help these patients. When DM is used for a specific cause, like ketogenic diet (KD), to control refractory epilepsy (RE), it is easy to evaluate benefits, but when it is used to improve the energy supply, those benefits are not so clear. Most authors make references to KD, anaplerotic, high medium-chain triglyceride and high-fat diets. Methods: Retrospective study of the DM in 9 patients, divided in 3 groups with different nutritional therapies(G1, G2 and G3), followed in our Pediatric Department. Results: Our sample has 78 % males, age varies between 4 and 14 years old, with partial defects in complex I, II and IV and in both (I and IV, I and II), and mDNA depletions. G1 (33 %) are severe neurodevelopment delayed. One patient with RE improved with KD, and the other two, severely undernourished, needed hypercaloric diet by gastrostomy tube. G2 (11 %) are obese(BMI > P95), miopatic, with metabolic syndrome and hypocaloric diet. G3 (55 %) has high-fat diet (50 % fat, 30 % carbs). 44 % are still on diet and refer benefits (100 % more muscle strength, 50 % better sleep quality, 50 % improve attention, 25 % less aggressive). Discussion: Recommendations for a diet therapy in CM often rely only on personal experiences from single cases or small case series (class C

evidence). There is weak evidence of benefits of the nutritional therapy, yet all of the parents of G3 patients referred increase muscle strength, which can be explained due to reduced lactate/pyruvate ratios that leads to less fatigue. We need a well-planned prospective study to determine adequate nutritional therapy for the heterogeneous needs of these patients.

P-112

UK IMD Professional Consensus on the use of soya infant formula in galactosaemia Portnoi P 2, MacDonald A 1 1

Birmingham Childrens Hospital, Birmingham, United Kingdom, Galactosaemia Advisory Board, GSG, Birmingham, United Kingdom

2

Background: There is a limited choice of suitable infant formulae (e.g. soya, amino-acid, casein hydrolysate) for galactosaemia infants. For many years’ soya infant formula (SIF) has been the first-line formula used by most UK paediatricians and dietitians. In 2003 and 2013, the UK Committee on Toxicity (COT) stated that SIF should not be used in galactosaemia infancy due to concerns about the mild oestrogenic effects caused by its phytoestrogen content and particular vulnerability in early infancy. This has caused ongoing concern amongst UK inherited metabolic disease (IMD) professionals. Aim: The Medical Advisory Group (MAP) of the Galactosaemia Support Group (GSG) aimed to establish consensus on the use of SIF in galactosaemia from UK IMD professionals. Methods: Dietitians from the GSG reviewed literature on phytoestrogens on SIF. Issues were discussed and opinion sought from IMD professionals over 6 months and ended with a debate and vote at a British Inherited Metabolic Disease Group (BIMDG) 2015 meeting. Results: 83 % (n = 53/64) of the BIMDG audience members voted in favour of continuing to use SIF for infants with galactosaemia. This led to the following statement: “The Medical Advisory Group of the GSG and the BIMDG recommend that soya continue to be used as the first choice low galactose infant formula in galactosaemia. Whilst it is accepted that it does contain a source of phytoeostrogens and it is associated with a mild risk of oestrogenicity, potential disadvantages of other formula far outweigh any concerns of SIF’s phytoestrogen content. SIF is widely used by other countries for galactosaemia and it has been safely given to infants with galactosaemia for over 40 years. Whilst it is important to provide information to caregivers about the phytoestrogen content of SIF, they should also be reassured about its safety for infants with galactosaemia.” Discussion: The majority of IMD health professionals approved the continued use of SIF in galactosaemia.

P-113

Branched chain amino acid concentrations and Fischer ratio are elevated in ketosis Scholl-Buergi S 1, Zlamy M 1, Michel M 1, Pichler K 2, Ertl C 1, Moshammer E 1 , Haberlandt E 1, Karall D 1 1 Pediatrics, Medical University, Innsbruck, Austria, 2Pediatrics, Medical University, Vienna, Austria

Background: The aim of this retrospective study was to investigate the correlation of ketone body concentrations (total ketone bodies, ß-hydroxybutyric acid) and branched chain amino acids (BCAA) in plasma of patients with ketogenic diet (KD). Methods: 29 patients (14 female, 15 male; 99 measurements in total) were included in this study. All patients were started on a KD (mostly modified Atkins diet) because of therapy resistant epilepsy (n = 27) or glucose transporter 1 deficiency syndrome (n = 2). Blood samples were taken during routine clinical visits (irrespective of nutritional status). Ketone body concentrations were measured by an enzymatic assay (Wako Chemicals, Germany). Concentrations of plasma amino acids were measured by ion exchange chromatography with post column derivatisation with ninhydrine (Biochrom 30+,

S90 Laborservice Onken, Germany). Ratio of BCAA to aromatic amino acids was calculated (Fischer ratio; ref: 2.1–4.0). Ketosis was defined as total ketone body concentrations above 90 μmol/L. Results: Ketosis could be detected in 87 measurements (in 26 patients), isoleucine concentrations >100 μmol/L were detectable in 44/87 (in 22 patients); leucine >176 μmol/L in 49/87 (in 22 patients); valine >317 μmol/L in 48/87 (in 20 patients), Fischer ratio >4.0 in 82 measurements (in 26 patients). Additionally, a significant positive correlation was observed between Fischer ratio and total ketone body or β-hydroxybutyric acid concentrations (Pearson correlation, p < 0.0001), but not between concentrations of isoleucine, leucine or valine and total ketone body or β-hydroxybutyric acid concentrations. Discussion: Ketosis leads to (1) an elevation of BCAA concentrations in plasma, (2) an elevated Fischer ratio and (3) significant correlation of Fischer ratio and total ketone body or β-hydroxybutyric acid concentrations. This may be caused by an inhibition of the BCAA degradation due to ketosis, but the exact mechanism is not understood to date.

P-114

Dietetic management of infants screened positive for maple syrup urine disease (MSUD) on expanded newborn screening (ENBS)—a single centre’s experience. Skeath R H 1, Cawtherley S 1, Hunn V 1, Stafford J 1, Batzios S 1, Chakrapani A 1, Cleary M A 1, Davison J 1, Grunewald S 1, Dixon M 1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 control children liked sweet tasting foods, with preference for sweet fruit and vegetables. PKU children demonstrated challenging food neophobia, with no particular preference for bitter tasting foods associated with the taste of Lamino acids. Aim: In an observational extension study, we evaluated the influence of parental food choice and their neophobia on their children’s taste preferences and food neophobia. Methods: Male and female parents/carers of 35 children with PKU and 35 age/ gender matched control children, completed a neophobia and food frequency questionnaire. Results were compared with the same questionnaires completed for their children. Results: PKU children were rated as more food neophobic than their parents (child 35 vs mother 43, p = 0.003; vs father 47, p < 0.0001) but the reverse was observed for control children (child 46 vs mother 35; vs fatherfather 34, p < 0.0001). Parents of PKU children offered their children more fruits and vegetables than they ate themselves. PKU children consumed sweet potato (p = 0.0008), strawberry (p = 0.001) and apple (p = 0.02) more frequently than both parents. PKU children also consumed sweets (child 6/week [w] vs mother 1/w, p < 0.0001; father, 2/w p = 0.003) and sweet drinks (child 9/w vs mother 2/w, p < 0.0001; father 5/w, p = 0.008) more commonly than their parents. Control children ate more similarly to their parents with the exception of sweets, which were eaten more by children (3/w) than their mothers (1/w, p = 0.02). Parents in both groups consumed lemon and coffee flavours more frequently than their children. Discussion: In PKU, children’s food preferences and food neophobia did not appear to be influenced by their parents eating habits or food neophobia. The PKU child’s food neophobia may be associated with a fear of eating high protein foods and their preference for sweet foods may be influenced by availability of sweet foods low in phenylalanine.

1

Great Ormond St Hosp for Children NHS FT, London, United Kingdom

Background: In January 2015 newborn screening for MSUD was introduced in England following a 1 year pilot study. Supporting laboratory, clinical and dietetic management guidelines were written (www.bimdg.org). Dietetic guidelines include daily targets of 120 kcal/kg, 3 g/kg branched-chain amino acid (BCAA)-free protein equivalent (PE), 200–400 mg isoleucine (ISO) and valine (VAL) and leucine (LEU) restriction to maintain blood LEU 150–300 μmol/L. We summarise our dietetic management of babies screened positive for MSUD. Methods: A review of our cohort diagnosed with MSUD on ENBS since July 2013. Results: Eight infants screened positive for MSUD; n = 6 classic MSUD (one sibling), n = 1 intermediate MSUD, n = 1 E3 deficiency. Age at diagnosis was 4–12d, quantitative bloodspot LEU ranged from 87 to 3967 μmol/L (median 2361 μmol/L). All were admitted to hospital. All classic MSUD except the sibling (n = 5) were symptomatic and transferred to the paediatric intensive care unit (PICU), ventilated and given continuous veno-venous haemofiltration (CVVH). All five received IV dextrose and lipid, n = 4 tolerated some BCAA-free PE feed via NG tube. During CVVH ISO and VAL supplementation ranged from 50 to 200 mg/d. After 16–36 h on CVVH blood LEU fell to 247–660 μmol/L, ISO 12– 90 μmol/L, VAL 93–251 μmol/L. Enteral feeds were adjusted to provide LEU plus BCAA-free PE (100–300 mg LEU/d, 2–3 g PE/kg, 120 kcal/kg). All made full neurological recovery. Those not admitted to PICU were treated from diagnosis with 200–400 mg LEU/d or free diet + BCAA-free PE, 0–200 mg/d ISO and VAL. Treatment was guided by daily blood BCAAs. Discussion: In the UK BCAA-free PE, ISO and VAL are only available as enteral formulas. Achieving the guideline targets for energy, BCAA-free PE, ISO and VAL was difficult due to poor feed tolerance, fluid restrictions and periods of nil by mouth in the acute setting. LEU levels fell within 36 h on CVVH; LEU should be introduced immediately with optimal doses of ISO and VAL.

P-115

A comparison of food preferences and neophobia in parents of children with and without phenylketonuria (PKU) Evans S 1, Daly A 1, Chahal S 1, Ashmore C 1, MacDonald J 1, MacDonald A 1 1

Birmingham Children’s Hospital, Birmingham, United Kingdom

Background: In a case control study, we demonstrated that both PKU and

P-116

Transitioning protein substitutes in childhood PKU: 15 years of experience Evans S 1, Daly A 1, MacDonald J 1, MacDonald A 1 1

Birmingham Children’s Hospital, Birmingham, United Kingdom

Background: The progression of protein substitutes (PS) (liquid vs semisolid) in childhood is not documented in phenylketonuria (PKU). It is our practice to use an infant formula PS in early infancy, progress to semi-solid PS during weaning (WPS) and transfer to a liquid pouch by school age. There are no published data describing the progression of PSs during childhood in PKU. We report our experience when children were given a WPS in PKU. Methods: Retrospective data from dietetic records was reviewed from 31 PKU patients (boys n = 16), diagnosed by newborn screening, who all were given a WPS between 2001 and 2009. Data was collected on age commenced WPS, length time on WPS, prescription of WPS by age, issues with administration of WPS and age of transition to third PS. Results: Following use of infant PS formula, mean age commenced WPS was: 4.8 m (3.2–6.9). Mean daily protein from WPS increased steadily by about 0.2– 0.3 g/kg/m in the first 9 m then slowed to an increase of 0.1 g/kg/m to 2y (2.8 g/kg/day), aiming for a total protein equivalent intake of 3 g/kg/day. Girls remained on WPS longer: 6.3y (1.7–11.8y) vs boys 4.6y (1.2–9.6y). Median number of times a third stage liquid pouch PS was tried before accepted was 3 (1–8); 39 % (n = 12) took >1 m (6w–2.2y) to transition; and 6 subjects remained on WPS (aged 6.9–10.6y; 4 female). Some negative behaviours were experienced in the first 2 years of life with WPS but were also experienced with food, but not at the time of WPS introduction. Issues included rejection episodes during teething: WPS 45 % (n = 14), food refusal 42 % (n = 13); illness: WPS 58 % (n = 18), food refusal 55 % (n = 17); and when well: WPS 81 % (n = 25), food refusal 71 % (n = 22). Discussion: Transitioning between age-appropriate PS is challenging as PKU children dislike change. Any negative behaviour associated with WPS appeared associated with normal weaning behaviour and coincided with illness and teething. Guidelines for transitioning PS in childhood require development. Conflict of Interest declared.

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Dietary practice in treatment of MSUD in 10 Italian centres Salera S 1, Bernabei S M 2, Dianin A 3, Di Mauro A M 7, Gugelmo G 2, Fasan I 6 , Gallo G 2, Musiani C 4, Pozzoli A 5, Pretese R 8, Tarrini G 4, Tursi S 8, Zucchi C 9, Zuvadelli J 10 1 Ped Dept, IRCCS Policlinico, Milan, Italy, 2Bambin Gesu, Children Hospital, Rome, Italy, 3Inherit Metab Dis Unit, Dept of Ped, Verona, Italy, 4S Orsola Malpighi Hosp, Bologna, Italy, 5Piacenza Hosp, Piacenza, Italy, 6Dept Pediatrics Uni Hosp, Padova, Italy, 7Univ Hosp Giovanni XXIII, Bari, Italy, 8 Metab Unit, San Gerardo Hosp, Monza, Italy, 9Gaslini Hosp, Genova, Italy, 10 Cl Dep of Ped, San Paolo Univ Hosp, Milan, Italy

Background: Dietary treatment of maple syrup urine disease (MSUD) is described in literature, but there are few data about dietary practice. The aim of this work is to collected the data in Italian centres. Methods: We sent a questionnaire to the dietitians of 18 metabolic centres focusing on: 1) how leucine contents of food was calculated, 2) plasma leucine levels considered normal in well-being status, 3) supplementation with valine (V) and isoleucine (I) during decompensations. Results: 10 of 18 centres answered the questionnaire. 1) The amount of leucine in food is calculated in different ways: 2/10 centres do not consider leucine content of low protein foods and fruit with protein content < 1 g/ 100 g; 6/10 consider only low protein foods with a protein content >1 g/ 100 g; 2/10 consider leucine content of all foods. 8/10 interviewed dietitians use exchange system of equivalent based on leucine content of foods. Databases used for the calculation are different among centres. 2) Maximum tolerated plasma leucine level in well-being conditions is 200 umol/L for 0–5 years of age and 300 umol/L over 5 years in 4/10 centres; other 4 centres consider acceptable more elevated values; 1/10 keeps leucine values more restricted (up to 180) at every age and 1/10 centre did not answer. 3) In emergency treatment, 6/10 centres increase V and I intake and 8/10 BCAA free mixture. Discussion: Dietary practices vary widely among Italian metabolic centres. We do not know if it is the same in other countries and think that it would be useful to uniform the dietary practices.

P-118

The use of vegetal protein in glycogen storage disease (GSD) type III Paci S 1, Montanari C 1, Re Dionigi A 1, Zuvadelli J 1, Morgano A M 1, Banderali G 1 1

Ped Dept, Univ Milan, S Paolo Carlo ASST, Milan, Italy

Background: GSD III is due to deficiency in glycogen debranching enzyme. GSD IIIa is the most frequent phenotype (85 %) and causes liver disease, skeletal myopathy and hypertrophic cardiomyopathy in the long-term follow-up even in well-controlled patients. New therapeutic options have been successfully tested (a ketogenic diet with addition of synthetic ketone bodies, a high protein very-low-calorie diet, a high-fat high-protein diet with a progressive stop of uncooked cornstarch UCCS), and showed significant improvement particularly in cardiomyopathy. Case Report: Eight months ago one of our adult patient affected by GSD IIIa presented a rapid and serious worsening of cardiac function (ejection fraction EF = 25 %). We decided to change his nutritional schedule from frequent night-and-day meals, followed by the assumption of UCCS (55 % carbohydrate, 22 % protein, 23 % fat), to a high-fat (53 %) high-protein (29 %) and low-carbohydrate (18 %) dietetic treatment, with a progressive interruption of UCCS. He maintained a good glycemic and metabolic control. We recommend extra-virgin olive oil as unique type of relish, but the great change was the introduction of nuts (such as walnuts, almonds, pistachios, nuts, etc), capable to satisfy the increased requests both of fats (LC-PUFA over saturated) and proteins, accepted with great compliance by our patient as snacks during the day.

Results: Only few months after of the introduction of the new dietary treatment, we observed a dramatic improvement in clinical manifestation and in biochemical/instrumental parameters, particularly in heart function (EF = 45 %, with a relevant reduction of IVS thickness and outflow obstruction), but a long-term follow-up is necessary to confirm the efficacy of this new approach. Discussion: This suggestion opens new therapeutic options regarding the use of vegetal protein in inborn errors of metabolism, a group of diseases that often share the necessity of a specific nutritional intervention.

P-119

Nutritional status in patients with phenylketonuria under glycomacropeptide diet Pinto A 1, Almeida M F 1 2, Ramos P 1, Rocha S 3, Guimas A 3, Ribeiro R 3, Martins E 3, Bandeira A 3, MacDonald A 4, Rocha J C 1 5 6 1

Centro de Genetica Medica, CHP, Porto, Portugal, 2UMIB ICBAS UP, Porto, Portugal, 3Unid Doen Heredit Metab, CHP, Porto, Portugal, 4Birmingham Child Hosp, Birmingham, United Kingdom, 5Faculdade de Ciencias da Saude, UFP, Porto, Portugal, 6Cent Health Technol Serv Res (CINTESIS), Porto, Portugal Background: Glycomacropeptide-based protein substitute (GMP) is an alternative to traditional L-amino acid supplements (AA) used in the dietary management of phenylketonuria (PKU). There are limited data reporting nutritional status markers in patients treated with GMP. In a retrospective study, we report the nutritional status of PKU adults taking AA and GMP. Methods: 11 PKU patients aged 27 ± 10 y (1 HPA, 4 mild PKU, 3 classical PKU and 3 late diagnosed) were studied. Patients were evaluated at two different time points as part of an annual review: 1) taking AA and 2) GMP. Data on anthropometry, body composition, biochemistry and nutritional intake was collected at each review. Metabolic control (blood phenylalanine [Phe] and tyrosine [Tyr]) was analyzed before (mean time 13 ± 5 months) and after GMP introduction (mean time 13 ± 7 months). Good metabolic control was defined when median blood [Phe] was < 8 mg/dL. Results: Mean GMP contribution to the total protein substitute (PS) intake was 57 % (27–100 %), providing an additional 34 ± 12 mg/day Phe. Nutritional intake, anthropometry and body composition measurements were similar between two evaluation time points. Median blood Phe did not change (8.6[5.6– 12.3] vs. 9.0[5.8–14.6] mg/dL; p = 0.594), although the number of blood Phe concentrations within target range improved (36 % vs. 46 %). From the 1st to the 2nd evaluation, mean blood Tyr increased (52.0 ± 19.2 vs. 63.2 ± 25.6 μmol/L; p = 0.033), resulting in decreased blood Phe/Tyr (10.4[7.1– 23.0] vs. 7.5[5.6–20.3];p = 0.041). All biochemical markers remained stable after GMP introduction, except for lower A1C hemoglobin (%) (5.1 ± 0.4 vs. 5.0 ± 0.3; p = 0.020). Discussion: The part use of GMP to total PS intake does not affect nutritional status in patients with PKU. Phe control was not adversely affected and an increased blood Tyr and a decreased blood Phe/Tyr were observed after GMP introduction. The reduced A1C hemoglobin after GMP introduction warrants the need for future studies. Conflict of Interest declared.

P-120

Hypertrophic cardiomyopathy improved by high-fat low-carbohydrate diet in a glycogen storage disease type III patient Kumru B 1, Hismi B 2 1 Div Nutrition and Diet, Child Hosp, Gaziantep, Turkey, 2Div Metab Dis, Tepecik Edu Res Hosp, Izmir, Turkey

Background: Glycogen storage disease type III (GSD III) is an inherited metabolic disease caused by deficiency of the glycogen

S92 debranching enzyme amylo-1,6-glucosidase and results in the accumulation of abnormal glycogen (‘limit dextrin’). In subtype IIIa hepatic tissue, skeletal and cardiac muscle tissue is affected, while in subtype IIIb only hepatic tissue is affected. Cardiac storage of limit dextrin causes a form of cardiomyopathy, which resembles primary hypertrophic cardiomyopathy on cardiac ultrasound. Current treatment of GSD III is based on frequent high-carbohydrate meals that have no effect on the cardiomyopathy. Recently, ketogenic diet was reported to be effective in few cases (n = 5). Case report: A 6-year-old boy with GSD IIIa developed left ventricular hypertrophy at 4.3 years of age. A diet with high fat (50 %), low carbohydrate (30 %) and high protein (20 %) was introduced. Cessation of cornstarch resulted in hyoglycemia for which modified cornstarch has to be used. Additional protein powders were used to achieve target protein consumption. Results: After 18 months, prominent echocardiographic and biochemical improvement was observed (CK 1628> > 1125 U/L; left ventricular outflow gradient 35> > 20 mmHg; interventricular septum thickness 21> > 10 mm; posterior wall thickness 18> > 11 mm) and his fatigue resolved. Discussion: Here we report a GSD IIIa patient with hypertrophic cardiomyopathy who improved clinically and objectively on a diet with high protein, high fat and low carbohydrates. Hyperinsulinism induced by a classical high-carbohydrate GSD diet may inhibit lipolysis and the energy availability for the heart. High protein diet might reduce limit dextrin accumulation in myocardial cells by triggering gluconeogenesis. This diet approach could be a beneficial treatment for GSD III with cardiomyopathy.

P-121

Special low protein foods intake in patients with phenylketonuria Ramos P C 1, Almeida M F 1 2, Pinto A 1, Rocha J C 1 3 4 1 Centro de Genetica Medica, CHP, Porto, Portugal, 2UMIB ICBAS UP, Porto, Portugal, 3Faculdade de Ciencias da Saude, UFP, Porto, Portugal, 4Cent Health Technol Serv Res (CINTESIS), Porto, Portugal

Background: Despite therapeutical advances, a natural protein and phenylalanine (phe)-restricted diet, supplemented with protein substitutes and special low protein foods (SLPF) remains the mainstay treatment of phenylketonuria (PKU). Excessive consumption of SLPF may have a role in overweight etiology, even though there is no scientific data to support this. Our study aimed to investigate energy intake contribution from SLPF in overweight and non-overweight early treated PKU patients. Methods: A sample of 73 patients (17.0 ± 8.0 y; 1–33 y; 43 % females) was studied. Total daily energy intake (kcal/day) and specific contribution from SLPF (%) were calculated based on food history, collected at the annual routine nutritional status evaluation. Overweight was defined using WHO criteria, both in pediatric and adult patients. Good metabolic control was defined as median blood [phe] < 6 mg/dL or < 8 mg/dL in patients < or >12 y, respectively. Results: Overweight was present in 21 patients (29 %; aged 18 ± 7 y; 52.4 % female; 42.9 % classical PKU), while 52 patients were considered eutrophic (71 %; aged 17 ± 8 y; 38.5 % female; 34.6 % classical PKU). The majority of patients (58 %) were under good metabolic control, as well as 61.9 % of overweight patients and 56 % of nonoverweight patients. No differences were found between median blood [phe] of patients with and without overweight (7 ± 2.8 vs 7 ± 3.2; p = 0.881). Daily energy intake (2318 ± 464 vs 2203 ± 468; p = 0.341), SLPF energy intake (470 ± 307 vs 440 ± 303; p = 0.713) and energy intake contribution from SLPF (23 ± 11 vs 23 ± 13; p = 0.977) were similar among overweight and non-overweight patients. Discussion: In our study, energy intake from SLPF was similar in overweight and non-overweight patients with PKU. While overweight in

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 PKU is still under debate, a detailed and longitudinal food pattern characterization would be welcome. Considering the multifactorial etiology of overweight, careful interpretation of food history is needed in PKU.

P-122

Anthropometric evaluation in patients with fatty acids oxidation defects and glycogen storage diseases requiring a high carbohydrate diet Gallo G 1, Maiorana M 1, Bernabei S 2, Bellucci F 2, Taurisano R 1, Deodato F , Bevivino E 1, Cotugno G 1, Dionisi-Vici C 1

1

1 Div Metab, Bamb Gesu Child Hosp , Rome, Italy, 2Div nutr, Bambino Gesu Child Hosp, Rome, Italy

Background: Hypoglycemia is a characteristic feature of fatty acids oxidation (FAO) defects and of glycogen storage diseases (GSDs). To prevent hypoglycemia, patients are treated with fasting avoidance, combined in selected diseases with slow release carbohydrate and/or MCT oil. A potential side effect of these dietary regimens is the development of overweight and insulin resistance (IR), leading to ‘metabolic syndrome’. In order to evaluate the occurrence of metabolic syndrome in patients with FAO and GSDs, we evaluated anthropometric indexes and insulin resistance. Methods: We retrospectively analyzed data from 58 patients, 27 with FAO (1 CPT1, 3 CPT2, 2 CACT, 6 VLCAD, 8 MTP, 7 MCAD) and 31 with GSDs (17 type Ia, 4 type Ib, 10 type III). Overweight was defined as >110 % (obese >120 %) index body weight (IBW) percentage. IR was defined as Homa Index >2.5 and/or glucose/insulin ratio < 6. Results: Overall, 13/58 (22.4 %) patients were overweight, of whom 5/58 (9 %) were obese (3 with GSDs and 2 with LC-FAO defect). IR was found in 8/25 patients (75 %), 6 with FAO and 2 with GSDs, mainly in patients >12 y. When stratified for age categories (1 < 6y, >6 < 12y, >12y), the glucose/kg/min intake was similar in FAO e GSDs. Discussion: The prevalence of overweight in our cohort was similar to the Italian pediatric population (22.4 % vs. 20.9 %). However, overweight was more frequent in GSDs, whereas IR was more frequent in FAO. Most of overweight patients were in two age categories, 1–6 y and >12 y. No correlation was found between glucose intake for age category and development of overweight/obesity. In conclusion, our patients on a high carbohydrate normocaloric diet did not presented with increased risk of overweight and metabolic syndrome, although overweight was more frequent in GSDs.

P-123

Weaning patterns and practices in PKU: a longitudinal case-control study Evans S 1, Daly A 1, Ashmore C 1, Chahal S 1, MacDonald A 1 1

Birmingham Children’s Hospital, Birmingham, United Kingdom

Background: There are no published, evidence based weaning guidelines in phenylketonuria (PKU). For all infants, it is important to transition from a diet that is exclusively liquid to solid foods. Aim: In a longitudinal, prospective, controlled study, the weaning development was studied in PKU infants gradually transitioned to a phenylalanine (Phe)-free semi-solid second stage protein substitute (SSPS) from a Phe-free infant formula (PFIF), at weaning commencement. Methods: 17 PKU and 20 control infants matched for age, gender, birth order and mother’s educational level were reviewed monthly from weaning start to 12 m of age. Anthropometry, weaning development, 24-h dietary intake,

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 gastro-intestinal symptoms, maternal anxiety and coping strategies were recorded. Results: Median age for weaning PKU: 4 m (range: 3–6 m), control: 5 m (range: 4–7 m). PKU infants achieved developmental milestones of finger feeding (6.6 vs 5.9 m) and progression onto normal family foods (8.0 vs 7.2 m) later than controls but ate from a spoon earlier (9.3 vs 9.7 m); although these were not statistically significant. At 12 m age, PKU infants consumed 70 ml/day less ‘formula’ (p = 0.05), and fewer solid meals/day than controls (4 vs 5; p = 0.02). In the PKU group, protein intake from semi-solid PKU protein substitute increased by 0.5 g/kg/day every 3 m. By 12 m, the SSPS provided 80 % (2 g/kg/day protein) and PFIF 20 % (0.5 g/kg/day protein) protein equivalent intake. By 12 m weight gain between both groups was equivalent (median: 10.3 kg, range: 7.7–12.8; z-score: 0.5–0.6) and there was no significant difference between z-scores for weight, length or BMI between groups. Gastrointestinal symptoms decreased with age in both groups but PKU infants experienced more burping, retching and constipation in the early stages of weaning. Maternal anxiety was similar and low in both groups. Discussion: The feeding development and nutritional status of infants with PKU given a SSPS is similar to healthy control children in the first year. Conflict of Interest declared.

P-124

Normal growth and bone mineral density reported in 38 PKU children taking conventional amino acid protein substitutes

Background: Dietary management of phenylketonuria (PKU) has allowed prevention of severe mental retardation over the last decades. The strict low-phenylalanine (Phe) diet needs careful evaluation of nutritional status. Our study aims to evaluate nutritional parameters at different ages of a cohort of PKU patients treated with Phe-restricted diet. Methods: 40 early-treated PKU patients (25 M/15 F), followed-up since 6 months to 6 years, were enrolled. According to Phe levels at diagnosis, 35 patients’ were classified as classical (>1200 mol/L), 5 as mild PKU (Phe 600–1200 mol/L). All patients were treated with Phe-restricted diet and had good adherence to diet (mean Phe < 400 umol/l). Growth parameters were evaluated at 6 months, 12 months, 3 years and 6 years together with metabolic control parameters. Patients were classified according to national BMI reference tables. Results: Overall 78 % of our cohort had normal weight, 13 % overweight, and 9 % underweight, none were obese. Overweight was more frequently observed at 3 and 6 years (17,5 and 12,5 % respectively), underweight was diagnosed more frequently at 6 months of age (12,5 %). 31 patients who had normal weight at 6 months highlighted an increasing of growth rate, which was more evident at 3 years of age. Discussion: The majority of our PKU patients had normal growth parameters. A tendency to overweight is detectable in early childhood with a higher peak of incidence around 3 years of age. These data confirm that growth fashion of PKU children is similar to normal population. A greater awareness of this tendency should guide decisions on caloric intake and promotion of regular physical activity also in this group of patients.

Daly A 1, Chahal S 1, Evans S 1, MacDonald A 1 P-126 1

Birmingham Childrens Hospital, Birmingham, United Kingdom

Background: Lower bone mineral density (BMD) has been documented in phenylketonuria (PKU). It is not known if this is an inherent part of the disease or secondary to dietary treatment. A low natural protein diet devoid of dairy foods and reliance on a phenylalanine-free L-amino acid supplement (L-AA) (commonly providing 50–80 % of total protein intake) has increased concerns about long term bone health in this population. Aim: to determine if BMD, bone mineral apparent density (BMAD), weight, height, BMI z scores and percentage body fat is different in a group of treated children from normal standards. Methods: 38 children with well controlled PKU (median blood phenylalanine [Phe] for previous 12 months was 300 μmol/L (90–590) (diagnosed by newborn screening) had DEXA scan measurements recording BMD g/cm3 BMAD g/cm3 whole body BMD g/cm3, total percentage body fat, weight, height and BMI. There were 22 boys and 16 girls, median age 9y (5–16y). Results: There were no significant differences observed between girls (g) and boys (b) for median z scores. Height: (g, 0.01; b, 0.04) weight: (g, 0.7; b, 0.7) BMI: (g, 0.9; b, 0.6) BMD: (g,0.3; b, −0.3) BMAD: (g, 0.8; b, −0.2), whole body BMD (g −0.75; b −0.4). A significant difference was observed for total body fat (g, 29.4; b, 22.5 p = 0.004). Median dietary intake was: protein equivalent from LAA 60 g (60–80 g); natural protein, 5 g/day (3–30 g); energy, 90 % (70–129 %) of estimated average requirement, calcium 1200 mg/d and vitamin D, 13 μg/d. Discussion: There was a trend for males to have slightly lower BMD and BMAD but for the girls to have a higher total body fat. However, all children with PKU on a low Phe diet were growing appropriately and all had a normal bone density for age and size. Conflict of Interest declared.

P-125

Growth monitoring in PKU children: trend of nutritional parameters in infancy and early childhood Di Mauro A M 1, Corvasce S 1, Masciopinto M 1, Carella A 1, Ortolani F 1, Simonetti S 2, Armenise E 2, Tummolo A 1, Papadia F 1 1

Dept of Metabolic Dis, Child Hosp, Bari, Italy, 2Clin Path Lab Child Hosp, Bari, Italy

Application of the ketogenic diet in inherited metabolic diseases Marchio’ M 1, Bruni G 2, Belli F 2, De Leo S 3 1 Univ of Modena and Reggio Emilia, Modena, Italy, 2Diet Unit, Meyer Child Hosp, Florence, Italy, 3Dept of Clin Med, Umberto I Univ Hosp, Rome, Italy

Background: The ketogenic diet (KD) is the treatment of choice in the defects of glucose transporter type 1 (GLUT-1) and pyruvate dehydrogenase complex (PDHD). The objective of present work was to collect information on the use of the ketogenic diet in inherited metabolic diseases (IMDs) in Italy. Methods: A questionnaire of 26 multiple choice questions was sent to dietitians of 15 Italian centres specialized in the care of the IMDs. The answers were analysed with the tools of descriptive statistics. Results: The cumulative number of Italian KD patients was 79, including 54 with GLUT-1 D and 12 with PDHD. Other conditions were: glycogen storage disease type III (5), non ketotic hyperglycinemia (3), congenital hyperinsulinism (3) and respiratory chain complex I-III defects (2). The number of treated patients varied considerably across centres: three of them have in charge almost 80 % of the cases. At present, half of the centres have had patients treated with KD for more than 10 years. The minimum age of initiation of treatment was about 3 months, but in few KD was also initiated in adulthood. The most widely used diet was the classic diet, followed by the MCT and the modified Atkins diets. The most frequent side effects were high cholesterol and constipation. There was instead no increased occurrence of fatty liver, infections and stunting. For dietitians specialized in KD, the most useful tool is to regularly discuss their cases with other experienced colleagues. Valid work tools are also diet processing software, dedicated nutritional dossiers and training and updates. Discussion: The KD in IMDs may act as an etiologic treatment for some conditions and a symptomatic one for many others associated with epileptic encephalopathies. The extreme heterogeneity of cases and diseases highlights the value of working in a team of experts in metabolic disorders and the importance of a continuous sharing of experiences with all the centres using this therapeutic approach.

S94 05. Phenylketonuria: general

P-127 Assessment of nutritional parameters and micronutrient levels in classical phenylketonuria patients Kose E 1, Arslan N 1 1

Div Metab Dis, Dokuz Eylul Univ Hosp, Izmir, Turkey

Background: The aim of this study was to determine the growth parameters and micronutrient levels in phenylketonuria (PKU) patients and to compare with healthy controls (HC). Methods: Age- and sex-matched 112 PKU patients and 17 controls were enrolled. BMI-SDS and weight for height (WH) were calculated to define obesity and malnutrition, respectively. Hematologic and nutritional biochemical parameters were measured. Children who were using vitamin-mineral supplementation during the last 3 months were excluded. Results: Antropometric values were within normal levels in all HC. Although BMI-SDS did not differ between two groups, malnutrition and obesity were detected in 7.2 % and 5.4 % of PKU patients, respectively. Hemoglobin, albumin, calcium, phosphorus, zinc, iron, vitamin B12, vitamin E, 25-OH vitamin D and copper levels were not significantly different between patients and controls. Ferritin, folic acid, prealbumin and vitamin A levels were significantly higher in PKU patients than HC. There was no clinical sign of nutritional deficiency and no child with vitamin A deficiency in whole group. Vitamin E deficiency was detected in 3.3 % of PKU patients and 0 % of controls. Low ferritin levels were detected in 13.0 % and 37.5 % of PKU and control groups, respectively (p = 0.023). However, hemoglobin and iron levels and transferrin saturation index were not different between two groups. The other biochemical nutritional deficiencies were not significantly different. Mean phenylalanine (Phe) level of previous year was positively correlated with BMI-SDS and negatively associated with zinc and 25-OH vitamin D levels. Discussion: Higher Phe levels, an indicator of poor dietary compliance, have been found related to high BMI values and also zinc and 25-OH vitamin D deficiencies. Close monitoring of dietary compliance is mandatory to prevent obesity and micronutrient deficiencies in PKU patients.

P-128

National study to assess current practices of the management of phenylketonuria Jurecki E 1, Rohr F 2, Cederbaum S 3, Kopesky J 4, Sanchez-Valle A 5, Viau K 6 , Cohen-Pfeffer J 1 1 BioMarin Pharmaceutical Inc, Novato, United States, 2Boston Child Hosp, Boston, United States, 3Intellect and Dev Disabilities Res, Los Angeles, United States, 4Child Hosp Wisconsin, Milwaukee, United States, 5Univ of South Florida, Tampa, United States, 6Univ of Utah, Salt Lake City, United States

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 consistent with the ACMG guidelines of 120–360 μmol/L. For adults, the targeted ranges in the clinics were greater, with a mean upper limit of 445 μmol/L. Adherence to clinic-recommended target blood Phe levels was reported for 84 % of children 0–4 years, 70 % of children 5–12 years, 49 % of adolescents 13–17 years, 41 % of adults 18–29 years, and 31 % of adults ≥30 years. About 73 % of preconception women were within range. The majority (89 %) of clinics had treatment protocols in place for frequency of blood Phe testing. Discussion: These results from a national survey distributed to clinics across the US represent approximately half of the PKU patient population actively followed in clinic. The percentage of patients achieving their target blood Phe levels progressively decreased with age, suggesting compromised metabolic control with increasing age. Improving patient blood Phe control provides an opportunity to address a challenge in PKU management, with the goal to improve outcomes for these patients. Conflict of Interest declared.

P-129

Phenylketonuria (PKU) phenotype-genotype scoring and global phenotype differences Shen N 1, Burgard P 1, Trefz F K 1, Hoffmann G F 1, Blau N 1 1

Univ Child Hosp Metabol Center, Heidelberg, Germany

Background: Information from the BIOPKU database (www.biopku.org/ biopku/), based on the genotype and phenotype data of over 10,000 PAHdeficient patients from all over the world, suggests a heterogeneous global phenotypes distribution. In addition, more than 950 different PAH gene variations are tabulated in the locus-specific database (www.biopku.org/pah/), and these result in almost 2000 different genotypes. Methods: The BIOPKU database was linked with the PAHvdb and phenotypes, genotypes and BH4 responsiveness was calculated for Eastern Europe, Northern Europe, Middle Europe, Southern Europe, Middle East, Asia and the U.S. Based on functionally hemizygous genotypes, allelic phenotype value (APV) was assigned to each PAH gene variant, where applicable. Results: We found the global distribution of mild HPA, mild PKU and classic PKU to be approximately 17 %, 27 %, and 56 %, respectively. There were significant differences in the frequency of severe classic PKU versus mild PKU and mild HPA between the eastern and southern European countries, with classic PKU being more frequent in the Eastern Europe and mild PKU and mild HPA in the Southern Europe. Reporting of mild HPA was lowest in Asia and the U.S. Accordingly, BH4 responsiveness was more frequent in regions with milder phenotypes. Both the phenotype and BH4 responsiveness correlated with the severity of the genotypes. Discussion: Patients-based genotypes database, linked with the locus-specific database, is an invaluable tool to study both the PKU phenotypes distribution worldwide and the genotype-phenotype correlation. Allelic phenotype scoring predicts the severity of phenotypes. P-130 The analyses of PAH gene’s mutations among Kazakhs with phenylketonuria in Kazakhstan Salimbayeva D N 1, Berezina G M 1, Svyatova G S 1

Background: We assessed current phenylketonuria (PKU) management practices in clinics across the US and evaluated clinic adherence to American College of Medical Genetics and Genomics (ACMG) guidelines and patient adherence to their clinic recommendations. Methods: Dietitians and physicians with experience managing PKU developed a questionnaire that was sent to 130 metabolic clinics specializing in the treatment of individuals with PKU. Results: One third of metabolic clinics (44/130) qualified and reported on 3,772 patients with PKU. The mean targeted blood phenylalanine (Phe) ranges for children used in the clinics were reported to be

1

Sceintific center of obstetrics, gynecol, Almaty, Kazakhstan

Background: The etiologic factor of phenylketonuria (PKU) are mutations in the PAH gene in which over 800 mutations have been described. Frequency and spectrum of mutations have ethnic characteristics. Methods: We studied DNA of 31 Kazakh PKU patients from unrelated families. DNA was isolated with standard method. At first we performed molecular genetic studies of the most frequent mutations (R158Q, R252W, R261Q, R408W, P281L, IVS14 + 5G > T, IVS10-11G > A ? IVS12 + 1G > A,) by

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 PCR. If no mutations were found we searched more rare mutations in the PAH gene by direct automated sequencing. Results: The informative value of a kit for 8 common mutations in the PAH gene for Kazakhs was 38.7 %. After automatic direct sequencing we identified the following mutations: R243Q (0.290), R408W (0.161), P281L (0.097), IVS4 + 5G > T (0.048), IVS10-11G > A (0.032), A300S, W187X, R158Q, Y387H, IVC12 + 1G > A, IVS1014C > G, IVS12 + 1G > A, V230I, I65N (−0.016). The frequency of unidentified mutations after automatic direct sequencing decreased from 0.613 to 0.226. Most common mutations in PAH gene by type were missense (62.9 %), splice (12.9 %) and nonsense (1.6 %). Most mutations in PAH gene were located in E7 region (38.7 %), E12 (16.1 %), I10 and I4 (4.8 %), E6 and I12 (3.2 %), E11, E8, E5 and E3 (1.6 %). Discussion: We established a preliminary spectrum of mutations in PAH gene in Kazakh patients with PKU. The kit including the 8 most common mutations in the PAH gene was not informative for Kazakhs, because these mutations are more common in European populations. We analyzed type and location of mutations in the PAH gene in Kazakhs patients. Ethnicity must be considered for choosing the aimed at molecular genetic diagnosis of PKU patients, including prenatal diagnosis of PKU.

P-131

Absorption of casein glycomacropeptide (CGMP-20) and free synthetic amino acids (AA) in phenylketonuria (PKU) patients in connection with standardized meal Ahring K K 1, Jensen E 2, Lund A M 3, Nielsen K B 1, Jensen T G 4, Moller L B 1 1

The Kennedy Centre, Cph Univ, Copenhagen, Denmark, 2Arla Foods Ingredients (AFI), Viby J, Denmark, 3Genetic Clinic, Cph Univ Hosp, Copenhagen, Denmark, 4Aarhus Univ Hospital, Aarhus, Denmark Background: The primary purpose of this study was to investigate, if there were differences between absorption of CGMP compared to synthetic, free AA by comparing AA profiles before and after the consumption of the standard meal. Also, it was investigated as whether a small amount of phenylalanine (phe) in CGMP affects plasma phe. Methods: Patients (7 females, 1 male, age 15–48 (mean 33.25 + SD 11.21), weight 47–85 kg (mean 72.8 + SD 15.9) were asked to come in to clinic in the morning, fasting. They had been selected from the Kennedy Centre (KC) database from the following criteria: well treated from birth, age >15, and classical PKU. All patients had 4 identical visits in randomized order and had a new drink mixture (DM) at every visit, containing CGMP or AA. The DM were designed as follows: DM1: 100 % CGMP, DM2: 100 % AA (=DM1), DM3: 78.4 % CGMP + 19.6 % AA, DM4: 100 % AA (=DM3). The AA profiles were measured at 0 (fasting), 15, 30, 60, 120 and 240 min after the meal. Results: There was no significant difference between the DM when comparing AA profiles at fixed time intervals (results presented as Mean +/− SD): Time 0 (fasting): DM1: (809 + 431 μmol/l), DM2: (827 + 268 μmol/l), DM3: (410 + 430 μmol/l), DM4: (334 + 279 μmol/l). Time 15: DM1: (762 + 368 μmol/l), DM2: (872 + 343 μmol/l), DM3: (375 + 394 μmol/l), DM4: (334 + 279 μmol/l). Time 30: DM1: (819 + 393 μmol/l), DM2: (786 + 248 μmol/l), DM3: (377 + 396 μmol/l), DM4: (350 + 293 μmol/l). Time 60: DM1: (801 + 441 μmol/l), DM2: (790 + 280 μmol/l), DM3: (363 + 381 μmol/l), DM4: (334 + 279 μmol/l). Time 120: DM1: (757 + 386 μmol/l), DM2: (797 + 316 μmol/l), DM3: (406 + 426 μmol/l), DM4: (332 + 277 μmol/l). Time 240: DM1: (790 + 368 μmol/l), DM2: (856 + 289 μmol/l), DM3: (393 + 412 μmol/l), DM4: (326 + 272 μmol/l). Discussion: Absorption differences were non-significant (p > 0.05). A small amount of phe in CGMP (DM4) did not influence the blood phe significantly compared to DM3 (p > 0.05). Conflict of Interest declared.

P-132 Phenylketonuria intestinal microbiota: insights from Pahenu2 mice fed amino acid, glycomacropeptide and casein diets Ney D M 1, Sawin E A 1, Stroup B M 1, Murali S G 1 1

Dept Nutri Sci, Univ WI, Madison, United States

Background: Glycomacropeptide (GMP) is a glycosylated peptide isolated from cheese whey and made into low-phe foods for PKU. GMP is a prebiotic defined as a fermentable ingredient that stimulates a beneficial intestinal microbiota. Objective: To determine the impact of GMP, amino acid (AA) or casein diets on the cecal intestinal microbiota, short chain fatty acids (SCFA), immune responses, and the hepatic metabolomics profile. Methods: Weanling PKU (Pahenu2) and WT mice were fed isoenergetic AA, GMP or casein diets between 3 and 23 wks of age. The cecum content was collected for microbial DNA extraction to perform 16S microbiota analysis by Ion Torrent PGM sequencing and to measure SCFA by gas chromatography. Splenocyte T cell populations were assessed using flow cytometry. Global metabolic profiles were determined in liver using the Metabolon platform. Results: The GMP diet resulted in a reduction from 30 to 35 % to 7 % in Proteobacteria, genera Desulfovibrio, in both WT and PKU mice (p < 0.002) with genotype dependent changes in Bacteroidetes or Firmicutes. Cecal concentrations of the SCFA acetate, propionate and butyrate increased with GMP. Spleen mass was significantly larger in PKU mice compared with WT mice and significantly smaller in mice fed GMP. The percent of stimulated spleen cells producing interferon-gamma was significantly reduced in mice fed GMP. PKU mice displayed altered hepatic levels of metabolites derived from phe and tyrosine and known to be microbial origin. Discussion: The PKU genotype alters the intestinal microbiota. The prebiotic benefits of GMP include reduction in sulfate-reducing bacteria and increased SCFA known to improve intestinal barrier function and reduce inflammation. The prebiotic effects of GMP may explain the improved gastrointestinal symptoms reported by PKU subjects eating GMP medical foods compared to AA formula. Supported by USDA Hatch grant WIS01790 and NIH T32 DK007665. Conflict of Interest declared.

P-133

Neuropsychiatric comorbidities and concomitant medications in individuals with phenylketonuria: findings from the PKUDOS registry Waisbren S 2, Zambrano J 1, Grant M 3, Cheng B 1, Parker S 1, White D 4, Cohen-Pfeffer J L 1 1 BioMarin Pharmaceutical Inc, Novato, United States, 2Boston Child Hosp, Boston, United States, 3Drexel Univ, Coll Medicine, Philadelphia, United States, 4Washington Univ, St Louis, United States

Background: Phenylketonuria (PKU) is characterized by a defect in the metabolism of phenylalanine (Phe) that can lead to Phe accumulation and neuropsychiatric dysfunction. We report the neuropsychiatric comorbidities and medication use in individuals with PKU enrolled in an industry-sponsored registry. Methods: Data were extracted from the Phenylketonuria Demographics Outcomes and Safety (PKUDOS) registry for individuals with PKU who are (or have been) prescribed sapropterin dihydrochloride. Results: Of 1447 individuals with PKU enrolled in the registry, 25.7 % were noted to use at least one neuropsychiatric medication. A total of 763 individuals had information pertaining to anxiety. Of these, 193 individuals had symptoms of anxiety (60.6 % mild, 35.8 % moderate, and 3.6 % severe), and 47 (24.4 %) were noted to be taking anxiety medication at baseline or during the first year in the registry. Of the 695 individuals with data pertaining to attention-deficit/hyperactivity

S96 disorder (ADHD), 170 were reported to have symptoms of ADHD (39.4 % mild, 53.5 % moderate, and 7.1 % severe), and 85 (50.0 %) were noted to be taking ADHD medication at baseline or during the first year in the registry. Of the 752 individuals with data pertaining to depression, 133 were noted to have symptoms of depression (57.9 % mild, 36.1 % moderate, and 6.0 % severe), and 48 (36.0 %) were noted to be taking anti-depressant medication at baseline or during the first year in the registry. Discussion: Data from the PKUDOS registry suggest that individuals with PKU have neuropsychiatric comorbidities and often take adjuvant treatment. These data support assessment of neurocognitive and psychiatric status as part of routine PKU management. Conflict of Interest declared.

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Pro-oxidant and pro-inflammatory states in phenylketonuric treated patients Deon M 1 3, Sitta A 1, Faverzani J L 1, Guerreiro G B 1 3, Mescka C P 1 3, Coelho D M 1, Coitinho A 4, Wajner M 1 2, Giugliani R 1 2, Vargas C R 1 2 3 1 Medical Genetics Service, HCPA, Porto Alegre, Brazil, 2 Grad Prog Biochemistry PPGBioq, UFRGS, Porto Alegre, Brazil, 3Grad Prog Pharmac Sciences PPGCF, UFRGS, Porto Alegre, Brazil, 4Grad Prog Physiology PPGFisio, UFRGS, Porto Alegre, Brazil

Background: Phenylketonuria (PKU) has been associated with oxidative stress. Objectives: Considering that there are few studies focused on oxidative stress and inflammation directly in PKU disease, the aim of this study was to evaluate and correlate oxidative damage to biomolecules, pro-inflammatory cytokines, phenylalanine (Phe) and its metabolites (phenyllactic acid [PLA] and phenylacetic acid [PAA]) levels in urine and plasma from patients with PKU under dietary treatment. Methods: Random urine samples and plasma samples of 10 treated PKU patients and 10 healthy control subjects were analyzed. The parameters evaluated were: determination of urinary 15-F2t-isoprostane (isoprostane) and di-tyrosine (Di-Tyr), measurement of plasma interleukins 6 (Il-6) and 1β (Il-1β), and determination of urinary Phe levels and its metabolites (PLA and PAA). Results and Discussion: The levels of isoprostane, a biomarker of lipid oxidative damage, and the levels of Di-Tyr, a biomarker of protein oxidative damages, were significantly increased in patients with PKU under dietary treatment when compared to the control group. Our findings concerning the inflammatory cytokines Il-6 and Il-1β, both significantly increased in these patients, provide evidence that the proinflammatory state occurs. Furthermore, Il-1β was positively correlated with isoprostane. Di-tyr was positively correlated with Phe, as well as with PAA. These findings may suggest that the protein damage may be induced by Phe and its metabolite PAA in PKU. Moreover, our results indicate that pro-oxidant and pro-inflammatory states occur and are, in part, correlated in PKU treated patients. Financial support: CAPES, CNPq, FIPE/HCPA.

P-135

Study of group-I metabotropic receptors in a mouse model of phenylketonuria (PKU) Nardecchia F 1 2 3, Orlando R 2, Iacovelli L 2, Colamartino M 3 5, Fiori E 5, Leuzzi V 1, Piccinin S 6, Nistico’ R 6 7, Puglisi-Allegra S 3 5, Nicoletti F 2 4, Pascucci T 3 5 Dept of Pediatr and Child Neuropsychiatry, Sapienza Rome, Italy, 2Dept of Physiol and Pharmacol, Sapienza Rome, Italy, 3Dept of Psychology, Sapienza 1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Rome, Italy, 4IRCCS Neuromed, Pozzilli (IS), Italy, 5IRCCS Fondazione Santa Lucia, Rome, Italy, 6European Brain Research Institute, Rome, Italy, 7 University of Rome ‘Tor Vergata’, Rome, Italy Background: BTBR-Pah(enu2) mice represent a genetic model of PKU, which recapitulates the cognitive deficit associated with PKU in humans. Group-I metabotropic glutamate (mGlu1 and mGlu5) receptors have been implicated in the pathophysiology of neurodevelopmental disorders characterized by intellectual disability (ID) and autism. For example, the evidence by Mark Bear and Kimberly Huber that mGlu5 receptor-dependent hippocampal long-term depression is amplified in fmr1 knockout mice paved the way to the clinical development of mGlu5 receptor antagonists for the treatment of Fragile X syndrome. We decided to examine the expression of mGlu1 and mGlu5 receptors and the scaffolding protein, Homer, in different brain regions of PKU mice. Methods: Expression of Group-I mGlu receptors and Homer proteins was evaluated in different brain regions of male adult homozygous BTBRPah(enu2) and their wt counterparts by means of WB and rtPCR. Results: No significant variations of mGlu1 receptor levels were found in any brain region examined. In contrast, mGlu5 receptor protein levels were significantly increased in the hippocampus and striatum of PKU mice, as compared to wt mice. This increase was apparently caused by post-transcriptional modifications because no changes were detected in mRNA levels. Interestingly, levels of the long isoforms of the Homer protein, which link mGlu1 and mGlu5 receptors to intracellular signalling proteins, were reduced in the hippocampus of PKU mice. No changes were found in the levels of the short Homer isoform, Homer1a. Discussion: Our data suggest that changes in mGlu5 receptor expression and their scaffolding protein might be linked to ID associated with PKU. These data lay the groundwork for a functional analysis of mGlu5 receptors in PKU mice, and raise the possibility that drugs that either activate or inhibit mGlu5 receptors influence synaptic dysfunction and behavioural abnormalities in PKU mice.

P-136

Phenylalanine hydroxylase genotype phenotype association in the United States: a single center study Rajabi F 1, Rohr F 1, Wessel A 1, Martell L 1, Levy H L 1 2 Div Genetics Genomics, Boston Child Hosp, Boston, United States, 2Dept Pediatr, Harvard Med Sch, Boston, United States 1

Background: Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene. The correlation between genotype and clinical phenotype can be complex and variable, but very useful for accurately categorizing and prognosticating dietary tolerance and potential outcome. Methods: We reviewed medical records for 514 patients diagnosed with PKU or mild hyperphenylalanemia for PAH genotype, phenylalanine levels (at newborn screening, confirmation, and highest known), dietary phenylalanine tolerance, and sapropterin responsiveness. These parameters were used to assign classification into classical, moderate, mild, or non-PKU hyperphenylalanemia phenotypes. Results: Of the 514 patients reviewed, 373 had undergone PAH genotyping. 117 different mutations were identified on 724 PAH alleles comprising 337 (90.3 %) compound heterozygous cases, 17 (4.6 %) homozygous cases, 16 (4.3 %) heterozygous cases, and 3 (0.1 %) cases with no mutations found after genetic testing. The most frequent mutation R408W was present in 15.7 % of the 724 alleles; other frequent mutations were I65T (6.5 %), IVS12 + 1G > A (5.9 %), and R261Q (5.7 %). Previously unreported mutations include L48V, E205D, and c.1342_1345delCTCC. The L48V allele was heterozygous with D59V, and the E205D allele was heterozygous with A403V, both with a mild PKU phenotype. The c.1342_1345delCTCC allele was heterozygous with E280K in a moderate PKU phenotype. Discussion: This is the first large single center report of PAH mutations in the United States and can be compared with similar European multicenter reports. The mutation spectrum in the United States is understandably similar to the

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 European spectrum and reflects the known regional variability of metabolic phenotypes in the United States.

P-137

Molecular study of the PAH gene in PKU patients of the Canary Islands Valerio-Hernandez E 1, Ruiz Pons M 3, Murray-Hurtado M 2, Pena-Quintana L 4 , Santana-Rodriguez A 4, Salido-Ruiz E 2 1

Servicio Canario de Salud, Santa Cruz de Tenerife, Spain, 2Hosp Univ de Canarias, La Laguna (Santa Cruz de Tenerife), Spain, 3Hosp Univ Nuestra Sra de la Candelaria, Santa Cruz de Tenerife, Spain, 4Hosp Univ Insular MaternoInfantil, Las Palmas de Gran Canaria, Spain Background: Molecular study of the PAH gene in PKU patients (phenylketonuria, OMIM # 261600) and their mutational frequencies at global level, is contributing to a better understanding of PKU, improving important aspects such as prediction of the genotype-phenotype relationship or the therapeutic approach (BH4). We present the mutational spectrum (PAH gene) of the PKU patients in the Canary Islands and their phenotype-genotype relationship. Methods: A molecular study of the PAH gene was performed in 40 out of the 45 PKU patients monitored in the three reference hospitals (5 already had the mutational study performed in an external laboratory), with newly designed primers for the 13 exons. Results: A total of 45 PKU patients (42 families, 84 mutated alleles) were analyzed showing an incidence of 1:10,222. 24 different mutations, and a frequency of 66.6 % of compound heterozygotes were found. Mutation frequencies were: p.R408W (19 %); p.R68S (11.9 %); p.A403V (10.7 %); p.R176L (9.5 %); IVS10-11G > A (8.3 %). The remaining mutations showed frequencies < 5 %. For clinical phenotype (Phe levels at birth), benign forms were the most common (56.8 %). From the patients susceptible to BH4 treatment regarding their phenotype, 7 of them showed a sentitive genotype according to databases, 4 being currently under treatment. Discussion: PKU patients from the Canary Islands showed a high frequency of benign forms (Phe < 6 mg / dL), just like previous studies on the Iberian Peninsula. However, mutations and frequencies found are very different, with a dominant mutation (p.R408W) that is almost anecdotal in the Mediterranean region. We also revealed a new mutation (p.P409L) not described in the currently available bibliography. Nowadays, genetic analysis on PKU patients is mandatory, providing a good genetic counseling for patients, information about the use of other therapeutic tools and increase the current databases.

P-138

The first case of phenylketonuria with tyrosinemia type III Coskun T 1, Bilginer Gurbuz B 1, Pektas E 1, Yildiz Y 1, Dursun A 1, Sivri H S 1 , Tokatli A 1 1

Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey

Background: Phenylketonuria (PKU) is an autosomal recessive metabolic disease caused by either phenylalanine hydroxylase (PAH) or its cofactor BH4. PKU incidence varies widely all over the world. Turkey has the highest incidence of PKU (1:4000) due to high rate of consanguineous marriages. The high rate of consanguinity plays a role in the high frequency of other autosomal recessive diseases like tyrosinemia. Tyrosinemia type III is caused by deficiency of 4hydroxyphenylpyruvate dioxygenase (4-HPPD), an enzyme involved

in the catabolic pathway of tyrosine. We described the first case of PKU with tyrosinemia type III. Case Report: A 6-year-old boy was detected on routine newborn PKU screening (4.3 mg/dl). Parents were first cousins. He was born at term with caesarian section because of breech presentation weighing 3500 g. Serum phenylalanine and tyrosine levels were 41.5 mg/dl and 11.44 mg/dl, respectively at the time of diagnosis. A phenylalanine-restricted diet was started. At 43 days of life phenylalanine and tyrosine levels were 10.3 mg/dl, and 8.57 mg/dl, respectively. While the patient was off diet for 2 days, serum levels of phenylalanine, tyrosine and alpha-fetoprotein were found 0.4 mg/dl, 14.29 mg/dl, and 393 IU/ml, respectively. Urine organic analysis was normal. The patient was considered to have tyrosinemia in addition to PKU. Results: Genetic studies showed that the patient had previously described homozygous IVS4 + 5G > T splice mutation in PAH gene and novel homozygous p.G53S (c.157G > A) mutation in HPD gene. In silico analysis demonstrated this novel PAH gene mutation was the cause of the disease. Discussion: Such an association, described for the first time, indicates that the detection of one genetic disease does not exclude the possibility of presence of another one in the same patient in countries where close relative marriages are prevalent.

P-139

Characterization of synaptic molecular components involved in cortical E/I imbalance in ENU2 mice De Jaco A 4, Mango D 1, De Angelis F 4, Favaloro F L 4, Andolina D Colamartino M 3 5, Nistico’ R 2, Puglisi-Allegra S 3 5, Pascucci T 3 5

3 5

,

1 EBRI-European Brain Research Institute, Rome, Italy, 2Dept of Biology, Univ Tor Vergata, Rome, Italy, 3Dept of Psycho, Sapienza Univ, Rome, Italy, 4Dept of Bio and Biot Sapienza Univ, Rome, Italy, 5Santa Lucia Foundation, Rome, Italy

Background: Phenylketonuria (PKU, McKusick 261600) is the most common genetic metabolic disease, characterized by high levels of PHE in the plasma and variable neurological and mental impairments. The animal model of PKU, the PAH Enu2 mice, allowed studying mechanisms involved in the onset of cognitive delay. We have previously showed lower enzymatic activity of the enzyme responsible for serotonin (5-HT) biosynthesis and a reduction in dendritic spine densities in the brain of ENU2 mice. Here we investigated alterations at the functional and molecular levels, in the prefrontal cortex of ENU2 mice. Methods: We used ENU2 mice that represents a valid tool to identify molecular mechanisms underlying MR in PKU. Real time rt-pcr and western blot analysis from prefrontal cortex of ENU2 and control mice measured the expression of synaptic molecular components. Electrophysiological recordings (whole-cell patch clamp) analysed inhibitory and excitatory synaptic transmission from adult ENU2 and control mice. Results: Up-regulation of the messenger for the serotonin receptors 5HT2A was accompanied by lower protein levels in ENU2 in comparison to wild type (WT) mice. Neuroligin proteins were regulated in ENU2 compared to WT mice with higher messenger levels for NLGN1 (excitatory) and lower for NLGN2 (inhibitory). Analysis of the levels of each family member revealed higher NLGN1 and NLGN2 levels in ENU2 compared to WT mice. Functional data showed higher frequency of inhibitory transmission in ENU2 mice compared to WT and significant reduced frequency of excitatory transmission in ENU2 mice compared to WT. Moreover, E/I ratio was significantly reduced in ENU2 mice compared to WT. Discussion: Our data point toward an imbalance in the cortical E/I neurotransmission in prefrontal favouring inhibition. We believe that this inhibitory shift might be strictly linked to the synaptic immaturity observed in the PKU mice.

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Challenging yourself—the benefit of an outdoor activity experience for young people with phenylketonuria Cochrane B 1, Adams S 2, Wildgoose J 3 1 Royal Hosp for Child, Glasgow, United Kingdom, 2Royal Victoria Inf, Newcastle, United Kingdom, 3Bradford Teaching Hospitals NHS Trust, Bradford, United Kingdom

Background: Phenylketonuria (PKU) is an inherited metabolic disorder managed by a socially isolating and semi-artificial diet. Following a restrictive diet can lead to feelings of social isolation. Encouraging young people to develop skills to enable them to become confident adults is an important part of their management. To do so in a safe, controlled, fun environment will help them engage with others with PKU, become aware of differences in dietary management, try new foods, increase self-confidence, develop resilience and challenge themselves. They can then use this experience to help them mature and develop social and emotional awareness. A weekend in an outdoor centre in the Lake District with the ethos of giving young people skills to develop themselves, has been an activity offered to young people with PKU attending centres in Scotland and the North of England. Methods: To evaluate the value placed on the weekend by the families, a questionnaire with 11 open ended and fixed questions was sent to parents of the young people who attended. Results: Questionnaires were returned for 15 out of 27 (55.5 %) of the young people aged 9–18 years (mean 12.7 years) who were in the 2015 group with a mean attendance of 3.3 years (0–7 years). When asked why they wanted to go away with the group, the reasons given included: being with others who had same condition and they enjoyed the activities. Parent’s reasons included gaining independence and meeting with others with PKU. One parent’s comment was “We knew that it would help the girls accept and embrace their PKU”. Discussion: Many of the young people ask to return for the weekend in the following years and look forward to meeting up with the friends they have made. The young persons are reported by parents as being more independent following the weekend away, which in turn helps them with the choices in managing their condition and will stand them in good stead as they move into adolescence. Conflict of Interest declared.

P-141

Neurological and psychiatric disorders in a French cohort of adults with phenylketonuria Maillot F 1, Hassen-Khoja C 2, Charriere S 14, Feillet F 5, Astudillo L 6, Lavigne C 7, Arnoux J B 8, Odent S 9, Gay C 10, Schiff M 11, Thauvin-Robinet C 12, Mazodier K 13, Kuster A 15, Rigalleau V 16, Levesque H 17, De Parscau L 19, Besson G 18, Fouilhoux A 3, Gissot V 2, Caille A 2, Douillard C 4, Douillard C 4 1 Int Med Dept, Univ F Rabelais, Tours, France, 2CIC, Univ F Rabelais, Tours, France, 3Div Metab Dis, Univ Hosp, Lyon, France, 4Div Metab Dis, Univ Hosp, Lille, France, 5Div Metab Dis, Univ Hosp, Nancy, France, 6Int Med Dept, Univ Hosp Purpan, Toulouse, France, 7Int Med Dept, Univ Hosp, Angers, France, 8Div Metab Dis, Univ Hosp Necker, Paris, France, 9Div Genet, Univ Hosp, Rennes, France, 10DiV Pediatr, Univ Hosp, SaintEtienne, France, 11Div Metab Dis, Univ Hosp R Debre, Paris, France, 12Div Genet, Univ Hosp, Dijon, France, 13Int Med Dept, Univ Hosp, Marseille, France, 14Div Endoc, Univ Hosp, Lyon, France, 15Div Pediatr, Univ Hosp, Nantes, France, 16Div Endoc, Univ Hosp, Bordeaux, France, 17Int Med Dept, Univ Hosp, Rouen, France, 18Div Neurol, Univ Hosp, Grenoble, France, 19Div Pediatr, Univ Hosp, Brest, France

Background: Long term neurological and psychiatric manifestations of phenylketonuria (PKU) have been partially described. The aim of the study was to evaluate the frequency of neuropsychiatric disorders in adults with PKU.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Methods: Data were collected from a French prospective cohort of adults with PKU (ECOPHEN study). Neuropsychiatric outcomes and corresponding ICD10 codes were described. The Chi square test was used to compare the frequency of psychiatric disorders between classic and mild PKU. Results: 187 patients were included (127 w, 60 m, 27.6 ± 6.3y). Number of patients with classic PKU, mild PKU and hyperphenylalaninemia (HPA) were, respectively, 152, 26 and 9. 20 patients (10.7 %) had a history of neurological disorders, only in the classic PKU group. Main neurological features were migraines/headaches, tremors, myoclonus, and balance disorder. Corresponding ICD-10 codes were extrapyramidal and movement disorder, migraine, headache, other visual disturbances, and myoclonus. 48 patients (25.7 %) had a history of psychiatric disorders: 40 (26.3 %) with classic PKU, 6 (23.1 %) with mild PKU, and 2 (22.2 %) with HPA. Psychiatric features were depression, anxiety, anxio-depressive disorder, bulimia, and sleep disorders. ICD-10 codes were major depressive disorder, brief psychotic disorder, phobic anxiety disorder, eating disorders, bulimia nervosa and hypersomnia. There was no significant difference in percentages of patients suffering from psychiatric disorders between the three groups. Discussion: This study shows that neuropsychiatric manifestations are not uncommon in PKU. As expected, neurological complications were only described in individuals with classic PKU. Such findings suggest that a neurological examination should be performed on a regular basis during the long-term follow-up of these patients. Frequency of psychiatric disorders, although it was lower in our cohort as compared to previous studies, shows that mental healthcare support may be needed in adults with PKU.

P-142

Novel biomarkers to monitor trace element status in children with phenylketonuria Cochrane B 1, Armstrong J 2, Stefanowicz F 3, Duncan A 3, Talwar D 3, Robinson P 1, Gerasimidis K 2 1 Royal Hosp for Child, Glasgow, United Kingdom, 2Univ of Glasgow, Glasgow, United Kingdom, 3Royal Inf, Glasgow, United Kingdom

Background: Phenylketonuria (PKU) is a disorder of protein metabolism, requiring a lifelong low phenylalanine diet, complemented by a phenylalaninefree amino acid supplement. This very restricted, semi-artificial diet can give rise to micronutrient deficiencies. Clinical management guidelines of these patients advise an annual nutritional blood screen. Previous studies have found low levels of serum ferritin, selenium and zinc. In addition, standard screening utilises biomarkers in plasma, which are likely to be affected by recent intake, and in some cases by systemic inflammation. Erythrocyte biomarkers are more likely to be reflective of long term stores and are unaffected by inflammatory processes. Methods: Patients aged from 2.7 to 17.6 years (mean 7.9, median 5.9) attending the Royal Hospital for Children in Glasgow, UK, were recruited from outpatient clinics. Plasma micronutrient measurements were obtained for 46 patients, and 25 erythrocyte samples were analysed for copper, selenium and zinc at the Scottish Trace Element and Micronutrient Research and Diagnostic Laboratory. These were compared using z-scores derived from a cohort of healthy children from the same area. Results: The children with PKU exhibited significantly lower z-scores for plasma zinc (p = 0.001), while the z-scores for erythrocyte zinc were significantly higher (p < 0.001) than for the reference cohort. Plasma copper z-scores showed no significant difference (p = 0.191), nor did erythrocyte copper z-scores (p = 0.475). Plasma selenium z-scores in this group were no different than the mean (p = 0.856), but erythrocyte selenium was significantly lower (p < 0.001). Discussion: The discrepancy between the plasma and erythrocyte measures for zinc and selenium highlights the limitations of using plasma biomarkers to assess long term trace element status. Use of erythrocyte biomarkers may identify deficiencies or excesses not seen in plasma. Conflict of Interest declared.

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Is the gut microbiota associated with clinical phenotype in phenylketonuria patients? Oliveira F P 1 3, Mendes R H 1 3, Dobbler P T 4, Dias P D C 1 3, Vairo F P 2, Refosco L F 2, Roesch L F W 4, Schwartz I V D 1 2 3 Federal University of Rio Grande do Sul, Porto Alegre, Brazil, 2Med Gen Serv, Clin Hosp Porto Alegre, Porto Alegre, Brazil, 3Bas Res Adv Inv Neu, Clin Hosp Por Ale, Porto Alegre, Brazil, 4Federal University of Pampa, Sao Gabriel, Brazil

which has not been found yet in oriental populations, was frequently found in this study. We also observed that BH4 responsiveness was associated with specific genotypes (R53H, R241C, R408Q and T278I), suggesting there are some correlations between phenotype and genotype. 6Pyruvoyltetrahydropterin synthase (PTPS) deficiency was associated with genotypes (c.272A > G, c.347A > G, c.259C > T, c.616A > G, c.155A > G). DISCUSSION: In our study, classic PKU (n = 160), BH4 responsive PKU (n = 28), and benign hyperphenylalaninemia (n = 10) have been characterised in Korea.

1

Background: Phenylketonuria (PKU) is an inborn error of metabolism characterized by high blood phenylalanine (Phe) and wide clinical heterogeneity with patients classified in classical or mild PKU according to Phe levels or Phe intake tolerance. Human gut harbors 1013–1014 microbial cells whose compositional/functional features have shown significant differences in many diseases. Aim: to compare the gut microbiota of PKU patients with different PKU types and blood Phe levels. Methods: Stool samples were collected from 22 PKU patients on treatment (median age: 7.08y IQ: 3.14–15.25; median Phe blood level: 404.14 μmol/L IQ: 288.43–573.05; mean Phe intake: 484.2 ± 275.4 mg/day). Bacterial DNA was extracted and 16S rRNA gene was sequenced by PGM Ion TorrentTM. Microbiota analysis was performed with the STAMP software. Results: Mild PKU patients (340.31 μmol/L IQ: 248.06–484.41; Phe intake: 539.2 ± 298 mg/day) showed greater abundance of Lachnospiraceae family (p < 0.05) than classical PKU patients (578.9 μmol/L IQ: 412.6–808.8; Phe intake: 410 ± 236.5 mg/day). PKU patients with blood Phe level < 360 μmol/L (284.35 μmol/L IQ: 166.1–290.1; Phe intake: 388.5 ± 309.3 mg/day) showed decrease of Rikenellaceae, Ruminococcaceae and increased of Lachnospiraceae family (p < 0.05) compared to patients with blood Phe level >360 μmol/L (485.65 μmol/L IQ: 416.24–642.81; 543 ± 68.3 mg/day). Discussion: The Rikenellaceae and Ruminococcaceae decrease found in patients with blood Phe level < 360 μmol/L is associated with Crohn’s disease and non-alcoholic fatty liver disease, respectively, but our patients didn’t show any of these conditions and moreover increase of Lachnospiraceae is a marker for healthy gut microbiota and negatively associated with inflammatory bowel disease. Our results suggest that treated PKU patients differ in few bacterial groups in gut microbiota and healthy gut microbiota may be associated with less severe phenotypes in PKU patients.

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Molecular characterisation of hyperphenylalaninemia in Korea

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Psychosocial adjustment of children and adolescents with phenylketonuria in Korea Lee J 1, Lee D H 1 1

Soonchunhyang University Hospital, Seoul, Republic of Korea

Background: Phenylketonuria (PKU) is one of the most common inherited metabolic disorders. Even with the advent of early and aggressive onset of treatment, several studies have reported an increased prevalence of psychiatric and behavioral symptoms in adults. Methods: The Korean version of the Child Behavior Checklist (K-CBCL) was used to assess the psychopathology of 50 children with PKU and 50 healthy comparisons. Recent and mean phenylalanine levels and demographic information were collected through a retrospective chart review. Results: Compared with healthy comparisons, children with PKU showed significant differences in social, school and aggressive behavior, externalizing problem and total behavior scores in the K-CBCL. Significant differences in the total competence scale (p < 0.001), total behavior scale (p = 0.004), attention (p = 0.031) and emotional lability (p = 0.031) were found between groups in clinical and nonclinical spectrum. As the age of diagnosis progressed, higher significance of score for anxiety, depression, thought problem, attention, emotional lability was observed in the K-CBCL. In addition, internalizing problem (p = 0.025), thought problem (p = 0.016), delinquent behavior (p = 0.006) were correlated with recent and mean phenylalanine levels in subjects with PKU. Internalizing problem (p = 0.004), attention (p = 0.008) and social problem (p = 0.016) were related with variability of blood phenylalanine levels. Discussion: Children with PKU may experience various and serious psychosocial problems compared to healthy children. Recent and mean phenylalanine levels can be the risk factors of psychiatric manifestations in PKU patients. Variability of phenylalanine levels is related to internalizing, attention and social problems. Treatment strategies that enhance stability of the blood phenylalanine levels should seriously be considered.

Lee D H 1 1

Soonchunhyang University Hospital, Seoul, Republic of Korea

Background: Hyperphenylalaninemia (HPA) is a term referring to the condition of accumulation of phenylalanine in body fluid and tissue, due to defect of phenylalanine conversion to tyrosine. A major cause is the lack of phenylalanine hydroxylase (PAH), or its cofactor, tetrahydrobiopterin (BH4). Methods: Clinical, biochemical, and genetic analyses were done retrospectively from January 1999 to July 2015 in Soonchunhyang University Hospital. Results: 195 patients were confirmed as affected by classic PKU (n = 160), BH4 responsive PKU (n = 28), benign hyperphenylalaninemia (n = 10), PTPS deficiency (n = 11) and DHPR deficiency (n = 1). Several mutations reoccurred with high frequency including R243Q (18 %), Y204S (12 %), IVS4-1G > A (7.8 %), Y356X A (7.8 %), R241C (7.8 %), A259T (6.1 %), T278L (6.5 %) from 256 independent alleles. Although some common characteristics of allele frequency and distribution were identified among oriental populations, several distinctive characteristics were revealed in Korean patients. Although the R413P allele is the most prevalent form (30.5 %) in Japanese patients, we detected it in only six alleles (2.3 %). The A259T allele,

P-146

Oxidative status and serum paraoxonase-1 activity in phenylketonuric patients Kumru B 1 2, Bagci C 2, Hismi B 3, Kaplan D S 2 1 Div Nutrition and Diet, Child Hosp, Gaziantep, Turkey, 2Dept Physiol, Health Sci, Gaziantep Univ, Gaziantep, Turkey, 3Div Metab Dis, Tepecik Edu Res Hosp, Izmir, Turkey

Background: Paraoxonase-1 (PON1), is an important antioxidant enzyme against oxidative stress. PON1 protects lipids in HDL and LDL against oxidation. The PKU diet therapy can be considered as vegetarian-like. Also, this vegetarian-like diet can be defined as ‘non-atherogenic’ because of the reductions of animal lipids and cholesterol and the increased intake of dietary fiber. To our knowledge, one study has evaluated PON1 activity in PKU. The authors suggest that future PKU oxidative stress research should include PONs

S100 activity. In this work, we aimed to compare the influence of blood Phe levels on oxidative status and PON1 activity in two groups of treated PKU patients, one with well controlled and the other one with high Phe blood levels. Methods: 20 classical PKU patients were classified into two groups according to their mean Phe levels. Group A consisted of 10 patients (mean age 4.2 ± 2.8 year, mean Phe levels 5.0 ± 2.6 mg/dl), with good dietary adherence. Group B included 10 patients (mean age 5.4 ± 2.4, mean Phe levels 13.8 ± 2.6 mg/dl), with poor dietary adherence. Control group consisted of 10 healthy children (mean age 5.8 ± 3.2). We evaluated various parameters including total antioxidant status (TAS), total oxidant status (TOS), PON1, HDL, LDL, and total cholesterol (t-Chol) in serum of PKU patients and controls. Results: No significant differences between patients and controls were observed in the levels of TAS, TOS, PON1, HDL, LDL, t-Chol. Discussion: Since PKU patients receive amino acid mixture enriched in antioxidant vitamins, minerals and trace elements, there is no significant difference in terms of TAS and TOS levels. There is no relationship between antioxidantoxidant status and Phe plasma levels in PKU. Besides this, PKU patient diet records showed no intake of animal proteins in all patient groups. Unless patients eat animal protein, PON1 and lipid profile levels remain unchanged. Therefore PKU patients diet record should be monitored closely.

P-147

Psychological well-being of early and continuously treated phenylketonuria patients Spiess N 1, Thiele A G 1, Rohde C 1, Arelin M 1, Ceglarek U 2, Kiess W 1, Beblo S 1 1 Div Metab Dis, Univ Child Hosp, Univ Hosp, Leipzig, Germany, 2Inst Lab Med, Clin Chem, Mol Diag, Leipzig, Germany

Background: Patients with phenylketonuria (PKU) depend on a lifelong, Pherestricted diet. Former studies showed heterogeneous impact of insufficient metabolic control on psychological wellbeing. We performed a crosssectional study to assess emotional functioning and psychological distress of children and adolescents with PKU. Methods: Behavioral attributes were evaluated by the Strengths and Difficulties Questionnaire (SDQ; 5 scales: hyperactivity, emotional symptoms, conduct problems, peer problems, prosocial behavior; two versions: selfassessment for ages 11–17y; parent-reported for age 4–17y) in relation to metabolic control. Inclusion was restricted to early and continuously treated PKU patients without concomitant diseases. Data were compared to reference values. Results: A total of 34 patients (17f/17 m, aged 4 to 17 years) with classical PKU (n = 15), hyperphenylalaninaemia (HPA, n = 7) and tetrahydrobiopterin (BH4)responsive PKU (n = 12) participated. The entire cohort revealed a median total SDQ score of 8 (range 2–25) by self-assessment (normal: 0–15) and of 7 (range 1–17) by parent-assessment (normal: 0–13). Four female patients (12 %) graded borderline or abnormal regarding the total score. Values beyond the norm in emotional symptoms scores were found in six female patients (18 %). Five patients, aged 11 to 17 years, scored above the reference range on the subscale “prosocial behavior”. Mean of median dried blood phenylalanine concentration in all patients was 309 μmol/l (SD: 163). No relation between insufficient metabolic control and higher SDQ values were detected. Discussion: The preliminary analysis revealed a low percentage of psychological difficulties in this patient cohort. Interestingly, parents judged their children’s behavioral attributes better in comparison to self-reports. This indicates that parents might underestimate the burden of the disease for their child.

P-148

Executive functioning in adults with phenylketonuria: associations with plasma phenylalanine levels, plasma homovanillic acid, and striatal dopamine receptor density Huijbregts S 1, Boot E 2, Jahja R 3, Hollak C 4, Bosch A 5, Van Amelsfoort T 6, De Sonneville L 1, Nederveen A 7, Van der Meere J 8, Bakermans A 7, Nieman D

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 10

, Rubio-Gozalbo E 9, Brouwers M 9, Hofstede F 11, De Vries M 12, Janssen M , Van der Ploeg A 14, Langendonk J 15, Bour L 13, Van Spronsen F 3, Booij J 2

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1 Clin Child Adolesc Studies, Leiden Univ, Leiden, Netherlands, 2Nucl Med, Amsterdam Medical Center (AMC), Amsterdam, Netherlands, 3Metab Dis, Beatrix Children’s Hosp, UMCG, Groningen, Netherlands, 4Internal Med, AMC, Amsterdam, Netherlands, 5 Pediatrics, AMC, Amsterdam, Netherlands, 6Psychiatry and Psychology, UMCM, Maastricht, Netherlands, 7 Radiology, AMC, Amsterdam, Netherlands, 8Dev Clin Neuropsychol, Groningen Univ, Groningen, Netherlands, 9 Gen Metab Dis, UMCM, Maastricht, Netherlands, 10Psychiatry, AMC, Amsterdam, Netherlands, 11 Metab Dis, Wilhelmina Children’s Hosp, Utrecht, Netherlands, 12Metab Dis, UMC St Radboud, Nijmegen, Netherlands, 13 Neurology, AMC, Amsterdam, Netherlands, 14Lysosomal and Metab Dis, Erasmus Med Ctr, Rotterdam, Netherlands, 15Internal Med, Erasmus Med Ctr, Rotterdam, Netherlands

Background: The behavioral phenotype of children with early-treated phenylketonuria (PKU) is characterized by deficits in executive functioning (inhibitory control, working memory, cognitive flexibility). This study investigated whether a similar cognitive profile could be observed in adults with PKU, despite early diagnosis and continuous treatment. It was also examined to what extent cerebral monoaminergic deficits were related to impairments in executive functioning in adults with PKU. Methods: 57 PKU patients (mean age 27.7 years ± 6.0, range 18.7–40.0 years) and 57 healthy controls (HC, mean age 25.7 ± 5.6, 18.1–40.8 years) performed computerized executive function (EF) tasks. Historical pPhe-levels were collected and blood samples were taken on the day of testing to determine current plasma homovanillic acid (pHVA) and pPhe-levels. In vivo striatal D2/3 receptor (D2/3R) availability was measured using [123I]iodobenzamide single photon emission computed tomography (SPECT) a in subsample of 15 adults with PKU and 12 HC. Results: Repeated measures analyses of variance showed that PKU patients performed more poorly than healthy controls on tasks when working memory [ps: 0.037, 0.013, and 0.034] or inhibitory control [p = 0.037] were required, and that those with pPhe-levels above 360 μmol/l during childhood (0– 12 years) performed the worst. No significant correlations between current pPhe levels and EF were observed. D2/3R availability was higher in PKU compared to HC (p = 0.032), and pHVA levels were lower in PKU (p < .001). D2/3R availability correlated positively with error rate during a working memory task (r = 0.49, p = 0.037), and with error rate during the cognitive flexibility task (r = 0.57, p = 0.016). Discussion: The neuropsychological profile of adults with PKU resembles that of children with PKU, and is characterized by EF deficits. Evidence was found for reduced dopamine availability in PKU and for this hypodopaminergic state to be related to the extent of EF impairment.

P-149

Improvement in maternal PKU outcomes with development of a nurse/ dietitian led clinic Stenson C M 1, Crushell E 1, Hayes A 1, Knerr I 1, Losty E 1, Macauley M 1, Monavari A A 1, Muhammad A 1, O’Regan M 1, Treacy E 1, Hughes J A F 1 1

National Centre IMD, Dublin, Ireland

Background: All patients with phenylketonuria (PKU) in the Republic of Ireland attend the National Centre for Inherited Metaboilc Disorders (NCIMD). The incidence of PKU in Ireland is 1:4500 live births. There is a large patient cohort following 50 years of newborn screening. Elevated maternal phenylalanine is teratogenic. A nurse/dietitian led clinic for patients with PKU was introduced in 2004 with a special focus on education and support for female patients with regard to pregnancy in PKU.The importance of planning a pregnancy with good biochemical control prior to and throughout is emphasised regularly. Methods: A retrospective review of outcomes in maternal PKU was undertaken between 1997 and 2005. We sought to compare the outcomes following the introduction of the nurse/dietetian led clinic. All pregnancies in women with

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 PKU presenting between 2006 and 2015 were reviewed. The Clinical Nurse Specialists and dietitians had the following data on record for each pregnancy: planned or unplanned, initial presenting phe level, all phe levels during pregnancy and birth growth parameters (head circumference [HC], weight and length). Results: In our recent study 82 women had 127 pregnancies resulting in 104 live births, 56 (54 %) were planned. Planned pregnancies had significantly lower initial phe levels (347.8+/−233.6) compared to unplanned pregnancies (586.8+/−274.6). All growth parameters in the planned pregnancy cohort were significantly higher than in the unplanned cohort. 10 % of babies in the unplanned cohort had HC < 2nd percentile. In the initial study 36 % of pregnancies were planned with over 20 % of babies in the unplanned cohort having HC < 2nd percentile. Discussion: There has been a marked increase in the number of women planning pregnancy with overall improvement in growth parameters for all babies born to women with PKU since the introduction of the nurse/dietician led clinic.

P-150

European guidelines on diagnosis and treatment of PKU Van Spronsen F J 1, Van Wegberg A M J 1, Ahring K 2, Belanger-Quintana A 3, Blau N 4 5, Bosch A M 6, Burlina A 7, Campistol J 8, Feillet F 9, Gizewska M 10 , Huijbregts S C J 11, Kearney S 12, Leuzzi V 13, Maillot F 15, Muntau A C 16, Trefz F 4, Van Rijn M 1, Walter J H 17, MacDonald A 14 1 Div Ped Metab Dis, UMC Groningen, Groningen, Netherlands, 2Dept of PKU, Kennedy Centre, Glostrup, Denmark, 3Metab Dis Unit, Dept Peds, Hosp Ramon y C, Madrid, Spain, 4Univ Child Hosp,Dietmar-Hoppe Metab Cent, Heidelberg, Germany, 5Univ Child Hosp Zurich, Zurich, Switzerland, 6 Dept Peds, Div Metab Dis, AMC, Amsterdam, Netherlands, 7Div Inh Metab Dis, Dept Peds, Univ Hosp, Padova, Italy, 8Neuroped Dept, Hosp S ant Joan de Deu, Barcelona, Spain, 9Dept Peds, Hopital d’Enfants Brabois,CHU, Nancy, France, 10Dept Peds, Pomeranian Med Univ, Szczecin, Poland, 11 Dept Clinic Child Adolescent Studies, Leiden, Netherlands, 12Clinical Psych Dept, Birmingham Child, Birmingham, United Kingdom, 13Dept Peds, Child Neurol, Psych Sapienza Un, Rome, Italy, 14 Dietet Dept, Birmingham Child, Birmingham, United Kingdom, 15CHRU de Tours, Univ French Rabelais, Tours, France, 16Univ Child Hosp, UMC HamburgEppendorf, Hamburg, Germany, 17Medicine, Manchester Academic HS Centre, Manchester, United Kingdom

Background: Phenylketonuria (PKU) management differs widely across Europe. Guidelines have been developed aiming to optimize PKU treatment. Methods: 19 European experts developed these guidelines according to the Appraisal of Guidelines, Research, and Evaluation method. Literature search, critical appraisal and evidence grading were conducted according to the Scottish Intercollegiate Guidelines Network method. The Delphi-method was used when there was no or limited evidence available. World-wide external consultants revised the guidelines. Results: The cornerstone of treatment is the diet, but some patients may benefit from tetrahydrobiopterin (BH4) and other treatment strategies are at various levels of development. A total of 70 recommendations were formulated, of which the 10 most crucial will be addressed. Discussion: BH4 deficiencies need to be excluded in all patients. Followup and treatment for life is advised if untreated blood phenylalanine (Phe) > 600 mmol/l. If untreated Phe < 360 mmol/l, treatment is not recommended for PKU or maternal PKU. If untreated Phe 360–600 mmol/l, treatment is necessary for < 12 years and maternal PKU. Treatment targets are 120–360 mmol/l for 0–12 years and 120–600 mmol/l afterwards, except for pregnancy, where 120–360 mmol/l is advised. Annual nutritional, clinical and biochemical review is necessary for each PKU patient, regardless of therapy and especially in patients with poor metabolic control. In children < 12 years with 50–100 % of Phe levels > target range significant effort should be directed to gain acceptable control including increased blood phe measurements, re-education, and/or psychology consultation, sometimes with hospital admission and /or consultation with social services and child safeguarding measures. Conflict of Interest declared.

P-151

Socio-demographic findings in French adults with phenylketonuria: the ECOPHEN study Hodges P 2, Hassen-Khodja C 3, Charriere S 13, Douillard C 4, Bonafe L 1, Feillet F 5, Astudillo L 6, Lavigne C 7, Arnoux J B 8, Odent S 9, Gay C 10, Schiff M 11, Bonafe L 1, Mazodier K 12, Kuster A 14, Rigalleau V 15, Levesque H 16, De Parscau L 18, Besson G 17, Caille A 3, Maillot F 2 1 Center Mol Dis, Univ Hosp, Lausanne, Switzerland, 2Int Med Dept, Univ F Rabelais, Tours, France, 3CIC, Univ F Rabelais, Tours, France, 4Div Metab Dis, Univ Hosp, Lille, France, 5Div Metab Dis, Univ Hosp, Nancy, France, 6 Int Med Dept, Univ Hosp Purpan, Toulouse, France, 7Int Med Dept, Univ Hosp, Angers, France, 8Div Metab Dis, Univ Hosp Necker, Paris, France, 9Div Genet, Univ Hosp, Rennes, France, 10Div Pediatr, Univ Hosp, Saint-Etienne, France, 11Div Metab Dis, Univ Hosp R Debre, Paris, France, 12Int Med Dept, Univ Hosp, Marseille, France, 13Div Endoc, Univ Hosp, Lyon, France, 14Div Pediatr, Univ Hosp, Nantes, France, 15Div Endoc, Univ Hosp, Bordeaux, France, 16Int Med Dept, Univ Hosp, Rouen, France, 17Div Neurol, Univ Hosp, Grenoble, France, 18Div Pediatr, Univ Hosp, Brest, France

Background: Few data exists about social outcome in treated patients with phenylketonuria (PKU). The aim of the study was to describe educational level and social integration in French adults with PKU. Methods: Data were collected from a prospective cohort study of PKU adults (ECOPHEN, 2013–2020). Marital status and professional occupation were assessed. Educational attainment was defined using ISCED. Social vulnerability was measured by “EPICES” questionnaire (deprivation threshold 30.17). Chi square tests were used to compare distributions of marital status and educational level categories between classic and mild PKU. A Student’s t-test was used to compare EPICES scores between classic and mild PKU. Results: 187 patients diagnosed with PKU were included (127 w, 60 m, age 27.6 ± 6.3y, 152 classic PKU, 26 mild PKU and 9 HPA). In total, the marital status single 47.1 %; married/cohabitation 50.3 %; divorced/separated 2.1. About educational attainment using ISCED, 6 patients reached level 1, 48 level 2, 37 level 3,44 level 5 and 49 levels 6 +. 61.7 % patients were in either full-time or part-time employment. Distributions of marital status categories and educational level categories were similar between classic PKU and mild PKU. Regarding the EPICES score, it was 27.2 ± 16.6 for classic PKU, 20.4 ± 16.0 for mild PKU and 18.6 ± 14.0 for HPA. The proportion of patients with an EPICES score > 30.17 was higher in classic when compared to mild PKU Discussion: The present study suggests that social integration of adults with PKU is suboptimal. Education attainment (at levels 1, 2, 5 and 6 +) and employment rate were lower in PKU in comparison to the general population (INSEE 2014). These data may be related to a suboptimal neurological outcome, but such a relationship has to be further studied. Concerning the “EPICES” score, a clear difference was found between classic and mild PKU, which implies that classic PKU patients are more at risk of social vulnerability.

P-152

Monitoring the renal functions of patients with phenylketonuria Pazdirkova R 1, Kolsky A 1, Hedelova M 2, Krotka J 2, Zikmund J 1, Spicka J 2 1 Dept of Ped, 3rd Fac of Med Charles Univ, Prague, Czech Republic, 2Dept of Biochem Inst of Lab Dg, Prague, Czech Republic

Background: Neurocognitive functions of people suffering from the metabolic disorder phenylketonuria (PKU) are impaired without treatment. The therapy of PKU is based on reaching optimal levels of phenylalalanine (Phe) in plasma. In recent years, a possible correlation between PKU and kidney damage has been discussed. Impairment of renal function is explained by a higher intake of amino acid mixtures without phenylalanine, which comprise up to 90 % of the PKU diet.

S102 Methods: 23 (15 male, 8 female, ages 19–44 yrs) patients with classic phenylketonuria on PKU diet taking the amino acid mixture (protein intake 0,9– 1,0 g/kg/d) were included. The renal functions have been investigated within the framework of regular ambulatory check-ups. Values of serum creatinine measured by enzymatic method and cystatin C were used for calculation of glomerular filtration rate (eGFR) according to CKD-EPI equation. Proteinuria was determined from a 12-h night time collection of urine. Protein/creatinine index (PCR) was obtained from a one-time sample of urine. Results: Two of 23 patients had lowered levels of eGRF, calculated from the values of S-creatinin (according to CKD-EPI equation) . Creatinine serum concentrations were within normal ranges in all patients. When using the calculated eGFR from the values of S-cystatin C, lowered eGFR (1–1.49 ml/s/1,73 m2) were discovered in four patients (17 %), phase 2 as per K/DOQI. Six patients (26 %) had borderline proteinuria (150–300 mg/d), three patients (13 %) had PU higher than 300 mg/d. Two of them had an elevated PCR. Discussion: Lowered glomerular filtration estimated by means of serum cystatin C and proteinuria occurs in PKU patients. Hence, PKU may influence the function of kidneys. We will continue to collect data for further evaluation. Patients with PKU need lifelong monitoring, including the examination of renal functions.

P-153

Moving beyond phenylalanine levels to further optimize PKU management Van Vliet D 1, Berry G T 2, Blau N 3, Federico A 4, Feillet F 5, Gizewska M 6, Hertecant J 7, Jung-Klawitter S 3, Moseley K D 8, Rutsch F 9, Trefz F K 3, Usurelu N 10, Van Spronsen F J 1 1

UMCG, Beatrix Child Hosp, Div Metab Dis, Groningen, Netherlands, 2Div Genet Genomics, Boston Child Hosp, Boston, United States, 3Dept Ped, Univ Heidelberg, Heidelberg, Germany, 4Dept Med Surg Neur, Univ Siena, Siena, Italy, 5 Dept Ped, CHU Nancy, Nancy, France, 6 Pomeranian Medical University, Szczecin, Poland, 7Dept Ped, Tawam Hosp, Al-Ain, United Arab Emirates, 8Gen Div, Dept Ped, USC, Los Angeles, United States, 9Dept Gen Ped, Muenster Univ Child Hosp, Muenster, Germany, 10Instit Mother Child, Chisinau, Moldova, Republic of Background: The combination of early diagnosis and initiation of treatment has resulted in near-normal outcome for PKU patients. However, at least some patients still show mild neuropsychological, mood, and behavioral problems, even if plasma phenylalanine (Phe) is only mildly elevated. On the other hand, some untreated and late-diagnosed classical PKU patients have been reported to have escaped from severe intellectual disability and seizures. By reviewing these cases, we aim to provide further understanding of the differences in brain vulnerability between PKU patients and the possible underlying mechanisms. Methods: We performed a literature search on late-diagnosed classical PKU patients with a (near) normal IQ and are trying to identify additional (not reported) PKU patients who have been diagnosed (and previously untreated) after the age of 1 year, with blood Phe ≥1200 μmol/l, and an IQ of ≥80. Results: Thus far, we have identified >30 PKU patients (both reported and unreported) who fulfill the inclusion criteria. Of them, most were diagnosed because of an affected PKU sibling or because of children showing the maternal PKU syndrome. Most patients who have escaped from severe intellectual disability do not show other brain symptoms of untreated PKU. However, many of these patients do show behavioral and some neuropsychological issues. Discussion: The fact that late-diagnosed PKU patients who have escaped severe intellectual disability (as well as early and continuously treated adults) may show other cerebral features suggests that various pathophysiological mechanisms could underlie different PKU symptoms. Further understanding of the mechanisms underlying the different outcomes of PKU patients is necessary to further optimize PKU management and treatment. To solve such a puzzle, we need more well-described PKU patients who are late-diagnosed with blood Phe >1200 μmol/l, and have escaped from severe intellectual disability showing an IQ >80.

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Psychodiagnostic evaluation in classical PKU patients: possible requirements to improve patients’ care Vendemiale M 1, Settembre M S 1, Cornacchia D 1, Carella A 1, Di Mauro A M 1, Ortolani F 1, Piccinno E 1, Masciopinto M 1, Papadia F 1, Tummolo A 1 1

Dept of Metabolic Dis, Child Hosp, Bari, Italy

Background: Phenylketonuria (PKU; OMIM 261600) is an inborn error of metabolism characterized by high plasma phenylalanine (Phe) and low tyrosine levels. The cornerstone of treatment is the low-Phe diet, although dietetic adherence is one of the major therapeutic challenges, especially in adolescents and young adults. Our study aims to compare psychological issues of patients with normal and low diet adherence and to characterize special issues that may represent a possible target for intervention to improve patients’ care. Methods: Thirty early-treated classical PKU patients, aged between 13 and 25 years, were enrolled. They underwent psycho-diagnostic assessment using self-report questionnaires and interview. According to Phe levels on dried blood spot they were divided into low adherent (A) and adequate adherent to diet group (B), using 400 μmol/L as cut-off value for blood Phe concentration. Results: Depression, low self-esteem and perception of strong impotence were the most common symptoms in group A. Anxiety is more frequent in group B (23 % vs 11.8 %), although in this group an internal locus of control was frequently detected with positive way of dealing and secure attachment. The family adaptation evaluation interview revealed that balanced families are more frequent in group B, whereas in group A an intermediate phenotype is more frequently observed. Discussion: In our group a poor adherence to diet has led to internalization symptoms with low family cohesion. The strict adherence to diet was linked to a higher tendency to anxiety with a more stable familiar support. Our data support the hypothesis that the disease acts as psychological stress both for patients and their families independently from diet adherence. A focused psychological intervention enforcing a positive coping, internal locus of control and secure attachment could represent a valid strategy to improve disease management.

P-155

Living with phenylketonuria: the relationship between executive functioning, quality of life and adherence to protein substitutes Hofman D L 1, Dye L 1, Dickinson J 1, Lawton C L 1 1

University of Leeds, Leeds, United Kingdom

Background: Adherence to the phenylketonuria (PKU) diet is especially important during childhood and should be maintained through adulthood to protect patients from neuropsychological dysfunction. However, the strict PKU diet imposes a burden on patients and their families and has been associated with dietary non-adherence, especially in adolescents and young adults. This study aimed to better understand (1) factors contributing to different levels of adherence to protein substitutes and (2) the executive functioning (EF) in daily life settings and quality of life (QoL) impacts experienced by adult PKU patients as a result of different levels of protein substitute adherence. Methods: Adult PKU patients were invited to complete an online survey which included questions on potential contributors to (in)adherence (e.g. SES), the Morisky 8-item Medical Adherence Questionnaire MMAS-8, a PKU specific QoL Questionnaire and the Behavioural Rating Inventory of Executive Function–Adult version (BRIEF-A). Results: Responses obtained to date indicate that patients with higher adherence experience a lower overall and emotional impact of PKU, as well as fewer symptoms (e.g. tiredness, anxiety). Furthermore, all EF-related behaviours measured using the BRIEF-A were worse in patients with lower adherence. BRIEF-A scores on planning/organizing and inhibition seemed to be the most accurate discriminants of adherence level.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Discussion: Overall, in spite of following a strict diet with protein substitutes, adherent PKU patients seem to experience less of an impact on their QoL than patients with a lower adherence level. It is unclear whether deficits in subjective measures of EF in daily life settings in adult PKU patients are caused by or are a cause of decreased protein substitute adherence. BRIEF-A is a measure of behaviours associated with EF, not actual EF. Therefore, a cohort of respondents will be invited to complete an online cognitive test battery aimed at assessing several EFs.

P-156

Case–control study of neuropsychological results in patients with mild hyperphenylalaninemia 1

Belanger-Qunitana A , Zamora B

BMI between 25.0 and 29.9 kg/m2, grade I obesity as BMI of 30.0–34.9 kg/m2 and grade II as BMI > 35 kg/m2. The high cardiovascular risk is defined by a waist circumference > 102 cm in men or > 88 cm in women. Results: We evaluated 25 adult PKU patients (age: 18–47 years old), 16 women and 9 men, referred to the unit of adults metabolic disease. 10/25 had normal BMI. 9/25 were overweight; 6/25 had obesity (5/6 grade I and 1/6 grade II). Analysing the waist circumference, 6/25 women were found at risk for overweight/obesity associated disease; no man was found to have a highrisk waist circumference. Discussion: In PKU adult patients women appear to have a superior risk of metabolic disease and complications related to abdominal obesity. Conflict of Interest declared.

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2

PKU patients under dietary treatment: evidence of selenium deficiency Div Metab Dis, Hosp Ramon y Cajal, Madrid, Spain, 2 Div Ped Neuropsychology, Hosp 12 Octubre, Madrid, Spain 1

Background: Patients with forms of PAH deficiency who maintain phenylalanine (Phe) concentrations under target levels ( +2 at the age of 18. Results: The study included 180 PKU patients (98 women and 82 men). The medium height z-scores for both men and women shows a normal growth pattern. Nutritional parameters were evaluated in order to determine their relation with growth and weight. The overweight and obesity rates were superior in the centers with higher reported phenylalanine intake. Discussion: PKU patients should have an adequate final physical development. It is important to collect longitudinal growth data throughout childhood and adolescence in PKU that considers any change in growth in relationship to dietary patterns.

Alterations of myelin basic protein in juvenile PKU mice

P-160

Vulnerability and resilience to phenylalanine in PKU patients Manti F 1, Nardecchia F 1, Paci S 4, Chiarotti F 3, Carducci C 2, Dalmazzone S 4 , Giordano L 4, Cefalo G 4, Banderali G 4, Leuzzi V 1 1 Dept Ped Child Neur Psyc, Univ Sapienza, Rome, Italy, 2Dept Molec Med, Univ Sapienza, Rome, Italy, 3Centre Epid Surv Health Prom, Nat Inst H, Rome, Italy, 4Ped Dept, Univ of Milan, S Paolo e Carlo, Milan, Italy

Background: We conducted a longitudinal study to explore the cognitive outcome in early treated PKU (ETPKU) patients in order to assess individual resilience and vulnerability to phenylalanine (Phe). Methods: 65 ETPKU patients were assessed for IQ twice during their lifetime (age range at 1st and the 2nd assessment was 7–13 and 17– 25 years, respectively). We considered the index of dietary control (IDC) from two periods: from the start of the diet to the day of the 1st assessment (IDC0-1) and the interval between the two evaluations (IDC1-2). Patients were subgrouped according to their metabolic control choosing as cut-off for good metabolic control 360 μM for the 0– 1 period and 600 μM for the 1–2 period. IQ was evaluated using the Wechsler Intelligence Scale. Results: The within-group comparison showed that patients who had IDC12 < 600 μM obtained a significantly better cognitive outcome compared to those who had a poor metabolic control in the same period (p = 0.0015), independently from the IDC0-1. 5/19 ETPKU with a good life-long metabolic control (IDC0-1 < 360 μM and IDC1-2 < 600 μM) worsened their cognitive functioning. 18/25 ETPKU with a poor long-life metabolic control (IDC01 ≥ 360 μM and IDC1-2 ≥ 600 μM) remained stable or improved their IQ. Discussion/Conclusion: Results of this study confirm the relevance of a good metabolic control from the second decade of life onwards for the cognitive outcome in PKU patients; however, the Phe concentrations that are safe in this period are still debated. In our sample the 26 % of PKU patients who maintained good life-long metabolic control anyway showed a significant decrease of IQ and the 72 % of patients with life-long poor metabolic control kept stable or improved their IQ suggesting that there must be individual factors conferring vulnerability or resilience to Phe. This is an important topic to be further investigated for tailored dietary treatment.

Sasso V 2, Viscomi M T 2, Valzania A 1 2, Leuzzi V 3, Puglisi-Allegra S 1 2, Pascucci T 1 2 1 Dept of Psycho, Sapienza Univ, Rome, Italy, 2Santa Lucia Foundation, Rome, Italy, 3Dept of Neurops, Sapienza Univ, Rome, Italy

Background: White matter alterations is a common feature in young adult early treated phenylketonuria (PKU) patients. Disorders of myelin development have been found also in animal model of the disease. A direct link among elevated levels of phenylalanine, phenylpyruvate and phenylacetate and myelin alterations could not be found. The chronological trends of brain dopamine and myelin protein increase follows a similar pattern in normal mouse, so suggesting a possible link between neurotransmitter synthesis and axonal maturation. In a previous study we found a relevant depletion of biogenic amines in the brain of PKU mouse aged PND 14 to 21. The aim of the present study was to investigate brain myelination in PKU mice during this period of rapid brain maturation. Methods: BTBR wild Type (WT) and untreated ENU2 mice were sacrificed at different time points (PND 14 and PND 21) and brains were examined by Western blotting or immunofluorescence in order to assess the expression level of myelin basic protein (MBP), a protein essential for the CNS myelination process. Results: Western blot analysis showed significant differences in the MBP level between ENU2 and WT mice. Furthermore, densitometry analysis of confocal material confirmed that the MBP staining in the ENU2 mice brains was significantly lower than in WT mice. Discussion: Different mechanisms may affect both biogenic amines and MBP synthesis: a) they may depend on the availability of LNAA whose transport is impaired by high levels of blood Phe; b) low levels of dopamine and serotonin may affect brain maturation, also involving myelin maturation; or c) they may be the result of independent pathogenetic processes affecting different components of brain maturations in the same developmental period.

P-162

Neuropsychological and quality of life outcomes in untreated adults with mild hyperphenylalaninemia with phenylalanine levels between 360 and 600 μmol/L Feigenbaum A 1 2, Wilson A 1, Nagy L 1, Siriwardena K 3, Nasr E 1, Kerr E 1 1 Hosp for Sick Children and Univ of Toronto, Toronto, Canada, 2Rady Childrens Hospital and UCSD, San Diego, United States, 3 Stollery Childrens Hospital, Edmonton, Canada

Background: Thresholds to treat hyperphenylalaninemia have been historically discrepant, with the majority of clinics treating phenylalanine (PHE) levels >360 μmol/L. There is no consensus about the neuropsychological function and quality of life of untreated patients with mild hyperphenylalaninemia (MHP), with PHE levels 360–600 μmol/L. Further well designed studies of functioning in untreated individuals with MHP are needed to clarify findings of the limited studies that show variable outcomes in this population. Objective: To determine whether neurocognitive, psychological, and quality of life outcomes for adults with MHP, as defined by untreated PHE levels between 360 and 600 μmol/L, are within the normal population values. Methods: Eligible subjects were administered a comprehensive neuropsychological battery measuring IQ, executive function, and quality of life. Statistical analysis was performed using z-tests and interclass correlation coefficients. Results: 10 subjects were studied. No difference was found in full-scale IQ scores using the Wechsler Adult Intelligence Scale when compared to Canadian normative data. On the Beck Depression and Anxiety Inventories, 10 % of patients reported moderate depression, and 40 % reported mild to moderate anxiety. CANTAB cognitive testing showed deficits in executive

S105

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 functioning, working memory, and reaction time when compared to age and gender normative data. Discussion: The sample size was limited but these results warrant further research into the potential adverse effects of untreated MHP with PHE levels >360 μmol/L on executive function and emotional regulation. The CANTAB measure is shown to be a useful measure in this population. Improved treatment options, including better nutritional products to aid with diet adherence and pharmaceutical options, may make management of patients with MHP more feasible. Conflict of Interest declared.

P-163 Control status of phenylketonuria (PKU) patients born after 1995: a single center experience in Japan Ogawa E 1, Ishige M 1, Takano C 1, Usui H 1, Owada M 2, Fuchigami T 1, Takahashi S 1 1

and to evaluate the association between blood Phe levels and possible effects of hyperphenylalaninemia. Subjects were children aged between 6 and 16 and followed by Child Metabolism Department due to MHP with blood Phe levels between 2 and 10 mg/dl when on an unrestricted diet, who were not treated with Phe-free amino acid formulas. Study group consisted of 45 children and control group consisted of 20 healthy children. All children were evaluated by experts of child mental health department. Structured and half structured interviews are conducted and self-report inventories are given to children and their parents. Regression and MANOVA were run on SPSS 20.0. Results: Healthy controls had higher scores compared to children with “grey zone” mild HPA on Perceptual Reasoning Index and Full Scale IQ Score. In addition, these results suggest that the degree of hyperphenylalaninemia was positively correlated with behavioural problems. Discussion: Our study supports the proposal that “grey zone” mild HPA (Phe: 6–10 mg/dl) should be treated to avoid neurocognitive and behavioural problems. Based on the result of this study, we anticipate that future studies with more patients might indicate that even lower cut-off level should be set to initiate treatment.

Dept Ped Child Health, Nihon Univ, Tokyo, Japan, 2Tokyo Health Service Association, Tokyo, Japan

P-165

Background: The necessity of lifelong treatment first appeared in Japanese PKU treatment guideline in 1995. Twenty years later in 2015, the national health policy on intractable diseases was revised to extend public financial assistance to PKU treatment beyond the age of twenty. Objective: To describe the control status of PKU patients born after 1995 and discuss the positive influence of 2015 revision of the national health policy on continuing PKU treatment into adulthood. Methods: Clinical and laboratory data were collected retrospectively for PKU patients born after 1995 and referred to Nihon University Hospital. Results: 31 PKU patients (male: 12, female: 19) have been referred to our hospital. 17 are classical and 14 are mild, with 9 possible tetrahydrobiopterin responders. 23 patients were referred to us before the age of 1, and 95 % of them showed optimal control during the first 3 years of life, with >75 % of phenylalanine (Phe) concentrations in target range. Although fluctuation of Phe concentration increased with age, most remained within the target range. There was no short stature or intellectual impairment. Overweight was seen in patients with erratic Phe concentrations. Underweight was observed in adolescent girls with good control. Only one female patient is currently lost to follow up. Another female patient had a period of discontinuation but has resumed treatment with the 2015 revision of the national health policy on intractable diseases, which extends public financial assistance to PKU treatment beyond the age of 20. Discussion: Phe control has been satisfactory during childhood and adolescence in our institute. Revision of the national health policy is expected to support lifelong PKU treatment.

Effects of irregular amino acid mixture intake on macro- and micronutrient status of adult patients with phenylketonuria

06. Phenylketonuria: treatment, BH4

P-164 Preliminary results of the study relevant to ‘evaluating neurocognitive functions of untreated children with hyperphenylalaninemia’ Evinc S G 1, Foto Ozdemir D 1, Pektas E 1, Oktem F 1, Yildiz Y 1, Tokatli A 1, Coskun T 1, Karaboncuk Y 2, Sivri H S 1 Hacettepe University, Ankara, Turkey, 2Baskent University, Ankara, Turkey

1

Background: Previous studies indicate that mild hyperphenylalaninemia (MHP) with blood phenylalanine (Phe) levels between 2 and 6 mg/dl does not affect children’s cognitive development and executive functions, and therefore treatment is not needed for these children. Conflicting opinions exist about the need to treat “grey zone” MHP, defined as blood Phe levels between 6 and 10 mg/dl on an unrestricted diet. However, recently it has been discussed that MHP may have effects on cognitive abilities, executive functions and attention. This study aimed to compare the psychological, social, behavioral, mental and cognitive functions among healthy children and children with MHP

Hochuli M 1, Bollhalder S 1, Thierer C 1, Baumgartner M R 2 1 Div Endo and Diabetes, Univ Hospital, Zurich, Switzerland, 2 Div Metabolism, Univ Child Hospital, Zurich, Switzerland

Background: Adult phenylketonuria (PKU) patients often reduce the dosage of amino acid mixture (AAM). Effects of reduced AAM intake on nutritional status and laboratory parameters of adult PKU patients were evaluated in a prospective cross-sectional study. Methods: Macro- and micronutrient intake was calculated in a cohort of 20 adult patients (17 classical, 3 moderate PKU, mean age 33y), based on a structured 4-day food record. Blood samples for assessment of amino acid profiles, micronutrients and vitamins were collected as part of the routine care. Patients were classified in two groups: (A) regular AAM intake or (B) AAM intake below theoretical requirements. Results: Mean Phe concentrations were similar in both groups (A 650 ± 283; B 760 ± 350 mmol/l, ns). AAM intake was 0.73 ± 0.1 g/kg in group A, and 0.38 ± 0.28 g/kg in group B. Total protein intake was below the recommended amounts in 60 % of patients in group B, versus 7 % in group A (p = 0.03), mean deviation from theoretical requirements: group A +31.1 ± 27 %, group B −0.72 ± 35 % (p < 0.05). However, group B consumed a higher proportion of natural protein (59.9 ± 23 % vs 32.7 ± 12 %, p = 0.002). Fat intake was higher in group B (39 ± 9 % vs 31 ± 6 %, p = 0.03), mainly from saturated fats. Supply of calcium, vitamin B12, folate, selenium and zinc was significantly lower in group B, but only selenium, folate and vitamin B12 intake was clearly below theoretical requirements. However, mean serum concentrations of all these analytes, as well as plasma amino acids remained within the normal range in both groups, although vitamin B12 was lower in group B, but within normal limits. Plasma tyrosine concentrations correlated with AAM intake, and hydroxyprolin correlated with the amount of natural protein consumed. Discussion: Relaxed AAM intake resulted in insufficient supply of protein, vitamin B12 and selenium, despite a compensatory increase in consumption of natural protein. Care has to be taken to insure adequate nutrient supply in the follow-up of adults with PKU. Conflict of Interest declared.

P-166

Role of the Phe/Tyr ratio in assessment of tetrahydrobiopterin—responsiveness in phenylketonuria Smon A 1, Groselj U 1, Zerjav Tansek M 1, Repic Lampret B 1, Kobe H 1, Battelino T 1 2

S106 1

Univ Child Hosp, UMC Ljubljana, Ljubljana, Slovenia, 2Faculty of Medicine, Univ of Ljubljana, Ljubljana, Slovenia Background: Phenylalanine (Phe) restricted diet can prevent the effects of hyperphenylalaninemia (HPA) on cognitive function. As an alternative to dietary treatment, tetrahydrobiopterin (BH4) was shown to be an effective and safe therapy in responsive patients. Limited amount of literature exists on the effect of BH4 on the phenylalanine/tyrosine ratio (Phe/Tyr) and its possible role in assessing BH4-responsiveness. Methods: In 32 selected patients after increased intake of natural proteins (2 g/kg body weight) the BH4 loading test was performed. Blood collection was at 96 h, 72 h, 48 h, 24 h, 16 h and just before administering BH4 (orally one dose of 20 mg/kg body weight) and 8 h, 24 h and 48 h after BH4 administration. All measurements were done from dried blood spots by LC-MS/MS. Results: Blood Phe and Phe/Tyr reached a plateau after 3 days of increased dietary intake of protein. Tyr values stayed roughly the same during the time. Of 32 patients 18 (56 %) responded to BH4, defined as a 20 % decrease of Phe 24 h after BH4 administration. Phe/Tyr between responders and nonresponders were statistically different at all times. In responders, the Phe/Tyr decreased in average of 64 % 8 h (p < 0.0001) and of 42 % 24 h (p = 0.0005) after drug administration. Phe values were the lowest 24 h after BH4 administration. Phe/Tyr did not show a decline in nonresponders. Discussion: Protein loading increased the value of Phe in blood, which kept rising until a plateau, whereas Tyr values were unaffected. There is a significant difference in Phe/Tyr between responders and nonresponders at all times. After BH4 administration the Phe/Tyr drops only in responders; the lowest was seen 8 h after BH4 administration. This is in disagreement with the values of Phe, which were the lowest 24 h after administration. Using Phe/Tyr to test for BH4 responsiveness could allow a quicker test for responsive patients. There is also a possibility that responsiveness could be defined by the Phe/Tyr.

P-167

Sapropterin dihydrochloride toxicity on developing reaggregated rat brain cell cultures Remacle N 1, Cudre-Cung H P 1, Do Vale Perreira S 1, Braissant O 2, Ballhausen D 1 Cent Mol Dis, Lausanne Univ Hosp, Lausanne, Switzerland, 2Biomedicine, Lausanne Univ Hosp, Lausanne, Switzerland 1

Background: Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism caused by phenylalanine hydroxylase (PAH) deficiency. Patients with mild or moderate PKU are often successfully treated with sapropterin dihydrochloride (SD) with a starting age going recently down to children below 4 years of age. SD is a pharmaceutical version of tetrahydrobiopterin (BH4), a cofactor of the deficient enzyme PAH. It is controversial whether this drug crosses the blood brain barrier. However, it has been shown that BH4 reaches the mouse brain after oral administration. Here we studied the effects of different SD concentrations on developing brain cells in a rat organotypic in vitro model. Methods: 3D organotypic rat brain cell culture aggregates derived from Sprague Dawley rat embryos at E15 were exposed to different concentrations of SD (50, 75 or 100 ng/ml) from DIV11 to DIV14 corresponding to early childhood. Changes in morphology and viability of different brain cell types were studied by immunohistochemistry. We investigated potential alterations of the GABA-ergic system by analyzing the expression of glutamic acid decarboxylase (GAD; the enzyme synthesizing GABA) as well as GABAAR. Results: Cell-type specific markers revealed swollen astrocytes and a decreased number of astrocytic fibers (50, 75 and 100 ng/ml) and oligodendrocytes (100 ng/ml). Immunofluorescence for activated caspase-3 revealed an increased apoptosis rate for all SD concentrations. We found decreased levels of GAD (100 ng/ml) and GABAAR expression (75 and 100 ng/ml). Discussion: Exposure of developing brain cells to SD leads to a significant toxicity on different brain cell types in our in vitro model. The altered expression levels of

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 proteins of the GABA-ergic system are similar to what is seen in attention deficit hyperactivity disorders (ADHD). Long term neuropsychological follow-up of patients treated since early age with SD seems to be indicated to study the potential correlation with ADHD. Conflict of Interest declared.

P-168

Genotype and basal blood phenylalanine predict BH4 responsiveness in phenylalanine hydroxylase (PAH) deficient patients Trefz F K 1, Blau N 1, Muntau A C 2, Feillet F 3, Belanger-Quintana A 4, Van Spronsen F J 5, Frauendienst-Egger G 6, Hoffmann G F 1 1 University Childrens Hospital, Heidelberg, Germany, 2University Childrens Hospital, Hamburg, Germany, 3CHU Brabois, Vandoeuvre les Nancy, Nancy, France, 4Hospital Ramon y Cajal, Madrid, Spain, 5University of Groningen, Groningen, Netherlands, 6Kreiskliniken Reutlingen, Reutlingen, Germany

Background: BH4 responsiveness can be predicted by genotypes bearing at least one mutation with some residual PAH activity. However, inconsistency of responsiveness is present and has not yet systematically been investigated. Methods: We evaluated BH4 loading results (20 mg/kg/bw over 24 h) in 309 infants (age 1–4 weeks) and calculated BH4 responsiveness using a semi-mechanisticallybased, nonlinear mixed-effect model. In 306 patients PAH genotype was available. Results: Positive correlation of responsiveness with baseline blood phenylalanine (Phe) was observed in genotypes p.[R261Q];[R261Q] (Phe 600–1400 μmol/l, n = 14) and p.[E390G];[any] (Phe 200–600 μmol/l, n = 10), a negative correlation with p.[L48S];[L48S] (Phe 400–1800 μmol/l, n = 8) and p.[A300S];[A300S] (Phe 250–1000 μmol/l, n = 4). Genotypes p.[Y414C];[any] showed responsiveness (Phe 300–1000 μmol/l) in all 15 patients, but with no correlation to Phe. Discussion: Optimal blood Phe concentration can be defined for BH4 responsiveness and is linked to the genotype. Since most patients are compound heterozygotes, where negative interallelic complementation may play a role, individual BH4 response data of patients with rare genotypes should be documented in a database. Calculating the slope for responsiveness provides a valuable tool for an objective measure of response and gives the possibility to compare in vivo and in vitro expression data (PAH landscaping). Conflict of Interest declared.

P-169

Lipid profile status and other cardiovascular risk factors in patients with hyperphenylalaninaemia Picans R 1, Aldamiz-Echevarria L 2, Vitoria I 3, Sanchez-Pintos P 1, De Castro M J 1, Hermida A 5, Leis R 4, Couce M L 1 1

U Diag Trat ECM, Dept Pediatr, HCU-USC, Santiago de Compostela, Spain, U Metabolism, Cruces Univ Hosp, Barakaldo, Spain, 3U Metabolopathies, H Univ La Fe, Valencia, Spain, 4U Gastr Nutr, Dept Pediatr, HCU-USC, Santiago de Compostela, Spain, 5U Diag Trat ECM, Dept Med Int, HCUUSC, Santiago de Compostela, Spain 2

Background: The diet used in patients with phenylketonuria (PKU) seems to reduce atherogenesis and confer protection to cardiovascular diseases although previous results were inconclusive. The aim of our study was to evaluate lipid profile and different parameters related with treatment in patients with hyperphenylalaninaemia (HPA) in order to optimize their monitoring. Methods: We conducted a cross-sectional multicentre study. 141 HPA patients were classified according to phenotype, type of treatment and dietary adherence. Annual median blood phenylalanine (Phe) levels, Phe tolerance, anthropometric measurements, blood pressure (BP) and biochemical parameters [total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), apolipoprotein A (ApoA), apolipoprotein B (ApoB), total homocysteine(Hcy), vitamin B12, high sensitivity Creactive protein (hsCRP)] were collected for each patient.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Results:: TC was lower in PKU than in mild-HPA (150 ± 31 vs. 164 ± 22 mg/ dL), and there was a significant inverse correlation between TC and Phe levels. HDL-C, LDL-C, ApoA and ApoB were also lower in PKU, but B12 was normal. Interestingly, PKU with good metabolic control displayed slightly higher HDL-C and LDL/ApoB ratio and lower Hcy concentration(5.6 vs. 6.99 μmol/L). Besides PKU had higher systolic BP, as indicated by quadratic correlation between median Phe levels and systolic BP (p = 3.77e−5) and diastolic BP (p = 8.14e−4). In overweight or obese PKU (24.11 %), TC, triglycerides, LDL-C, Hcy, hsCRP and BP were higher and HDL-C was lower. Discussion: Our data shows a correlation between healthy lipid profile and good adherence to PKU diet. However, lipid profile in overweight or obese PKU displayed an atherogenic profile and higher BP. Our study contributes to a better understanding of the relationship between phenotype, treatment and cardiovascular risk in HPA, which could be useful in improving follow-up strategies and clinical outcome.

P-170

Episomal minicircle-vectors expressing liver phenylalanine hydroxylase from its endogenous promoter for therapy of phenylketonuria

severe neurological symptoms are rare. Strict dietary changes may improve phenylalanine concentrations but it may take much longer to improve neurological symptoms. Four patients presented with unusual neurological complications that were most likely diet-dependent. Case Report: All 4 cases were females aged 24, 37, 36 and 53 years. Three were diagnosed with PKU at newborn screening and one was diagnosed at the age of 12 months. Two of them were off PKU diet due to learning difficulties or intentions to reduce body weight and two other were partially compliant with diet. On neurological examination all four patients had brisk reflexes, broad based gait and upgoing plantar reflexes. One patient had past medical history of stable syringomyelia. One patient tried baclofen and amitriptyline to help control her symptoms. Serum vitamin B12 levels was sufficiently replaced in all patients. Results: All patients had phenylalanine concentrations between 1200 and 2000 umol/L when repeatedly measured between metabolic clinic appointments over the last 3 years. In one case MRI scan did not show any spinal compression and CSF was reported as normal. Discussion: Spasticity in lower limbs may occur in patients with classical PKU after ceasing dietary restrictions or poor compliance with PKU diet. Due to the palatability and side effects of the amino acid supplements, many patients have difficulty complying with supplemented PKU diet. Long-term untreated B12 deficiency or underlying non-PKU-related neurological conditions warrant the neurology team’s attention and further specialist investigations. Conflict of Interest declared.

Viecelli H M 1, Schlegel A 2, Scherer T 1, Allegri G M 1, Heidelberger R 1, Schleef M 3, Cary H O 4, Haeberle J 1 5, Thony B 1 5 P-172 1

Div Metab, CRC, Univ Child Hosp Zurich, Zurich, Switzerland, 2Swiss HPB Transplant Cent, UZH, Zurich, Switzerland, 3PlasmidFactory GmbH, Bielefeld, Germany, 4Dept Mol Med Gen, OHSU, Portland, United States, 5 ZHIP and ZNZ, Zurich, Switzerland Background: A biological safe vector system for (hepatic) gene therapy of monogenic diseases would be a non-integrating (episomal), low-copy number, non-viral, naked DNA vector exhibiting robust and persistent expression of the therapeutic transgene from its natural tissue-specific promoter. We previously reported correction of phenylketonuria (PKU) in mouse liver upon hydrodynamic tail vein injection of a naked DNA/minicircle (MC)-based vectors expression phenylalanine hydroxylase (PAH) from a synthetic liver promoter. Methods and Results: Here we further characterized and developed MC vectors to treat PKU expressing PAH under more physiological conditions. Liver regeneration after partial hepatectomy of MC-corrected PKU mice exhibited hyperphenylalaninemia comparable to pre-treatment due to >95 % loss of vector-DNA and PAH activity confirming episomal maintenance of MC vectors. Replacing the synthetic liver promoter by the endogenous murine 3.6 kb Pah-upstream promoter-enhancer fragment in-frame with exon 1 conferred long-term therapeutic PAH expression from MC vectors comparable to viral or the synthetic liver promoters. At last, codon-optimization and inclusion of a truncated intron 1 in the Pah-cDNA allowed treatment under significant reduction of vector dosage and resulted in therapeutic MC-vector copy number 600 μmol/L received open-label PGVL at a randomized target dose (RD) of 20 or 40 mg daily, in preparation to enter PRISM-2. PRISM-2 had an 8-week, double-blind, placebo (PLB)-controlled randomized discontinuation trial (RDT). Subjects that reached RD and had ≥20 % blood Phe reduction from pre-treatment baseline were randomized to PGVL or PLB in RDT. Inattention (IA) was assessed with IA domain of the Attention Deficit Hyperactivity Disorder Rating Scale. Results: Of 261 subjects in PRISM-1, 203 entered PRISM-2 and 86 qualified for RDT. For RDT subjects, from pre-treatment baseline to RDT entry, mean (SD) blood Phe improved (PGVL, n = 58: 1318 [351] to 503 [520] μmol/L; PLB, n = 28: 1284 [356] to 536 [520] μmol/L) and mean (SD) IA improved (PGVL: 11.6 [6.7] to 5.9 [5.5] points; PLB: 9.6 [6.0] to 3.9 [4.1] points). At week 8 of RDT, mean (SD) blood Phe levels were maintained in the PGVL arm and increased in PLB arm (PGVL: 527 [557] μmol/L; PLB: 1385 [403] μmol/L; p < 0.0001). No difference in IA was observed between arms in the RDT. In the RDT, PGVL (n = 66) and PLB (n = 29) arms had similar rates of adverse events (AEs) (83 % and 93 %) and serious AEs (3 % and 4 %), with hypersensitivity AEs (39 % and 14 %, respectively) occurring more in the PGVL arm. For PGVL and PLB arms, most frequent AEs were arthralgia (14 % and 10 %), headache (12 % and 24 %) and fatigue (11 % and 10 %, respectively).

S108 Discussion: In RDT, the PGVL arm maintained blood Phe while PLB arm significantly increased blood Phe. There was an association from pre-treatment baseline to RDT entry of lower blood Phe and improved IA scores in subjects treated with PGVL. There were generally similar rates of overall AEs between treatment arms, except for increased hypersensitivity AEs with PGVL. Conflict of Interest declared.

P-173

Phase 3 PRISM-2 long-term extension evaluating efficacy and safety of pegvaliase for treatment of adults with phenylketonuria Levy H 2, Harding C 5, Longo N 6, Bilder D 6, Burton B 3, Zori R 4, Posner J 8, Thomas J 9, Olbertz J 1, Rosen O 1, Gu Z 1, Merilainen M 1, Weng H H 1, Dimmock D 7 1 BioMarin Pharmaceutical Inc, Novato, United States, 2Boston Child Hosp, Boston, United States, 3Lurie Child Hosp, Chicago, United States, 4Univ Florida, Gainsville, United States, 5Oregon Health and Science Univ, Portland, United States, 6Univ of Utah, Salt Lake City, United States, 7 Medical College Wisconsin, Milwaukee, United States, 8King’s College, London, United Kingdom, 9Univ Colorado School Med, Aurora, United States

Background: Pegvaliase (PGVL), PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase, converts phenylalanine (Phe) to trans-cinnamic acid and ammonia, and is a potential enzyme substitution therapy to lower blood Phe in phenylketonuria (PKU) patients. Methods: The ongoing PRISM-2 open-label long-term extension (OLE) enrolled subjects from PGVL phase 2 and phase 3 PRISM-1 studies. The OLE allowed individualized PGVL doses (5–60 mg daily) to optimize blood Phe. Blood Phe, inattention (IA; evaluated with IA domain of the Attention Deficit Hyperactivity Disorder Rating Scale) and safety were assessed at pre-treatment baseline (BL) and throughout the OLE. Results: At data cut (Jan 22 2016), 194 subjects were in OLE, with the majority (58 %) receiving PGVL at a daily dose of 40 mg (10 mg: 10 %; 20 mg: 18 %; 60 mg: 14 %). Mean (SD) change in blood Phe from pre-treatment BL to OLE was −415 (572), −801 (517) and −813 (556) μmol/L at OLE week 1 (n = 175), 17 (n = 109), and 41 (n = 90), respectively. Blood Phe < 360 μmol/L was achieved in OLE by 29 %, 58 % and 60 % of subjects at OLE week 1 (n = 175), 17 (n = 109), and 41 (n = 90), respectively. Mean (SD) change in IA score from pre-treatment BL to OLE was −3 (5), −5 (6), and −6 (6) points at OLE week 1 (n = 119), 17 (n = 73), and 41 (n = 74), respectively. The most frequent adverse events occurring in OLE were arthralgia (28 % of subjects), headache (24 %), and injection site reaction (23 %). Overall AE rates (events/ person-year) for any AE (19.4), SAE (0.1), and hypersensitivity AE (3.6) were fairly similar among dose groups. Discussion: Individualized PGVL long-term treatment was associated with sustained blood Phe reduction and sustained improved IA scores. Subjects had blood Phe reduction with individualized PGVL dosing of 5–60 mg daily and longer treatment duration, including those subjects unable to reach a blood Phe response in earlier PGVL studies. Overall safety profile of PGVL was fairly similar between dose groups. Conflict of Interest declared.

P-174

The sixth interim analysis of the Kuvan® Adult Maternal Paediatric European Registry (KAMPER): 9 pregnancies in PKU patients Feillet F 1, Lagler F B 2, Alm J 3, Muntau A C 4, Burlina A B 5, Belanger Quintana A 8, Alvarez I 7, Champigneulle A 7, Trefz F K 6 1 Hopital de Brabois Enfants, Vandoeuvre les Nancy, France, 2Paracelsus Medical University, Salzburg, Austria, 3Karolinska University Hospital, Stockholm, Sweden, 4Universitaetsklinikum Hamburg Eppendorf, Hamburg, Germany, 5Azienda Ospedaliera di Padova, Padova, Italy, 6University

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Children’s Hospital, Heidelberg, Germany, 7BioMarin Pharmaceutical Inc, London, United Kingdom, 8Hospital Ramon y Cajal, Madrid, Madrid, Spain Background: To describe the efficacy and safety of the use of sapropterin dihydrochloride in a group of 8 BH4-responsive pregnant women with phenylketonuria (PKU). Methods: Data were collected from the maternal sub-registry of KAMPER, which records data on the management, efficacy and safety of sapropterin in pregnant PKU patients. Results: Results of 8 patients (mean age 29.6; range 22.4–35.0, total of 9 pregnancies) are provided. From those, 4 were under a phenylalanine (phe)-restricted diet prior to becoming pregnant, and the other 4 were able to maintain blood phe under target range on unrestricted diets. Mean dose of sapropterin during pregnancy was 11.8 ± 5.9 mg/kg/day (range 3.3–21.0 mg) and the median duration of sapropterin use was 203 days. Blood phe data was available for 5 pregnancies; median values were within 120 and 360 μmol/L except for one patient who had a median value of 378 μmol/L only during her 1st trimester and another patient had a median value of 618 μmol/L only during her 2nd trimester. Three patients experienced a mild adverse event, one of which was considered related to study medication. All 9 of the pregnancies resulted in term live birth delivery of the infants with all infant conditions at birth reported as normal. An adverse event of mild severity was reported in 1 infant; jaundice together with ocular icterus, considered unrelated to sapropterin treatment. Of those patients for whom data was available, four breastfed; one remained on sapropterin and the other three did not while breastfeeding. Follow up data, obtained from 1 to 18 months after delivery, was available on 6 pregnancies, and no developmental issues were reported in the offspring. Discussion: In a small population of pregnant women with PKU, therapy with sapropterin during gestation was shown to be well tolerated and resulted in normal healthy births. Conflict of Interest declared.

P-175

The sixth interim analysis of the Kuvan® Adult Maternal Paediatric European Registry (KAMPER): interim results in PKU and BH4 deficiency patients Muntau A C 4, Lagler F B 2, Feillet F 1, Alm J 3, Burlina A B 5, Belanger Quintana A 9, Alvarez I 8, Champigneulle A 8, Trefz F K 6, Van Spronsen F J 7 Hopital de Brabois Enfants, Vandoeuvre les Nancy, France, 2Paracelsus Medical University, Salzburg, Austria, 3Karolinska University Hospital, Stockholm, Sweden, 4Universitaetsklinikum Hamburg Eppendorf, Hamburg, Germany, 5Azienda Ospedaliera di Padova, Padova, Italy, 6University Children’s Hospital, Heidelberg, Germany, 7University Medical Center of Groningen, Groningen, Netherlands, 8 BioMarin Pharmaceutical Inc, London, United Kingdom, 9Hospital Ramon y Cajal, Madrid, Madrid, Spain 1

Background: The KAMPER study is an observational, multi-centre drug registry designed to assess long-term safety of patients with hyperphenylalaninemia due to phenylketonuria (PKU) or BH4 deficiency treated with sapropterin up to 15 years. Methods: Data from 567 patients on growth, neurocognitive outcomes, dietary adherence, and long term sapropterin sensitivity are reported. Results: The sixth year interim analysis from 66 clinical sites in 8 countries included data from 523 PKU and 44 BH4 deficiency patients. The most common mutations identified in 377 PKU patients (n = number of patients with mutation on either allele) were p.Y414C (n = 73, 14.0 %), p.R261Q (n = 63, 12.4 %), and p.L48S (n = 60, 11.5 %), and for 3 BH4 patients were p.Y414C (n = 2, 4.5 %), PTS:IVS1-3C > G (n = 2, 4.5 %), and PTS:IVS3-37incC (n = 2, 4.5 %). Mean and median blood phe levels for patients 4–8 yrs were maintained near the recommended range of 120–360 μmol/L; older age groups had levels above this range, increasing with age though generally < 600 μmol/L. Dietary phe and natural protein intakes increased in all age groups by 1.5 to 2 times their intakes prior to sapropterin treatment. Blood Phe levels were generally lower in BH4 deficiency patients as compared to PKU patients. Median (Q1–Q3) sapropterin doses were 15.0 (10.3–19.0) mg/kg/day in PKU patients and 6.6 (5.0–12.0) mg/kg/day in BH4 deficiency patients. 428 AEs occurred in 174 PKU patients; 40 considered sapropterin related by investigators. The

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 most frequently reported were headache (5.2 %), nasopharyngitis (3.8 %), and abdominal pain (3.1 %). 12 BH4 deficiency patients experienced AEs; 4 experienced 6 SAEs, the rest were mild to moderate and none considered related to sapropterin. Discussion: Results show that sapropterin has a favourable safety profile. KAMPER provides a unique opportunity to gather a large collection of long-term follow up data related to sapropterin treatment in PKU and BH4 deficiency in 8 European countries. Conflict of Interest declared.

P-176

Pharmacological chaperones as an alternative treatment for phenylketonuria Aubi O 1, Underhaug J 1, Ying M 1, Knappskog P M 3, Hjelmdal E 1, Allegri G 2 , Thony B 2, Martinez A 1 1

Dept Biomedicine, Univ Bergen, Bergen, Norway, 2Dept Metab Dis, Univ Child Hosp, Zurich, Switzerland, 3C Med Gen-Mol Med, Haukeland Univ Hosp, Bergen, Norway Background: Phenylketonuria (PKU) is the most common inborn error of metabolism, caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Owing to the fact that the two current treatments of PKU, namely a strict phenylalanine (Phe) diet and the administration of sapropterin dihydrochloride present some pitfalls (neurodevelopmental or psychosocial problems and low responsiveness, respectively), new therapeutic strategies are accordingly needed. The use of LMW compounds that selectively bind to misfolded proteins and recover a native structure and function has proven to be successful in different diseases. For the particular case of PKU, the majority of the mutations in the enzyme PAH lead to misfolding. Hence, pharmacological chaperones (PC), acquired name for these molecules, could potentially be applied as therapeutic agents to correct this defect. We thus initiated a screening searching for stabilizers of PAH that could be developed into PC for PKU treatment. Methods: The screening consisted of a sequential multi-staged workflow comprising an initial high-throughput screening (HTS) of a commercial 10,000compounds library by differential scanning fluorimetry assay, a validation of the binding through surface plasmon resonance and an efficacy assessment in cultured cells constitutively expressing PAH. Subsequently, a further characterization of the mode and thermodynamics of the binding was performed. Result/Discussion: We obtained a total of 109 positive binders in the HTS which represent the 1.09 % of the entire library. In the aforementioned following stages, the number was reduced down to 2 candidates and the characterization phase resulted in the selection of our final hit compound. This compound displayed good affinity (KD ≈ 10 μM), enthalpically driven binding and excellent scores in cultured cells with an increase of 70.3 ± 9.7 % in PAH activity and 12.9 ± 1.5 % in PAH amount. Therefore, it is envisioned to be used as a PC for PKU after future derivatization.

P-177

A German multi-centre study of pregnancies of women with phenylketonuria (PKU) between 2000 and 2013—what do we know, what do we do, what comes out? Grohmann K 1, Maier E 2, Muehlhausen C 3, Ploeckinger U 4, Freisinger P 5, Das A M 6, Vom Dahl S 7, Rutsch F 8, Schwarz M 9, Leichsenring M 10, Spiekerkoetter U 11, Schick P 1, Buerger C 1, Gleich F 1, Burgard P 1 1

Univ Hosp Dept Gen Paediatr, Heidelberg, Germany, 2Dr von Hauner Child Hosp, Munich, Germany, 3Dept Pediatr, Univ Med Cent, Hamburg, Germany, 4 Cent Exc Rare Metab Dis, Charite, Berlin, Germany, 5 Child Hosp, Reutlingen, Germany, 6 Hannover Med School Paediatr Metab Med, Hannover, Germany, 7Univ Hosp Dept Gastroent, Hepat Infect, Duesseldorf, Germany, 8Univ Child Hosp Dept Gen Pediatr, Muenster, Germany, 9Practice

Int Med Metab Dis, Kaarst, Germany, 10Univ Child Hosp, Ulm, Germany, Univ Child Hosp Dept Paediatr Adolesc Med, Freiburg, Germany

11

Background: Strict metabolic control of pregnant women with PKU can prevent phenylalanine (phe)-induced embryofetopathy. Guidelines for treatment are based on evidence from carefully designed studies. Much less is known about how guidelines guide clinical practice. Methods: Retrieval of filed data in 11 German metabolic centres. Retrospective interviews with mothers. Standardized clinical and intellectual examination of children. Results: First pregnancies ending with childbirth in 80 women from a target population of 162 women with 280 pregnancies were analysed. Pregnancies were planned in 84 % and 78 % underwent preconceptional dietary training. Mean phe intake in the first trimester was 350 mg/day, and tripled afterwards. Target phe level was achieved in 90 % (64 % before conception, 26 % after conception). Preconceptionally, 42 % were off diet but reached target phe. With three exceptions (IQ = 69, 80, 80) childrens’ intellectual outcome was in the normal range (IQ: mean = 103.8, SD = 13.6). phe target levels (lower limit: 42–120 μmol/L; upper limit: 180–360 μmol/L), frequencies of blood phe control (1 to 12 times per month), outpatient appointments (0 to 13 per trimester), and the spectrum of parameters used for biochemical monitoring differed widely between centres. Discussion: Schemes for quality control of the clinical management of mild PKU are needed. Registries are not only necessary for understanding the natural history of treated IEM, but also for the growing number of pregnancies in women with IEM. Conflict of Interest declared.

P-178

Aggresomes formation and negative gain of function as alternative molecular mechanism in patients affected by phenylketonuria: implications for therapy Bonapace G 1, Moricca M T 1, Ceravolo F 1, Mascaro I 1, Strisciuglio P 2, Concolino D 1 1 Pediatrics, Faculty of Medicine, Catanzaro, Italy, 2Dept of Pediatrics University Federico I, Naples, Italy

Background: Phenylalanine hydroxylase (PAH also known as phenylalanine 4-monooxygenase EC 1,14,16,11) catalyzes the rate-limiting step in Lphenylalanine (L-Phe) catabolism in liver, using tetrahydrobiopterin (BH4) and dioxygen as additional cosubstrates. PAH mutations are associated with impairment of PAH activity, leading to accumulation of L-Phe in plasma, and neurological damage in untreated patients. Even though an early dietary treatment, consisting of a low protein diet with L-Phe restriction, can prevent the development of metabolic and pathological sequelae, the social burden of this treatment and reduced patients’ compliance, have led the search for new therapeutic approaches. To set a novel therapeutic approach for treating this disease we studied the aggregosome formation and the negative gain of function using the c.143C > T (L48S) PAH mutation as model. L48S is a well documented PAH misfolding mutation characterized by a progressive clinical worsening, with an inconsistent response to BH4. We hypothesized that in the patients carrying the L48S misfolding mutation in association with a second mutated allele that retains some residual enzymic activity the formation of solubile aggregates can take place and that these aggregates could trigger a generalized cell dysfunction explaining the observed worsening of the clinical phenotype. Methods: We transfected HEK 293 cells with PAH WT or PAH L48S and demonstrated the aggregosome formation and the loss of biological activity in the L48S transfected cells. Results: By using 1-deoxynojirimycin hydrocloride also described as a HSP70 inducers, we were able to decrease the amount of aggregated L48S protein in a dose response way in comparison to untreated cells, and to rescue the biological activity. Discussion: We discuss the possibility to study the aggresomes formation due to specific structural mutation as novel molecular mechanism in PKU with very interesting implications for the therapy.

S110 P-179

Neurobiological and functional benefits of a specific nutrient combination in phenylketonuria (PKU): proof of concept in the PKU mouse model Bruinenberg V M 1, Van der Goot E 1, Van Vliet D 2, De Vries M 1, Counotte D S 3, Kuhn M 3, Van Spronsen F J 2, Van der Zee E A 1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 P-181

Severe hyperhomocysteinemia leads to mitochondrial alterations in amygdala of rats Kolling J 1, Scherer E B 1, Siebert C 1, Dos Santos T M 1, Longoni A 1, Wyse AT S 1 1

UFRGS, Porto Alegre, Brazil

GELIFES, Univ Groningen, Groningen, Netherlands, 2Child Hosp, Univ Med Cent, Groningen, Netherlands, 3Nutricia Research, Utrecht, Netherlands 1

Background: The current method to manage PKU is a strict and early introduced diet of protein restriction and specific amino acid supplementation. However, even in treated PKU patients, the disorder manifests itself mostly at the level of the brain with changes in neurotransmitter metabolism, white matter integrity, synaptic functioning, and oxidative stress. One of the sites at which these four identified domains in PKU converge is the neuronal membrane. Therefore, the present study investigates the effects of a specific nutrient combination (SNC) containing precursors and cofactors for the synthesis of neuronal membrane on behavioral and neurobiological deficits in PKU mice (BTBR Pahenu2). Methods: To this end, we examined the impact of SNC in groups of 24 mice each (12 males, 12 females) with dietary treatment starting on postnatal day 31. The following six groups were compared: wild-type (WT), WT with SNC (WT + SNC), PKU high Phe (6.4 g/kg food), PKU high Phe + SNC, PKU low Phe (2.0 g/kg), PKU low Phe + SNC. Groups without SNC were fed an isocaloric control. The mice were tested in two learning and memory paradigms (novel object recognition [NOR] and spatial object recognition test [SOR]), and a motor performance test (balance beam) at three time points during the dietary intervention (3, 6 and 9 months after the start of the intervention). Results: Here we demonstrate that the diet containing SNC restored the ability of the PKU mice to learn the NOR, but SNC did not improve motor performance in PKU mice despite a significant lower performance compared to their WT litter mates. The SNC diet was nearly equally effective in PKU mice treated with high and low Phe in their diet. Discussion: Preliminary data show a beneficial effect of a SNC in a PKU mouse model. Future analysis of brain material will shed light on the mode of action of the SNC diet. Conflict of Interest declared.

Background: Severe hyperhomocysteinemia is caused by increased plasma levels of homocysteine (Hcy), a methionine derivative, and is associated with cerebral disorders. Creatine supplementation has emerged as an adjuvant to protect against neurodegenerative diseases, due to its potential antioxidant role. Here, we examined the effects of severe hyperhomocysteinemia on brain metabolism, and evaluated a possible neuroprotective role of creatine in hyperhomocysteinemia, by concomitant treatment with Hcy and creatine (50 mg/ Kg body weight). Methods: Hyperhomocysteinemia was induced in young rats (6-day-old) by treatment with homocysteine (0.3–0.6 μmol/g body weight) for 23 days, and then the following parameters of rat amygdala were evaluated: (1) the activity of the respiratory chain complexes succinate dehydrogenase, complex II and cytochrome c oxidase; (2) mitochondrial mass and membrane potential; and (3) the levels of necrosis and apoptosis. Results: Hcy treatment decreased the activities of succinate dehydrogenase and cytochrome c oxidase, but did not alter complex II activity. Hcy treatment also increased the number of cells with high mitochondrial mass, high mitochondrial membrane potential, and in late apoptosis. Importantly, creatine administration prevented some of the key effects of Hcy administration on the amygdala. Discussion: Our findings support the notion that Hcy modulates mitochondrial function and bioenergetics in the brain, and suggest that creatine might represent an effective adjuvant to protect against the effects of high Hcy plasma levels. Supported by CNPq.

P-182

Case report: potential hazards of betaine by a pyridoxine nonresponsive CBS deficiency Stepman H 1, D’Hooghe M 2, Stove V 1, Verloo P 3

07. Sulphur amino acid disorders

P-180 Maternal hypermethioninemia decreases brain-derived neurotrophic factor levels and the activities of energy metabolism enzymes in brain of rat pups Schweinberger B M 1, Rodrigues A F 1, Turcatel E 1, Pierozan P 1, Wyse AT S 1 1

UFRGS, Porto Alegre, Brazil

Background: In the present work we analyzed the effects of gestational hypermethioninemia on neuronal brain-derived neurotrophic factor levels and energy metabolism parameters (succinate dehydrogenase, complex II, and cytochrome c oxidase) in the brain from the offspring. Methods: Methionine (2.68 μmol/g body weight) or saline (control) was administered in Wistar female rats during all gestational period. After parturition, pups were killed at the 21st day of life for removal of encephalon. Results: Our results showed reduced brain-derived neurotrophic factor levels. Increased methionine levels during gestation were also able to reduce succinate dehydrogenase and complex II activities in the encephalon of the rat pups. Discussion: Our results are very important since they show that maternal hypermethioninemia may damage the brain during intrauterine life, which could prejudice the developing of central nervous system and cause behavior alterations to the offspring.

1 Lab Metab Dis, Ghent Univ Hosp, Ghent, Belgium, 2Dept Ped Neur, AZ SintJan Bruges-Ostend, Bruges, Belgium, 3Dept Ped, Div Ped Neur, Ghent Univ Hosp, Ghent, Belgium

Background: Cystathionine beta-synthase (CBS) deficiency is marked by the defective conversion of homocysteine to cystathionine. As the hallmarks are elevated homocysteine and methionine, therapy is focused on reducing the homocysteine and keeping the methionine range acceptable. By pyridoxine nonresponders, the therapy is mainly based on a methionine-low diet and betaine. By catalysing the formation of methionine, by giving a methyl group to homocysteine, concerns have been raised that betaine can elevate methionine to potentially harmful concentrations for the brain. However, only few case reports are described, starting with methionine concentrations of 1500 μmol/L in plasma. Case Report: A 2-year old boy with a sinus thrombosis and autistic manifestations was diagnosed with CBS deficiency. On diagnosis plasma levels of homocysteine were 264 μmol/L (RI 5–15) and of methionine 459 μmol/L (RI 11–76) . As methionine-restricted diet and pyridoxine were insufficient to control the homocysteine levels, betaine (2 times 1000 mg daily) was added. Results: In the follow-up of the child, agitated behaviour was marked. When the behaviour didn’t improve and the methionine concentration in plasma rose to 972 μmol/L, an MRI was performed. This showed diffuse white matter alterations with global diffusion restriction on ADC. Because of high concentration of methionine also in the cerebrospinal fluid (96 μmol/L with a RI of 1.4–6.7), betaine was stopped. There was also a deficiency of methyltetrahydrofolate in the cerebrospinal fluid, and folinic acid supplementation was

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 initiated. When methionine decreased in plasma, the behaviour of the child improved. Discussion: Our case describes that betaine may be associated with adverse effects and methionine concentrations of 1000 μmol/L. Special attention is required not only to homocysteine concentration, but also to methionine concentration in CBS deficient patients on betaine.

P-183

Characterization of a novel transgenic mouse model of CBS-deficient homocystinuria carrying the most common Qatari mutation p. R336C (c.1006C > T) Gallego-Villar L 1, Gupta S 4, Wang L 4, Yoshida S 1, Hyung-Ok L 4, Nasrallah G K 5, Ben-Omran T 3, Haberle J 2, Blom H J 1, Kruger W D 4 1 Div Metab Dis, Univ Child Hosp, Freiburg, Germany, 2Div Metab Dis, Univ Child Hosp, Zurich, Switzerland, 3Div Clin Gen Metab, Dept Ped, HMC, Doha, Qatar, 4Fox Chase Cancer Center, Philadelphia, United States, 5 Biomedical Research Center, Qatar Univ, Doha, Qatar

Background: Homocystinuria due to cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism caused by mutations in the CBS gene. The country of Qatar has the highest rate of CBS deficiency in the world (approximately 1 in 1,800 births) due to a specific founder mutation, p.R336C. Untreated homozygous patients for this mutation are clinically affected with severe mental retardation, thromboembolisms, ectopia lentis and bones abnormalities. Current treatment options are based on pharmacological therapy and life-long methionine-restricted diet. Compliance is poor in particular in adolescent and adulthood. Alternative treatment options are warranted. Here, we report the generation and characterization of a mouse model of CBS-deficient homocystinuria carrying the most common Qatari mutation p.R336C (c.1006C > T). Methods: Tg-R336C Cbs−/− mouse model in which the mouse CBS gene is inactivated and contains a transgene that expresses the human CBS p.R336C under the control of the zinc-inducible metallothionein promotor was generated as described previously (Wang L, et al. 2004). Serum metabolites and CBS activity in liver and kidney were measured using a Biochrom 30 amino acid analyzer. Protein levels in different tissues were analyzed by WB and tissue sections by immunohistochemistry. Results: This model exhibits a neonatal lethal phenotype in which the majority of mice die between 10 and 30 days of age. A surviving Tg-R336C Cbs−/− mouse has been characterized at 5 month of age. This animal exhibited alopecia and had extremely elevated serum homocysteine (438 uM). Analysis of liver showed that the R336C protein was expressed at near wild-type levels, but only had 12 % residual CBS activity compared to Tg-hCBS Cbs−/− controls. There was no sign of hepatopathy or renal damage in this mouse. Discussion: This mouse model will assist our understanding of the physiopathology of the disease in the Qatari population and improve our ability to evaluate alternative therapies in vivo.

P-184

Methylation of S-adenosyl-L-homocysteine hydrolase by PRMT1 impacts on the protein’s functional and structural properties Florindo C 1, Vieira J 1, Esse R 1, Ventura F V 1, Tavares de Almeida I 1, Castro R 1, Leandro P 1 1

iMedULisboa, Fac Pharmacy, Univ Lisboa, Lisboa, Portugal

Background: Protein methylation has been recognized to regulate protein’s function. This post-translational modification relies on Sadenosylmethionine (SAM) as the methyl donor, and on the activity of protein methyltransferases yielding the methylated protein and Sadenosylhomocysteine (SAH). Since SAH is an inhibitor of

SAM-dependent methyltransferases, excess SAH induces cell hypomethylation which has been associated with several diseases. SAH is further hydrolysed into L-homocysteine and adenosine by SAH hydrolase (SAHH). Interestingly, SAHH arginine residues have been suggested as a target for methylation by protein arginine methyltransferase 1 (PRMT1). Modulation of SAHH activity, by methylation, may constitute an additional control of intracellular SAH levels and cell methylation potential. In this work we aimed to confirm SAHH in vitro methylation and characterize, functionally and structurally, the unmethylated and methylated forms of the recombinant human protein (hSAHH). Methods: Human SAHH was produced in E. coli in fusion with a hexahistidyl tag and further purified by affinity chromatography. The purified hSAHH’s activity, binding capacity, susceptibility to limited proteolysis and thermostability were characterized. Methylation of hSAHH was performed according to standard protocols using SAM and PRMT1. Results: The hSAHH was obtained in high yield and with a purity grade >98 %. Differential scanning fluorimetry assays suggest that SAM is able to bind hSAHH. After hSAHH methylation and hydrolysis, HPLC quantification of released asymmetric dimethylarginine residues confirmed hSAHH as a target for PRMT1. Furthermore, methylated hSAHH presented lower catalytic activity and a more closed conformation. Discussion: By affecting enzyme activity and structural properties, hSAHH methylation by PRMT1 may constitute an additional cellular process for regulation of cell methylation potential. Work funded, in part, by iMed.ULisboa (FCT; UID/DTP/04138/2013)

P-185

Glucose-6-phosphate expression and activity is reduced by Sadenosylhomocysteine accumulation in HUVEC Caldeira Araujo H Almeida I 2

1 4

, Pimenta A 1, Rivera I

2 3

, Castro R

2 3

, Tavares de

1 CQM, University of Madeira, Funchal, Portugal, 2iMedUL, Fac Pharmacy, Univ Lisbon, Lisbon, Portugal, 3Dept Bioch Human Biol, Fac Pharmacy, Lisbon, Portugal, 4Fac Life Sciences, University of Madeira, Funchal, Portugal

Background: Hyperhomocysteinemia (HHcy) is an independent risk factor for vascular disease. While some studies indicate that homocysteine (Hcy) causes endothelial cell injury by promoting oxidative damage, others point to a reduction on the methylation potential, resulting from a decrease on the S-adenosylmethionine (AdoMet): S-adenosylhomocysteine (AdoHcy) ratio, as the pathogenic mechanism. Interestingly, recent studies showed that excess AdoHcy promotes oxidative stress in endothelial cells by decreasing glutathione peroxidase (GPX1) translation. GPX1 is an important antioxidant enzyme. Glucose-6-phosphate dehydrogenase (G6PD) is another important antioxidant enzyme, which increases its activity in normal vascular endothelium as a result of oxidant stress. Here we investigated whether a hypomethylation environment caused by intracellular accumulation of the Hcy precursor, AdoHcy, impacts G6PD expression and activity. Methods: Our study was conducted in human umbilical vein endothelial cells (HUVEC) cultured under normal and hypomethylating conditions created by a pharmacological approach. G6PD expression was evaluated by Western-blot and enzyme activity was determined spectrophotometrically, before and after 48 h incubation, in control or in culture medium supplemented with the pharmacological inhibitor of Sadenosylhomocysteine hydrolase, adenosine-2,3-dialdehyde. Results: Our results showed that under hypomethylated conditions, G6PD expression is reduced in 50 % when compared to control conditions, whereas its activity decreases from 48.71 to 39.15 Unit/min/mg. Discussion: In conclusion, our results show that a hypomethylating environment due to an increase in the AdoHcy pool impairs G6PD expression and activity in HUVEC.

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08. Other amino acid disorders

P-188

P-186

Decreasing mitochondrial ATPase6 and 8 genes’ expression in infantile Pompe patients

Molecular investigation of glutaric aciduria type 1 in Iran Houshmand M 3, Pirzadeh Z 1, Aryani O 2, Ghasemi F 2, Salehpour S H 1

Sanjari E 2, Bahreini F 1, Akrami M 1, Houshmand M 2

1

1

2

2

Shahid Beheshti University of Medical Sci, Tehran, Iran, Islamic Republic of, Medical Genetic Center, Tehran, Iran, Islamic Republic of, 3NIGEB, Tehran, Iran, Islamic Republic of Background: Glutaric acidemia, type I (GA I), was first described in 1975. The disease is caused by a genetic deficiency of the enzyme glutaryl-CoA dehydrogenase (GCD), which leads to the buildup of Glutaric acid in the tissues and its excretion in the urine of affected patients. GCD is involved in the catabolism of the amino acids lysine, hydroxylysine, and tryptophan. Over 200 cases of GA I have been reported in the medical literature. GA I is one of the most common organic acidemias and has an estimated incidence of about 1 in 50,000 live births. Because of the initial slow progression of clinical symptoms, GA I is frequently undiagnosed until an acute metabolic crisis occurs. Methods: A total of 25 unrelated patients suspected of having GA1 were investigated in our study. Genomic DNA was extracted from peripheral blood cells of the 25 probands which were biochemically and/or clinically and/or neuro-radiologically suspected to GA1. 15 of them had elevated glutaric acid in the urine organic acid test. PCR and direct sequencing of all 11 exons and their flanking region of the GCDH gene were examined. Some of them were investigated for known mutation in the other family members. Results: 15 patients had homozygous mutations and 10 patients were normal for GCDH gene. Discussion: 60 % known mutationn were found in our 15 patients. 80 % can be detected by 4 exons sequencing, so for molecular investigations exon 6, 7, 8, 10 are good choices for beginning of analysis. 33 % were mutations in exon 7, so because of the cost of genetic diagnosis we suggest that investigation begin with this exon. Pro 348 Leu was most detected (20 %). 40 % are new mutations which will be investigated for phenotype genotype correlations.

Medical School of Tehran University, Tehran, Iran, Islamic Republic of, NIGEB, Tehran, Iran, Islamic Republic of

Background: Pompe disease (PD) or glycogen storage disease type II is a rare neuromuscular genetic disorder. This disease has two forms classified by age of onset: infantile and late - onset. Previous studies show abnormal mitochondria function in Pompe patients but they did not focus on mitochondrial genes. This study compares infantile and adult patients simultaneously and evaluates mitochondrial variants in ATPase6 and ATPase8 genes and their expression in Iranian Pompe patients. Methods: Sequence of mitochondrial ATPase6 and ATPase8 genes and their expression were analyzed in 30 Pompe patients and 100 controls. All variants were analyzed by Finch TV software. The results were compared with controls and the Cambridge reference sequence (http://www.mitomap.org/) using the Clustal X program. The expression of ATPase6 and ATPase8 were evaluated by quantitative RT-PCR in patients and healthy controls. Results: This study showed that all mitochondrial variants in infants were different from adults (69 % in infants, 19 % in adult and 12 % in both groups), except three of them, A8860G, A8701G and A8524C. Expression of mtATPase6 gene was decreased in infantile patients significantly, while ATPase8 gene was decreased significantly in both patient groups compare to the controls. Discussion: Frequency of mitochondrial variants in infantile Pompe patients was more frequent than adult patients, and the type of that variants were different in both groups. Expression of mt-ATPase 6 and mtATPase8 genes were decreased in infantile groups compared with controls. Therefore, involvement of mitochondria and mitochondrial dysfunction in infantile Pompe patients is more severe than adults.

P-189 P-187 Mitochondrial D-loop variants and copy number in Pompe patients Houshmand M 2, Bahreini F 1, Modaresi M H 1, Akrami M 1

Oral glucose tolerance tests in Japanese citrin-deficient siblings before and after MCT-oil supplementation Otsuka H 1, Sasai H 1, Nakama M 2, Aoyama Y 3, Abdelkreem E 1, Kawamoto N 1, Kawamoto M 1, Ohnishi H 1, Numakura C 4, Hayasaka K 4, Fukao T 1 2

1

Medical School of Tehran University, Tehran, Iran, Islamic Republic of, NIGEB, Tehran, Iran, Islamic Republic of

2

Background: Pompe disease is a rare neuromuscular genetic disorder, with two classified forms: early and late-onset. For over two decades mitochondria have been recognized as an important contributor to an array of neuromuscular diseases. This study compared mitochondrial copy number and mitochondrial displacement-loop sequence in infantile and adult Pompe patients. Methods: Sequence of D-loop on mitochondria was analyzed by PCR and direct sequencing methods to detect possible variations in 28 Pompe patients and 100 controls in the retrospective study. Results of the study were compared with healthy controls and the Cambridge reference sequence. Real-time PCR was used to quantify mitochondrial DNA copy number. Results: Among 60 variants, 38 (63.3 %) were in infants, 14(23.3 %) in adults and 8(13.3 %) in both groups. Mitochondrial copy number in infantile patients was lower than adult patients (p < 0.05). A significant difference was seen between two groups in 11 SNPs. 318 in. CCC was seen in patients as a novel variant and six SNPs were found as normal variants in controls. There was inverse relationship between Mt copy number and Dloop variant number. Discussion: The 318 in. CCC was detected as a new mitochondrial variant in Pompe patients. Mt copy number had inverse correlation with D-loop variant number. A significant difference was seen between two groups in 11 SNPs.

1 Dept Pediatr, Gifu Univ, Gifu, Japan, 2Div Clin Genet, Gifu Univ Hosp, Gifu, Japan, 3Dept Biomed Sci, Chubu Univ, Kasugai, Japan, 4Dept Pediatr, Yamagata Univ, Yamagata, Japan

Background: Citrin deficiency, an inherited defect in the liver-type mitochondrial asparate/glutamate carrier isoform (citrin) may cause an impairment of glycolysis due to a decrease in the cytosolic NADH/NAD+ ratio. Cases and Methods: A Japanese boy, whose chief complaint was recurrent hypoglycemic episodes was suspected to have citrin deficiency due to characteristic food preference. His younger sister also had similar food preference. Both were diagnosed as citrin deficient by genetic analysis. For better understanding of their glucose metabolism, they underwent oral glucose tolerance test (OGTT) at 12 and 7 years of age. OGTT was repeated after 4 months of medium-chain triglyceride (MCT)-oil supplementation (10 ~ 20 ml/day). In OGTT, 1.75 g/kg glucose was used. Blood glucose, ammonia, lactic acid, pyruvic acid and insulin were measured before start, then every 30 min until 180 min. Results: In the first OGTT, they kept blood glucose levels until 180 min. At 210 min, they both vomited, felt ill, and their blood glucose decreased to 2.9 and 2.8 mmol/l in the brother and sister, respectively; both siblings improved after 20 % glucose infusion. In the second OGTT after MCT supplementation, there were no clinical symptoms or lowered blood glucose even after 240 min. Discussion: We report oral glucose tolerance test in siblings with citrin deficiency. In general, patients with citrin deficiency dislike too much

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 carbohydrate intake. We performed this test to uncover the cause behind the repeated hypoglycemia in these patients. Our results suggest that excess glucose intake can induce hypoglycemia, as well as fasting (ketotic) hypoglycemia, in patients with citrin deficiency and that MCT-oil supplementation may protect from such hypoglycemia.

P-190 Pyridoxine-dependent epilepsy (PDE): α-amino adipic semialdehyde (AASA) levels and development with triple therapy from day 6 onwards Lunsing R J 1, Heineman K R 2, Heiner-Fokkema M R 3, Bok L A 4, Struys E A 6, Van Rijn M 5, Derks T G J 5, Van Spronsen F J 5 1 Dept Paediat Neurol, Univ Med Center, Groningen, Netherlands, 2Dept Neurol, Treant Hosp, Stadskanaal Emmen, Netherlands, 3Dept Lab Med, Univ Med Center, Groningen, Netherlands, 4Dept Paediat, Maxima Center, Veldhoven, Netherlands, 5Dept Metab Dis, Univ Med Center, Groningen, Netherlands, 6Metab Unit, VU Univ Med Center, Amsterdam, Netherlands Background: PDE is caused by AASA dehydrogenase deficiency. Pyridoxine diminishes seizures but doesn’t normalize IQ. The usefulness of additional treatment with dietary lysine restriction and/or arginine supplementation (triple therapy) is debated. Urinary AASA excretion is considered the biochemical marker for therapy control. Aim: To evaluate the effect of triple therapy started at neonatal age. Case Report: A girl born at term had onset of seizures at day 1. At day 3, pyridoxine 30 mg/kg/d was started and seizures stopped. At day 6, lysine restriction and arginine supplementation were started. Patient underwent neurological follow-up. Development was estimated by ‘van Wiechenschema’ (vWS), which is based on development of healthy full-term-born children, and at 1 year by Infant Motor Profile (IMP). Urinary AASA was measured by LC-MS/MS. Results: Triple therapy gave no side effects. At day 4 she showed little spontaneous movements, at 4 months she was abrupt and stiff in her movements. Neurological examinations were normal from 4 months onwards. She scored age appropriate at the vWS. The total IMP score at 12 months was 92 (mean for that age 88, standard deviation 5); domains variation and performance were lower than average, domains adaptability, fluency and symmetry had high scores. AASA before starting therapy was 58 mmol/mol creatinine (reference 50,000 ug/L) just prior to diagnosis of HCC. 14 patients received NTBC. Urinary succinylacetone levels remained undetectable in all patients though blood NTBC levels of some patients (7) were below the ‘therapeutic range’. Under low-protein diet tyrosine levels were < 700 μM. 3 patients received liver transplant one of which was due to HCC. One child with HCC could not be transplanted and expired. Conclusion: There was a high rate of mortality and malignancy in our cohort, probably due to late diagnosis and treatment. Hepatosplenomegaly, failure to thrive and cirrhosis of liver were the most common features. Imaging should be performed as soon as there is an unexpected rise in AFP. Regular monitoring of SA, tyrosine and NTBC levels are essential. The ‘therapeutic range’ of NTBC has to be redefined.

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The biochemical findings suggested MAAI deficiency due to a defect in the GSTZ1 gene. The Illumina HiSeq platform was used to sequence the gene captured by the TruSight One Panel target enrichment system (Illumina). Analysis was performed with an in-house pipeline. Quality analysis of coverage data revealed a pattern consistent with a homozygous deletion of exons 3, 4, 5 and 6. This was confirmed by targeted microarray analysis (Affymetrix CytoScan HD array). This deletion has not been previously described in the literature or in any database; as it removes a big part of the gene it is likely to be pathogenic. Discussion: Although it is not clear whether MAAI deficiency has caused the neurodevelopmental problems, this case adds to our knowledge of the phenotype of MAAI deficiency.

Leucine levels in maple syrup urine disease (MSUD) from a single centre in the United Kingdom Gribben J 1, Billmore K 1, Slabbert A 1, Champion M P 2, Lemonde H 2, Mundy H 2 1 Dietetics Dept, St Thomas’ Hosp, London, United Kingdom, 2Ctr Inh Met Dis, Evelina Children’s Hosp, London, United Kingdom

Background: Optimal control of leucine concentrations in MSUD disease is essential for maximising neurocognitive outcomes. In 2014 Frazier et al. published guidelines recommending lowering the leucine treatment range to 75–200 μmol/L for patients ≤5 years and 75– 300 μmol/L for >5 years. In 2013, the UK Expanded Newborn Screening (ENBS) guidelines recommended the range 200– 400 μmol. In 2015, this was later reduced to 150–300 μmol/L and reported the typical leucine intake for classical MSUD as 300 mg/day (6 × 50 mg leucine exchanges). We have adopted these guidelines for our MSUD patients aged 5 years and under. Aim: To audit leucine monitoring results to determine if the lower treatment range was achievable. Methods: A 12 month retrospective review of all blood spot leucine levels (including during illness), number of 50 mg leucine exchanges per day and frequency of samples. All classical MSUD patients 5 years and under were included. Results: Six patients were identified (median age 4.75 years, range 0.7–8 years). All diagnosed in the neonatal period (median age 13 days, range 7– 17 days), median screening level 3635 (range 1153–4600 μmol/L). The mean of each patient’s 12 month leucine monitoring results was determined (median 210, range 178–290 μmol/L). The proportion of leucine concentrations below 300 μmol/L for each patient was also determined (median 75 %, range 60– 92 %). The median number of samples per patient received in 52 weeks was 64 (range 37–76). The median number of mean 50 mg leucine exchanges per day for each patient was 7 (range 5–13). The median peak leucine level during illness was 765 (range 554–895 μmol/L). Discussion: Our data shows that the UK ENBS lower treatment range of 150– 300 μmol/L is achievable without having to overly restrict leucine exchanges. P-194

A case of maleylacetoacetate isomerase deficiency Preece M A 1, Hardy C 1, Hutchin T 1, Santra S 2, Vijay S 2, Antoniadi T 3, McMullan D 3 1

Biochem Genetics, Children’s Hospital, Birmingham, United Kingdom, Clinical IMD, Children’s Hospital, Birmingham, United Kingdom, 3 Regional Genetics, Womens Hospital, Birmingham, United Kingdom 2

Background: Maleic aciduria leading to a diagnosis of maleylacetoacetate isomerase (MAAI) deficiency. Case Report: An 18 month old girl with developmental delay, difficult behaviour, irritability and autistic features. Results: Urine organic acids showed hydroxyketoheptanoate, diketoheptanoate and a trace of succinylacetone (SA). Further investigation for tyrosinaemia type 1 showed normal amino acids, liver function tests and coagulation. AFP was slightly increased (21kU/L: normal < 10) but lectin reactive-AFP was normal. Liver and brain MRI showed no significant findings. Plasma SA and urine 5-aminolevulinic acid were slightly increased (2.20umol/L; ref range < 0.1 & 22.1umol/mmol creat; ref range < 5.2 respectively). Apart from SA and metabolites, urine organic acids showed no phenolic acids but maleic acid was increased. Fibroblast fumaryl acetoacetase and sequencing of the FAH gene were normal. Treatment with low dose nitisinone (0.15 mg/kg/day) and a low tyrosine diet were commenced. The biochemical abnormalities resolved except AFP which remained slightly increased.

P-195

Hyperprolinemia as a clue in the diagnosis of a patient with a psychiatric disorder Duarte M 4, Moreira A 3, Antunes D 1, Ferreira C 5, Correia H 5, Sequeira S 2, Marques M 4 1 Genetic Dept, Hosp Dona Estefania, Lisbon, Portugal, 2Metab Unit, Ped Dept, Hosp D Estefania, Lisbon, Portugal, 3Neuroped Dept, Hosp Dona Estefania, Lisbon, Portugal, 4Child and Adol Psy Dep, Hosp D Estefania, Lisbon, Portugal, 5Unit Cyto Hum Gen Dept, INSA, Lisbon, Portugal

Background: Over the last few years, microdeletions of the 22q11.2 region responsible for DiGeorge syndrome, or velocardiofacial syndrome, have been increasingly related to neuropsychiatric disorders including schizophrenia and bipolar disorders. These signs seem to be related to certain genes located in the hemideleted region as the proline dehydrogenase (PRODH) and the catecholo-methyltransferase (COMT) genes. The PRODH or proline oxidase deficiency is responsible for hyperprolinemia type 1 (HPI) also causing psychiatric manifestations. Case Report: We describe a 17 year old boy with previous mild psychomotor and speech delay, mild cognitive impairment, and obsessive behaviours who started his adolescent psychiatric care presenting irritable mood and aggressive behaviour with schizophrenia symptoms that scored a “severely ill” level PANSS assessment. Symptoms got worse when he was treated with valproic acid and plasma aminoacids showing increase in alanine and proline, suggested a mitochondrial involvement of the proline metabolic pathway. Results: Mild dysmorphia suggested a possible 22q11.2 deletion genetically confirmed involving both the PRODH and COMT regions. HPI that can present with psychiatric features is however a recessive disorder and therefore the symptoms could not be solely explained by this genetic deletion. Additional investigations also showed disclosed a p.L289m (c.1865 T > A) mutation in the PRODH gene. Discussion: We believe that the association of this mutation together with the 22q11.2 deletion would lead to a decrease of functional protein. Although it may be difficult to diagnosis chromosomal abnormalities in patients with no clear malformations and mild dysmorphic features as in this patient we emphasize need to investigate the aetiology in patients with psychiatric symptoms, especially if they have other systemic manifestations such as developmental delay or psychotic symptoms, as it may be important in the management of the patients.

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A novel BCAT2 mutation causes hypervalinaemia/hyperleucineisoleucinaemia in a boy with a developmental disorder with autism Knerr I 1, Urquhart J 2, Hughes J 1, Rogers Y 1, Jones S 2, Lynch S A 3, Treacy E 1, Mayne P D 4, Banka S 2, Olpin S 5 1 Nat Centr Inher Met Dis TSCUH, Dublin, Ireland, 2Centre for Genomic Medicine, Manchester, United Kingdom, 3Genetics Department TSCUH,

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P-198

Background: The first step in the enzymatic breakdown of branched-chain amino acids (BCAA) is catalyzed by the two BCAA transferases (BCAT), BCAT1 and BCAT2. The pathophysiological consequences of hypervalinaemia/hyperleucine-isoleucinaemia remain to be elucidated. A possible underlying gene defect has only recently been reported in an adult with a confirmed mutation in the BCAT2 gene. Case Report: We present biochemical and molecular data along with fibroblast studies in the probable second confirmed human case of BCAT2 deficiency. Our patient is an adolescent boy who presented with developmental delay and autism. Metabolic work-up revealed profoundly elevated plasma BCAA concentrations (leucine 3446 microM/L, valine 3935, isoleucine 2774, alloisoleucine not detected). Maple syrup urine disease was ruled out. Results: Fibroblast studies showed reduced valine and leucine oxidation. Targeted Sanger sequencing revealed a novel homozygous in-frame deletion in the BCAT2 gene in this boy and heterozygous carrier status of a parent. Discussion: In describing the probable second patient with BCAT2 deficiency with a different phenotype compared to the first published case who had presented with headache and memory loss, our findings suggest that BCAT2 deficiency also causes a novel developmental disorder with autism. We were able to demonstrate residual enzyme activity in fibroblasts which could be a function of the isoenzyme BCAT1.

Resistant dystonia in cognitively normal child

P-197

Hyperglycinemia as diagnostic marker for iron-sulfur cluster pathway anomaly in two sisters with severe encephalocardiomyopathy and a novel GLRX5 missense mutation: a case report Van Noolen L 1, Benoist J F 7, Hardy G 1, Douchin S 6, Dubois F 4, Besson G 5, Jouk P S 2, Acquaviva C 3, Faure J 1, Corne C 1, Dieterich K 2 1 Biochimie Genetique Moleculaire, CHU Grenoble Alpes, France, 2Genetique Clinique, CHU Grenoble Alpes, France, 3 Maladies Hereditaires du Metabolisme, CHU Lyon, France, 4Neuropediatrie, CHU Grenoble Alpes, France, 5Neurologie Adulte, CHU Grenoble Alpes, France, 6Cardiologie Pediatrique et Foetale, CHU Grenoble Alpes, France, 7 BiochimieHormonologie, Hop Robert Debre, Paris, France

Background: Nonketotic hyperglycinemia (NKH) is characterized by deficient enzyme activity of the glycine cleavage system. Typical mutations (GLDC, AMT genes) are associated with a severe neonatal neurological spectrum. Variant NKH has been recently described with genetic mutations affecting lipoate biosynthesis and iron-sulphur cluster biogenesis. We report on two sisters with a severe neurological and cardiac phenotype of variant NKH caused by a novel homozygous GLRX5 missense mutation. Case Report and Results: Both sisters are born from Turkish consanguineous parents. The elder died at 4 months of age (moa) from acute cardiac failure. Plasma glycine level was elevated. Her sister had axial hypotonia from birth and presented at 4 moa with acute cardiac insufficiency. Cerebral MRI showed T2 hyperintense lesions in the periventricular white matter, the corticospinal tracts and the corpus callosum. The cerebellum was hypotrophic. Metabolic screening (lactate-pyruvate ratio, acid maltase, very long chain fatty acid, acylcarnitines) and mitochondrial investigations were normal. Glycine levels and glycine plasma/CSF ratio were high. Analysis of typical NKH genes was normal. After this episode she showed severely delayed psychomotor milestones, hypotonia and seizures. At 32 moa she had a fatal episode of acute cardiac failure. Post-mortem analysis of NFU1, BOLA3 and GLRX5 genes revealed the homozygous c.347G > A/ p.Gly116Asp mutation in GLRX5. Discussion: GLRX5 mutations have hitherto only been described in three patients with NKH and in two patients with adult-onset sideroblastic anemia. Though clinical signs differ from symptoms described with GLRX5 mutations to date, these have been reported with mutations in other genes of the ironsulfur cluster pathway. Interestingly the two sisters presented with microcytic anemia from birth not linked to iron depletion or thalassemia. Our findings expand the phenotypic spectrum of GLRX5 mutations.

El Habbas M 1, Jans J J M 2, Sechter C 3, Dessein A F 4, Cuisset J M 5, Dobbelaere D 1, Verhoeven-Duif N M 2, Mention K 1 1 Div Metab Dis, Lille Univ Hosp, Lille, France, 2Genetic Dept, Univ Medisch Cent Utr, Utrecht, Netherlands, 3Div End Dis, Besancon, France, 4Bio Lab, Lille Univ Hosp, Lille, France, 5Div Neuro Dis, Lille Univ Hosp, Lille, France

Background: Serine-deficiency disorders are caused by deficiencies in phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). L-serine biosynthesis plays an essential role in the development and functioning of the central nervous system. Congenital microcephaly, seizures and severe psychomotor retardation are symptoms of serine deficiency. Early diagnosis and treatment may be beneficial in preventing and ameliorating symptoms. Case Report: We report an atypical case of 3-phosphoglycerate dehydrogenase deficiency diagnosed at the age of 7 years in a patient who presented shortly after birth with hypotonia with developmental delay. At 1 year of age, he exhibited progressive spastic diplegia with dystonia of his lower limbs, not improving on neurological treatment. He had no microcephaly, no seizure and no cognitive impairment. The dystonias aggravated at the age of 7 years when metabolic work up was done. Results: Fasting amino acid chromatography revealed a serine deficiency in blood and cerebrospinal fluid (CSF). The activity of PGDH was reduced in fibroblasts. Genetic analysis revealed one heterozygous mutation in the PGDH gene. Treatment with serine (150 mg/kg/d) resulted in normalization of plasma serine values without improvement of spasticity and dystonia. Discussion: This case study highlights the atypical presentation; therefore, CSF and plasma serine levels should be measured in every case of spasticity, neurodevelopmental delay and dystonia as prompt treatment with L-serine may significantly impact the outcome of the disease.

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In vivo evidence that glycine disturbs MAPK signaling pathways and decreases Tau protein phosphorylation and synaptophysin content in rat brain Leipnitz G 1, Moura A P 1, Parmeggiani B 1, Grings M 1, Cardoso G M 1, Pletsch J T 1, Gasparotto J 1, Seminotti B 1, Moreira J C F 1, Gelain D P 1, Wajner M 1 2 Dept Bioq, ICBS, UFRGS, Porto Alegre, Brazil, 2HCPA, Porto Alegre, Brazil

1

Background: Nonketotic hyperglycinemia (NKH) is caused by deficient activity of glycine cleavage system leading to severe neurological impairment and glycine (GLY) accumulation in the central nervous system of patients. Since the mechanisms involved in the pathophysiology of brain injury observed in NKH are not totally established, we investigated the in vivo effects of GLY administration on signaling pathways and neuronal damage markers in rat brain. Methods: 30-day-old Wistar rats received a single intracerebroventricular injection of GLY (5 mmol) or NaCl (control group) and were euthanized 30 min or 24 h after the administration. Cerebral cortex and striatum were dissected and used to measure the immunocontent of synaptophysin, and the phosphorylation of Tau protein and of the mitogen-activated protein kinases (MAPK) p38, ERK1/2 and JNK. Histopathological analysis was also performed in rat brain in order to assess glial fibrillary acidic protein (GFAP), a marker of glial reactivity, and to evaluate myelin structure by fluoromyelin staining. Results: Our results showed that GLY decreased the phosphorylation of Tau in cerebral cortex and striatum 30 min and 24 h after its administration. Furthermore, synaptophysin content was decreased by GLY in striatum at

S116 30 min, and in cerebral cortex at 24 h after the injection. GLY also decreased p38, ERK1/2 and JNK phosphorylation 30 min after its administration in both brain structures evaluated. We further verified that GLY-induced decrease of p38 phosphorylation in striatum was attenuated by the pre-treatment with the NMDA receptor antagonist MK-801. In contrast, GFAP and fluoromyelin staining were not altered by GLY infusion. Discussion: It may be presumed that reduced phosphorylation of MAPK associated to neuronal injury induced by GLY may contribute, at least in part, to the neurological dysfunction observed in NKH. Supported by: CNPq, CAPES, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP IBN-Net and INCT-EN.

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Evaluation of dynamic thiol/disulfide homeostasis as a novel indicator of oxidative stress in maple syrup urine disease patients under treatment Zubarioglu T 1, Cansever M S 2, Kiykim E 1, Neselioglu S 3, Erel O 4, Aktuglu-Zeybek A C 1 1 Div Nutr Metab Dis, Ist Univ Cer Med Fac, Istanbul, Turkey, 2Cent Lab, Ist Univ Cer Med Fac, Istanbul, Turkey, 3Div Bio Chem, Ank Ata Edu Res Hosp, Ankara, Turkey, 4Div Bio Chem, Univ Yil Bey Med Fac, Ankara, Turkey

Background: Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by deficiency of branched-chain α-keto acid dehydrogenase complex. Although accumulation of toxic metabolites is associated with neurological symptoms, mechanisms underlying the brain damage remain unclear. Aim of this study is to evaluate thiol/disulphide homeostasis as a novel indicator of oxidative stress in MSUD patients under treatment. Methods: 20 patients with MSUD and 20 healthy individuals were included in the study. All the 20 MSUD patients had been regularly following up, treated with a protein restricted diet supplemented with specific formula and had a good metabolic control. Serum native thiol (−SH), total thiol (−SH + −S-S-) and disulphide (−S-S) levels were measured in all subjects. Disulphide/native thiol, disulphide/total thiol and native thiol/total thiol ratios were calculated from these values. Simultaneous blood sampling for plasma quantitative amino acid analysis was performed in both groups. Results: Any significant difference was not observed in −SH, −SH + −S-S-, −S-S levels between two groups. In addition no increase of disulphide/native thiol and disulphide/total thiol ratios which were indicators of oxidative stress was detected in patient group. Discussion: This study is the first study that evaluates dynamic thiol-disulphide homeostasis as an indicator of oxidative stress in MSUD patients. In recent studies, it has been claimed that oxidative stress should be responsible from neurotoxicity even in treated patients. Reduction in thiol levels and an increase in dynamic disulphide bonds were found to be associated with oxidative stress in several diseases. In our study, dynamic thiol/disulfide homeostasis status in our patient group showed that providing the good metabolic control in MSUD patients could prevent oxidative stress. Under regular follow-up and good compliance with diet, additional antioxidant therapies should not be necessary.

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Evolution of hereditary tyrosinemia type I: a description of eight cases Hazazi A 1, Khiari M E 1, Chaou M 1, Zeroual Z 1, Atek L 1, Tari S 1, Mouhand Oussaid A 1, Bouskia Y 1, Khati A 1, Benhalla K N 1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Results: During this period, we have collected 8 cases of TH I. 42 % were girls and 58 % of boys. The age of patients ranged from 1 month–5 years. The onset of symptoms was in 70 % of cases before the age of 2 months. The initial symptoms were: abdominal distension with vomiting (42 %), edema with ascites (40 %), hemorrhagic syndrome (18 %). The main clinical signs were: hepatomegaly (50 %), splenomegaly andedema and ascites syndrome (42 %), signs of rickets (28 %). All patients underwent laboratory tests (CBC, inflammatory balance, liver function, kidney function and viral serology) and dosage of tyrosine. The main biological features were: hepatocellular insufficienc (85 %) associated with moderate cytolysis syndrome (57 %) hypoalbuminemia (40 %). Renal function was preserved in our patients and viral serology was normal. alphaFP greatly was increased in 71 % of cases. All our patients had increased urinary tyrosine and succinylacetone. 57 % of patients with heterogeneous liver nodules on ultrasound data and portal hypertension signs in the most cases. No liver biopsy was performed. All patients were treated with nitisinone (0.6–1.2 mg/kg/d). Good response was noted within a period of 6 to 9 months with normalization of liver function but in long term, 50 % of cases presented cirrhosis with portal hypertension and in one case 2 relapses. A complicated patient died of a hepatoblastoma after transplant. Discussion: Tyrosinemia remains a challenge in our context, requiring urgent diagnosis and management.

P-202

Effect of chronic administration of L-tyrosine on brain-derived neurotrophic factor and nerve growth factor levels in the brain of rats treated with antioxidants Gomes L M 1, Carvalho-Silva M 1, Scaini G 1, Teixeira L J 1, Rebelo J 1, Schuck P F 2, Ferreira G C 3, Streck E L 1 1

Lab Bioenergetics, UNESC, Criciuma, Brazil, 2Lab IEM, UNESC, Criciuma, Brazil, 3Lab Neurochem, UFRJ, Rio de Janeiro, Brazil Background: Tyrosinemia type II is a rare disease caused by a mutation in the gene encoding the enzyme tyrosine aminotransferase (TAT), which is responsible for the metabolism of tyrosine. Thus, there is accumulation of tyrosine and/or their toxic metabolites. Studies show that patients affected by inborn errors of metabolism usually present with clinical cognitive deficit and oxidative stress; neurotrophins play a crucial role in memory and learning. In the present study we aimed to evaluate brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in brain of rats submitted to chronic administration of L-tyrosine and treated with antioxidants. Methods: Wistar rats were divided into 3 groups: control, L-tyrosine, L-tyrosine + antioxidants N-acetylcysteine (NAC) + deferoxamine (DFX). The animals received L-tyrosine (500 mg/kg of body weight, i.p., 12/12 h), NAC (20 mg/kg, s.c., 12/12 h) and DFX (20 mg/kg, s.c., once every other day) from the 7th to the 28th postnatal days (control group received saline solution). Twelve hours after the last administration, the animals were killed by decapitation; cortex, hippocampus and striatum were isolated for analysis of BDNF and NGF levels. Results: Chronic administration of L-tyrosine decreased BDNF levels in hippocampus and striatum; the antioxidants NAC + DFX were not able to prevent this effect. NGF levels were decreased only in striatum; the antioxidants NAC + DFX prevented NGF diminution in this brain area. Discussion: The results showed that chronic administration of L-tyrosine decreased neurotrophins levels in rat brain. We hypothesize that oxidative stress can interact with BDNF and NGF systems to modulate synaptic plasticity and cognitive function. The present results enhance our knowledge of the pathophysiology of hypertyrosinemia.

1

University Hospital of Beni Messous, Algeries, Algeria P-203

Background: Tyrosinemia (TH) I is a devastating disorder of childhood that causes liver failure, painful neurologic crises, rickets, and hepatocarcinoma. This disorder is caused by a deficiency of fumaryl acetoacetate hydrolase. It is a rare disorder considered as a orphan disease. Methods: This is a retrospective study of the children hospitalized in the pediatric department “A” over 10 years. The objective is to describe the clinical, biological and scalable characteristics of tyrosinemia.

Chronic administration of L-tyrosine alters energy metabolism parameters in brain of rats treated with docosahexaenoic acid Carvalho-Silva M 1, Gomes L M 1, Teixeira L J 1, Rebelo J 1, Santos M L C 1, Schuck P F 2, Ferreira G C 3, Streck E L 1

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Lab Bioenergetics, UNESC, Criciuma, Brazil, 2Lab IEM, UNESC, Criciuma, Brazil, 3Lab Neurochem, UFRJ, Rio de Janeiro, Brazil

P-205

Background: Tyrosine aminotransferase deficiency characterizes the inborn error of metabolism tyrosinemia type II, leading to increased levels of tyrosine and its by-products in plasma, resulting in eye, skin and neurological injuries. The mechanisms of brain injury are still not well known, but several studies suggest that impairment in brain energy metabolism is involved in the pathophysiology of tyrosinemia. Docosahexaenoic acid (DHA; C22: 6, an omega-3 fatty acid) plays important roles in the development and maintenance of the central nervous system. Thus, the present study aimed to assess DHA administration effects on the activity of mitochondrial respiratory chain complexes I, II, II-III, and IV, and creatine kinase in brain of young rats subjected to an animal model of hypertyrosinemia. Methods: Wistar rats were divided into 4 groups: control, L-tyrosine, DHA, and L-tyrosine + DHA. The animals received L-tyrosine (500 mg/kg of body weight, i.p., 12/12 h), and DHA (0.1 g/kg body weight by gavage, once a day) from the 7th to the 28th postnatal days (control group received saline solution). Twelve hours after the last administration, the animals were killed by decapitation; cortex, hippocampus and striatum were isolated for analysis. Results: We verified that L-tyrosine inhibited the activity of complexes I, II, IIIII and IV in striatum, and DHA prevented this effect in complexes I, II-III and IV. Creatine kinase activity was also decreased by L-tyrosine in striatum; DHA also presented a protective effect and reversed the enzyme inhibition. Discussion: We speculate that chronic administration of L-tyrosine impairs brain energy metabolism mainly in the striatum, and that omega-3 fatty acids may be an adjunctive treatment for patients with tyrosinemia type II.

Polymorphism of MTHFR A1298C, a reliable marker in North indian mothers with Down syndrome and its association with serum, RBC folate and serum homocysteine as risk factor and congenital heart defects

P-204

Behaviour and quality of life in tyrosinemia type 1 patients compared to phenylketonuria patients and healthy controls Van Vliet K 1, Van Ginkel W G 1, Jahja R 1, Daly A 2, MacDonald A 2, De Laet C 3, Cassiman D 4, Eyskens F 5, Korver-Keularts I 6, Goyens P J 3, McKiernan P J 2, Huijbregts S C J 7, Van Spronsen F J 1 1

Div Metab Dis, Bea Child Hosp, UMCG, Groningen, Netherlands, Birmingham Child Hosp, Birmingham, United Kingdom, 3Univ Child Hosp Queen Fabiola, Brussels, Belgium, 4Univ Hosp Gasthuisberg, Leuven, Belgium, 5Queen Paola Child Hosp, Antwerp, Belgium, 6Maastricht Univ Med Center, Maastricht, Netherlands, 7Univ Leiden, Leiden, Netherlands 2

Background: The clinical pictures of tyrosinemia type 1 (HT1) and phenylketonuria (PKU) differ completely, PKU leading to severe mental retardation and HT1 mainly to liver failure and liver cancer. However, when treated adequately, both diseases are associated with deficits in executive function, while PKU patients also tend to show problems behavioural problems. Here, we studied whether HT1 patients also show behavioural problems and issues in their quality of life (QoL). Methods: 10 HT1 and 10 age and gender matched PKU patients and healthy controls were included (age 7–13 years). All patients filled in the child behaviour checklist (CBCL) and eight of them also filled in a questionnaire regarding their QoL (TACQOL). We compared the 3 groups using Kruskal-Wallis tests and post hoc Mann–Whitney U tests. Results: The 3 groups differ on some domains of the CBCL questionnaire, HT1 patients showing more problems than controls on social problems (p = 0.009), attentional problems (p = 0.007), aggressive behaviour (p = 0.035), and externalizing behaviour (p = 0.029). When HT1 patients are compared to PKU patients, HT1 patients show more attentional problems (p = 0.019), aggressive behaviour (p = 0.029) and externalizing behaviour (p = 0.029). Regarding QoL, HT1 patients only report a significant lower outcome in the cognitive domain (cognitive functioning and school performance) if compared to healthy controls (p = 0.015). No further significant differences in the other domains (physical, motor, daily and social functioning and the occurrence of positive and negative moods), between the groups are found. Discussion: HT1 patients show more behavioural problems than PKU patients and healthy controls. However, this does not automatically lead to a difference in the QoL between HT1 patients and controls. Conflict of Interest declared.

Polipalli S K 1, Mohanty P K 1, Pandey S K 1, Kapoor S 1 1

MAMCollege, Lok Nayak Hospital, New Delhi, India

Background: Our study aimed to evaluate A1298C polymorphism as a risk factor for Down syndrome and its association with biochemical parameters and congenital heart disease. Methods: 81 mothers with babies having free trisomy 21 of North Indian ethnicity and 99 mothers who had no children with Down syndrome were evaluated. Fasting blood was collected and was used to determine plasma homocysteine, vitamin B12, folate (serum and RBC) and MTHFR A1298C polymorphisms were done by allele specific polymerase reaction and enzyme digestion. Homocysteine quantification was done by HPLC. Results: The prevalence of A1298C polymorphism in North Indian mothers of babies with Down syndrome cases and controls were 66.7 % vs. 39.4 % respectively. The heterozygous and homozygous genotype frequencies at 1298 position (AC and CC) among case and control mothers were (38.3 % vs. 22.2 % and 28.4 % vs. 17.25 %, respectively, OR = 3.10, 95 % CI 1.6–5.79, P = 0.002), showing a statistically significant difference between two groups. Low serum folate in 33.3 % of cases vs. 8.0 % in controls, the difference was significant (OR = 5.68, CI 95 % 2.41–13.4, P = 0.0001). Low RBC folate was found in 28.3 % of cases vs. 11.1 % in controls, which was significant (OR = 3.172, CI 95 % 1.43–6.99 P = 0.005). High serum homocysteine was found in 9.8 % of cases vs. 2.0 % in controls and the difference was statistically significant. The median intraquartile range serum homocysteine in cases was 10.2(Q1Q3, 8.2–12.0) vs. 7.6 (Q1-Q3, 5.7–10.1) in controls, showing higher serum homocysteine in cases. No relationship was observed among A1298C polymorphism and CHD (P value = 0.601). Discussion: MTHFR A1298C polymorphism is associated with 3 times higher risk of developing Down syndrome. Low serum, RBC folate and high serum homocysteine are significantly associated with MTHFR polymorphism and therefore the risk of occurrence of Down syndrome. Peri or preconceptional folate supplementation may therefore lead to a decline in Down syndrome births.

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Update on gluta mine synthetase deficiency, 11 years after the first reported case Spodenkiewicz M 1, Diez-Fernandez C 1, Haberle J 1 1

Div Metab, Univ Child Hosp, Zurich, Switzerland

Background: Glutamine synthetase (GS) deficiency was first described in 2005 and since then only three patients (unrelated) have been reported. This ultra-rare autosomal recessive disease is characterized by neonatal onset of severe epileptic encephalopathy and brain malformations, associated with low levels of glutamine in all body fluids and chronic moderate hyperammonemia. GS deficiency is caused by mutations in the glutamine ammonia ligase (GLUL) gene encoding for GS. Methods: We review the clinical and biochemical situation of all three patients reported with GS deficiency and summarize in addition what we have learned from treatment studies and from basic research on this primary defect of glutamine biosynthesis. Results: All known patients had a very poor neurological outcome and either died as neonates from multiple organ failure (2 patients) or survived into early childhood with severe psychomotor retardation (1 patient). They presented very low glutamine levels in all body fluids and had different homozygous missense mutations mapping in exon 6 of GLUL. Glutamine supplementation was only tried in one patient, with short-term benefit. NAD+ deficiency was found as an additional feature of the disease.

S118 Discussion: GS deficiency is an ultra-rare but probably underdiagnosed disorder that may escape diagnosis due to the non-standard way of detection, based on decreased plasma and urine glutamine levels. We propose to consider this differential diagnosis in all patients with severe encephalopathy, especially since treatment options exist, including glutamine and nicotinamide supplementation, which may alleviate the phenotype.

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Management of acute liver failure in tyrosinaemia type 1: urgent liver transplant or wait for nitisinone response? Pagliardini V 1, Calvo P L 1, Dellepiane M 1, Ceglie T 1, Biamino E 1, Pinon M , Porta F 1, Tandoi F 2, Romagnoli R 2, Spada M 1

1

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Dominant spastic paraplegia SPG9 is due to mutations in the ALDH18A1 gene, which encodes for Δ1-pyrroline-5-carboxylate synthetase (P5CS) Panza E 1 2, Escamilla-Honrubia J M 3, Marco-Marin C 3 4, Gougeard N 3 4, De Michele G 5, Morra V B 5, Liguori R 6 7, Salviati L 8 9, Donati M A 10, Cusano R 11 12, Pippucci T 2, Ravazzolo R 13 14, Nemeth A H 15 16, Smithson S 17, Davies S 18, Hurst J A 19, Bordo D 20, Seri M 2, Rubio V 3 4 1 Dept Hum Gen, Univ Utah, Salt Lake City, United States, 2Dept Med Surg Sci, Univ Bologna, Bologna, Italy, 3Inst Biomed Valencia CSIC, Valencia, Spain, 4CIBERER-ISCIII, Valencia, Spain, 5Dept Neur Rep Odontostom Sci, Univ Fed II, Naples, Italy, 6IRCCS Inst Sci Neur Bologna, Bologna, Italy, 7 Dept Biomed NeuroMot Sci, Univ Bologna, Bologna, Italy, 8 Clin Gen Unit, Univ Padova, Padova, Italy, 9IRP Citta della Speranza, Padova, Italy, 10A Meller Institute, Firenze, Italy, 11Inst Ric Gen Biome (IRGB), CNR, Cagliari, Italy, 12 Parco Tecn della Sardegna, Cagliari, Italy, 13 Med Gen Unit, G Gaslini Inst, Genova, Italy, 14DINOGMI Dep, Univ Genova, Genova, Italy, 15Nuffield Dept Clin Neurosci, Univ Oxford, Oxford, United Kingdom, 1 6 Dept Clin Gen, Churchill Hosp, Oxford, United Kingdom, 17 Dept Clin Gen, St Michael Hosp, Bristol, United Kingdom, 18 Inst Med Gen, Univ Hosp Wales, Cardiff, United Kingdom, 19Dept Clin Gen, Great Ormond St Hosp, London, United Kingdom, 20IRCCS AOU S Mart IST, Int Nac Ric Cancro, Genova, Italy

Background: Autosomal dominant hereditary spastic paraplegia SPG9, found in 2 families, presents early cataracts and upper motor neuron signs. ALDH18A1 is in the 4.78 Mb SPG9-linked region and its mutations had been associated to a recessive disorder with some traits of SPG9. The ALDH18A1 product P5CS is bifunctional (glutamate-5-kinase and reductase domains G5K and GPR), catalyzing ornithine/ proline biosynthesis first step. Methods: We searched for ALDH18A1 mutations and measured plasma amino acids in SPG9 patients, monitoring mutant P5CS production/ localization by fluorescence microscopy of GFP-tagged or immunostained P5CS in transfected cells and patient fibroblasts, respectively; and by fibroblast western blotting. Site-directed mutagenesis of recombinant human P5CS and of its E. coli surrogate G5K was used to determine the mutations effects, which were rationalized by in silico structural modeling. Results: We found ALDH18A1 exon 7 mutations c.727G > C (p.Val243Leu) or c.755G > A (p.Arg252Gln) (one in each family) in heterozygosis co-segregating with SPG9. These mutations did not abolish production or cause mislocalization of P5CS, but they caused P5CS function loss reflected in the aminogram and in enzyme activity assays of mutant recombinant human P5CS, also favoring dissociation of the P5CS hexamer to dimers. Discussion: The dominant inheritance of SPG9 may be due to dominant negative effects resulting from the need for an architecturally perfect P5CS oligomer. Thus, ALDH18A1 loss-of-function mutations could be associated with dominant inheritance or recessive inheritance depending on whether they disturb P5CS oligomeric architecture or not, as suggested by the results of structural modeling. Detailed comparison of P5CS deficiency and ALDH18A1-associated spastic paraplegia phenotypes is needed. Grants: Telethon Fondation Onlus, GGP06209 & GGP10121 (AMS, Gubbio) & It. Min. Health (EP&MS); MEC-BFU2011-30407 & GV-Prometeo II/2014/ 029 (VR).

1 Dept of Ped, University of Torino, Torino, Italy, 2Liver Transplant Center, Univ Torino, Torino, Italy

Background: Tyrosinaemia type 1 (TT1) is a disease caused by a deficiency of fumarylacetoacetase. The disorder is characterized by progressive liver failure and high risk of hepatocellular carcinoma. Nitisinone (NTBC) is an inhibitor of an enzyme that is upstream of FAH and causes a reduction of the flux through this metabolic pathway, leading to a decrease of the toxic metabolites. Case report: A previously healthy 3 months male presented to the emergency room for sudden onset of swollen abdomen with ascites, fair general condition, severe hypoglycemia and profound coagulopathy. Within 2 h the patient’s condition acutely worsened, with the necessity of resuscitation and intubation. Results: Urgent metabolic work-up revealed high urinary levels of succinylacetone (SA) consistent with a diagnosis of TT1 and NTBC was started at 2 mg/kg/die. Clinical conditions improved, plasma and urinary SA normalized but severe hepatic failure persisted for more than 1 week and the Liver Transplant Centre was consulted. As the neurological conditions were stable, the transplant was delayed. Finally, 12 days after the first dose of NTBC, hepatic synthesis restored. Discussion: Early diagnosis of TT1 is crucial and prompt high dose NTBC administration represents a life-saving treatment in cases presenting with acute liver failure. Data published in literature do not distinguish non-responder patients from late-responder ones. The response is usually rapid: coagulation improves within 48 h and all patients should respond within 1 week. Our patient developed a hepatic stupor, in which liver failure was caused by a functional impairment due to an acute metabolic insult rather than cellular necrosis. Biochemical response to NTBC was immediate, but hepatic function took 12 days to recovery. The innovative contribute of this case report lies in the unusual latency of liver response to NTBC that support a careful waiting medical approach in order to avoid radical surgery.

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A novel homozygous LIAS mutation that causes glycine encephalopathy Salvador C L 1, Stray-Pedersen A 3 4, Woldseth B 1, Baylor-Hopkins Center for Mendelian Genomics U S 4, Backe P H 5, Hoyer H 6, Svendsen M 6, Rasmussen M 2, Morkrid L 1 1 Nat Unit Lab Diagn IEM, Oslo Univ Hosp, Oslo, Norway, 2Div Paed and Adolesc Med, Oslo Univ Hosp, Oslo, Norway, 3Nat Unit NBS, Oslo Univ Hosp, Oslo, Norway, 4Dept Mol Hum Gen, Baylor College of Med, Houston, United States, 5Dept Med Bioch, Univ of Oslo, Oslo, Norway, 6Lab Med Gen, Telemark Hosp, Skien, Norway

Background: During the last years mutations in several genes causing variant glycine encephalopathy have been described, including LIAS, BOLA3, GLRX5 and LIPT2. LIAS encodes lipoic acid synthetase. Lipoic acid is a cofactor for several enzymes, including the glycine cleavage system, but also BCKDH, PDH and 2-KGDH. Case Report: A 10 y old female patient, consanguineous parents from Asia. A brother died 9 months old; probably encephalopathy. She has hypotonia and pes equinovarus, apraxia, loss of strength and balance, mild dysmorphism and intellectual disability. Sensorineural hearing loss was diagnosed at age 3. Brain MRI was normal in 2010. Result: Amino acids showed persistently increased levels of glycine in CSF, plasma and urine, and low levels of valine, isoleucine and leucine. CSF-gly 23 μmol/L (3–10), P-gly 617 μmol/L (110–440), P-val 77 (140–330), P-ile 20 (33–100) and P-leu 39 (70–160). Normal lactate concentration was found in CSF and blood, but pyruvate was slightly increased in CSF. PDH enzyme

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 activity in fibroblasts was decreased (0.34 nmol/min/mg protein; ref 0.7–1.1). Whole-exome sequencing revealed disease-causing variants: 1.L IAS(NM_194451):c.745C > T(p.R249C), homozygous, both parents carriers;glycine encephalopathy (OMIM#614462). Structural analysis based on the crystal structure of lipoyl synthase 2 from Thermosynechococcus elongatus (PDB code 4U0P) indicate that R249 is located in close vicinity of the iron-sulfur cluster responsible for the reductive cleavage of Sadenosylmethionine. 2. TRPS1(NM_014112):c.46C > T(p.R16W), heterozygous de novo; trichorhinophalangeal syndrome (OMIM#190350). Discussion: We report a patient with elevated glycine and low levels of branched chain amino acids, decreased PDH activity and a novel homozygous LIAS mutation that causes glycine encephalopathy. Her phenotype is relatively mild. Low levels of branched-chain amino acids are to our knowledge not reported earlier in LIAS deficiency, but has been found in LIPT2 deficiency.

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High prevalence of tyrosinemia type I in Chechen Republic in Russia Radzhabova G M 1, Baydakova G V 1, Melikyan L P 1, Bychkov I O 1, Dzhunidova L L 2, Zakharova E Y 1

tyrosine concentrations. To assess the influence of any metabolic alterations, we compared liver transplanted patients with patients treated with NTBC. Methods: All patients performed the Amsterdam Neuropsychological test battery (ANT). 19 patients were treated with NTBC (male: 15, mean age: 12.8 years) and 6 had liver transplantation (OLT) (male: 2, mean age: 16.4 years). Five of these six OLT patients received NTBC before the OLT. Different tasks measuring the percentage of errors and reaction time of core EF (inhibition, working memory and cognitive flexibility) and SQ (face recognition [FR] and identification of facial emotions [IFE]) were performed. Differences on the tasks were studied with Mann–Whitney U tests. Results: Patients with OLT had a significant lower percentage of errors than patients still on NTBC with the easiest part of the FR task, presenting frontal pictures (p = 0.003). The overall percentage of errors on this task was also lower in OLT patients (p = 0.015). On the IFE task, OLT patients made significantly less mistakes when they had to identify persons who were happy (p = 0.030). On the feature identification task, measuring working memory, NTBC treated patients had a significant longer reaction time (p = 0.047). No significant differences on the other tasks were found. Discussion: Differences in results could be due to differences in treatment modality, although the results should be considered with caution due to the low number of patients and the difference in age between the two groups. Still, these data indicate that brain function in NTBC treated patients requires further study to investigate if this is associated with NTBC (and diet) related parameters. Conflict of Interest declared.

1 FSBI Res Cent for Med Genet, Moscow, Russian Federation, 2State Budg Inst Mater Hosp, Grozniy, Russian Federation

Background: Tyrosinemia type I (TH1) is an inborn autosomal recessive disorder of tyrosine catabolism caused by defective activity of fumarylacetoacetate hydrolase and mutations in FAH gene. The frequency of TH1 is approximately one in 100,000 to 120,000 births. Several regions of the world have a higher expected frequency of TH1 due to the increased frequency of certain pathogenic variants resulting from a founder effect: In Norway, Finland and province of Quebec, Canada the birth prevalence is estimated at 1:74,000, 1:60,000 and 1:16,000 live births, respectively. The frequency of TH1 in whole Russia and in separate regions of Russia is not known. Among 24 patients diagnosed with TH1 in Russia, 8 patients were origin from the Chechen Republic and all of them had an identical genotype of c.1025C > T / c.1025C > T. Patients and methods: To directly estimate frequency of TH1 in Chechen population the simple PCR-RFLP analysis for p.Pro342Leu mutation was developed. DNA was extracted from dried blood spots of 296 randomized derived from newborns from the Chechen Republic. Results and discussion: Mutation p.Pro342Leu was found in 7/296 blood spots from newborns from the Chechen Republic in heterozygous state. The carrier frequency is 1:85. Estimated frequency of TH1 in Chechen population is 1:28570, which is one of the highest frequencies of TH1 in the world. Newborn screening in this region could be recommended for early detection this treatable inherited disease.

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Executive functions and social cognition in transplanted versus NTBC treated tyrosinemia type 1 patients Van Ginkel W G 1, Jahja R 1, Daly A 2, MacDonald A 2, De Laet C 4, Cassiman D 5, Eyskens F 6, Korver-Keularts I 7, Goyens P J 4, McKiernan P J 2, Huijbregts S C J 3, Van Spronsen F J 1 1

Beatrix Child Hosp, UMCG, Groningen, Netherlands, 2Birmingham Child Hosp, Birmingham, United Kingdom, 3Univ Leiden, Leiden, Netherlands, 4 Univ Child Hosp Queen Fabiola, Brussels, Belgium, 5 Univ Hosp Gasthuisberg, Leuven, Belgium, 6Queen Paola Child Hosp, Antwerp, Belgium, 7Maastricht Univ Med Cent, Maastricht, Netherlands Background: Tyrosinemia Type 1 (HT1) patients show neuropsychological deficits on executive functions (EF) and social cognition (SQ). It is hypothesized that these deficits could be related NTBC treatment causing high blood

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Evaluation of choline acetyltransferase and acetylcholinesterase activities in brain of rats submitted to chronic administration of L-tyrosine and treated with antioxidants Gomes L M 1, Carvalho-Silva M 1, Scaini G 1, Deroza P F P 4, Malgarin F 2, Zugno A I 4, Rico E P 5, Schuck P F 2, Ferreira G C 3, Streck E L 1 1 Lab Bioenergetics, UNESC, Criciuma, Brazil, 2Lab IEM, UNESC, Criciuma, Brazil, 3Lab Neurochem, UFRJ, Rio de Janeiro, Brazil, 4Neurolab, UNESC, Criciuma, Brazil, 5Lab Neurotox, UNESC, Criciuma, Brazil

Background: Tyrosinemia type II is a rare autosomal recessive disease caused by deficiency of hepatic tyrosine aminotransferase and is associated with neurologic and development difficulties in numerous patients. In the present study, we investigated the in vivo influence of antioxidant treatment (NAC and DFX) on the inhibitory effects, provoked by chronic administration of L-tyrosine, on the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in cerebral cortex, hippocampus and striatum of rats. Methods: Wistar rats (7 days) received L-tyrosine (500 mg/kg i.p.) or saline twice a day for 21 days, and were also supplemented with Nacetylcysteine (20 mg/kg s.c.) twice a day and deferoxamine (20 mg/kg s.c.) every 2 days. Twelve hours after the last injection, the animals were sacrificed by decapitation, their brains were quickly removed, and the hippocampus, striatum and cerebral cortex were collected for ChAT and AChE assays. Results: Our results showed that L-tyrosine administration decreased the activity of ChAT in hippocampus and cerebral cortex, while AChE activity was increased in hippocampus, striatum and cerebral cortex. Moreover, the co-administration of NAC plus DFX was able to prevent the inhibition of ChAT only in cerebral cortex, as well as the increased of AChE activity in hippocampus and striatum, but not in cerebral cortex. DISCUSSION: The results from the present study demonstrate a marked increase in AChE activity and a decrease in ChAT activity following the administration of L-tyrosine, suggesting that L-tyrosine administration may alter cholinergic synapses, and this may be involved in the pathophysiology of brain damage found in patients affected with tyrosinemia type II. Moreover, the present results also indicate a possible neuroprotective role for NAC/DFX as a potential adjuvant therapy to the patients with tyrosinemia type II.

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BCAA in brain of the offspring supplemented with DHA when compared with offspring supplemented with vehicle. Discussion: Our results demonstrate that DNA damage induced by BCAA was prevented by the administration of DHA only during the postnatal period. Thus, the results presented in this work, complemented by other existing studies, support the idea that omega-3 fatty acids play an important role in the corresponding and necessary antioxidant capacity in the central nervous system, suggesting that the omega-3 fatty acids might be an important adjunctive therapy in MSUD.

Maple syrup urine disease as a neurodegenerative disease: pathophysiological mechanisms Streck E L 1, Scaini G 1, Tonon T 2, Souza C F M 2, Margutti AV 3, Camelo-Jr J S 3, Amorin T 3, Seda J 4, Schwartz I V D 2 1 Lab Bioenergetics, UNESC, Criciuma, Brazil, 2BRAIN Lab, HCPA, Porto Alegre, Brazil, 3Dept Pediatrics, USP-RP, Ribeirao Preto, Brazil, 4Hosp Sirio Libanes, Sao Paulo, Brazil

Background: Maple syrup urine disease (MSUD) is an inherited disorder caused by deficient activity of the branched-chain α-keto acid dehydrogenase complex involved in the degradation pathway of branched-chain amino acids and their respective α-keto-acids. Because previous reports suggest that MSUD patients present severe neurological symptoms and brain abnormalities, we evaluated neurodegenerative markers in plasma from 10 MSUD patients during the dietary treatment. Methods: Using Luminex xMap technology, we measured plasma levels of neurodegenerative markers [Brain-derived neurotrophic factor (BDNF); platelet-derived growth factor AA and AB/BB (PDGF-AA and -AB/BB); neural cell adhesion molecule (NCAM); plasminogen activator inhibitor-1 total (PAI-1 total); cathepsin D] in 10 treated MSUD patients with the classic form, and 9 healthy individuals. Results: Our results showed a significant decrease of BDNF and PDGF-AA levels in MSUD patients. On the other hand, NCAM and cathepsin D levels were significantly greater in MSUD patients as compared to the control group, while no significant changes were observed in levels of PAI-1 (total) and PDGF-AB/BB between control and MSUD groups. Discussion: The present findings corroborate with previous studies demonstrating that neurotrophic factors and lysosomal protease may contribute, along with other mechanisms, to the intellectual deficit and neurodegeneration observed in MSUD.

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Effect of docosahexaenoic acid administration during pregnancy or postnatal period on DNA damage in brain of rats submitted to chronic administration of branched-chain amino acid Morais M O S 1, Scaini G 1, Strapazzon G 4, Damiani A P 4, Schuck P F 2, Ferreira G C 3, Andrade V M 4, Streck E L 1 1 Lab Bioenergetics, UNESC, Criciuma, Brazil, 2Lab IEM, UNESC, Criciuma, Brazil, 3Lab Neurochem, UFRJ, Rio de Janeiro, Brazil, 4Lab Biol Cell Mol, UNESC, Criciuma, Brazil

Background: Maple syrup urine disease (MSUD) is an autosomal recessive inherited disorder that affects branched-chain amino acid (BCAA) catabolism. MSUD patients usually present a variable degree of mental retardation and other neurological symptoms, but the mechanisms underlying the neurotoxicity of this disorder are poorly known. Considering that docosahexaenoic acid (DHA, C22: 6, an omega-3 fatty acid) have beneficial properties in the central nervous system, the present study aims to evaluate a possible preventive effect of the treatment with DHA during pregnancy or postnatal period on DNA damage elicited by BCAA in brain of the offspring. Methods: Pregnant Wistar rats were treated daily with DHA (0.8 g/kg) or vehicle from gestation day 5 until day 21. After, offspring rats (PD 7) were submitted to chronic administration of BCAA pool or saline twice a day for 21 days, and were also supplemented with DHA (0.1 g/kg) once a day. Twelve hours after the last administration the offspring were killed by decapitation; the brains were quickly removed, and the hippocampus, striatum and cerebral cortex were collected for Comet assay. Results: Our results showed that supplementation with DHA during the prenatal period did not prevent the DNA damage in the MSUD offspring’s brain. On the other hand, we showed a marked decrease in DNA damage induced by

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Maple syrup urine disease (MSUD)—metabolic decompensation after liver transplantation (LTx) Harnacke K 1, Murko S 2, Grabhorn E 1, Santer R 1 2 1 Dept Pediatr, Univ Med Cent Eppendorf, Hamburg, Germany, 2Metab Lab, Univ Med Center Eppendorf, Hamburg, Germany

Background: MSUD is caused by mutations in genes coding for subunits of the branched-chain (BC) alpha-keto acid dehydrogenase complex, an enzyme involved in the metabolism of BC amino acids isoleucine, leucine, and valine. It has been estimated that 9–13 % of whole body enzyme activity is located in liver, which is considered to be enough, so that LTx is a means to abolish metabolic decompensations in MSUD patients. Only very recently, repeated metabolic decompensation have for the first time been described in an MSUD patient after living-related LTx from a heterozygous donor (Al-Shamsi et al., JIMD Rep. 2016). Here, we report similar findings in a patient after non-living, non-related donor LTx. Methods: Case study Results: This German boy was detected in neonatal screening but developed metabolic crisis neonatally with coma and seizures, and required dialysis treatment. Diagnosis of MSUD was based on extremely elevated BC amino acids in blood (maximum Xleu 3131,8 μmol/l), elevated alloisoleucine, and a very high excretion of typical metabolites in urine (2OH-isovaleric acid, 2-oxoisocaproic acid, 2-OH-isocaproic acid, 2oxoisovaleric acid, 3-oxo-3Me-valeric acid). At age 10 mo, he received a post-mortem split liver organ which resulted in a dramatic improvement of metabolic control, almost normal blood concentrations of BC amino acid, only traces of allo-isoleucine, and a complete normalisation of urinary excretion of organic acids (all < 2 μmol/l). At age 6 y, during a period of tonsillitis, he developed recurrent vomiting, severe dehydration, and dizzyness. He showed severe ketoacidosis and a markedly elevated excretion of 2-OH-isovaleric acid (966,9 μmol/l), 2oxoisocaproic acid (massive), 2-OH-isocaproic acid, 2-oxoisovaleric acid (82,3 μmol/l), 3-oxo-3Me-valeric acid (267,4 μmol/l). Discussion: This case illustrates that enzymatic activity even in segments of a putative wild-type donor must not be sufficient in severe catabolic situations in MSUD patients.

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Investigation of inflammatory profile in MSUD patients: benefit of Lcarnitine supplementation Mescka C P M 2 4, Guerreiro G B 2 4, Donida B 2 3, Marchetti D 2 3, Jacques C E D 2 3, Coelho D M 2, Coitinho A S 5, Wajner M 2 3, Dutra-Filho C S 3, Giugliani R 2, Vargas C R 1 2 3 4 1 Faculdade de Farmacia, UFRGS, Porto Alegre, Brazil, 2Serv Gen Med, HCPA, UFRGS, Porto Alegre, Brazil, 3PPG em CB-Bioquimica, UFRGS, porto Alegre, Brazil, 4PPGCF-UFRGS, Porto Alegre, Brazil, 5PPG em CBFisiologia, UFRGS, Porto Alegre, Brazil

Background: Maple syrup urine disease (MSUD) is a metabolic disorder caused by a deficiency of the branched-chain α-keto acid dehydrogenase

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 complex activity, which leads to the accumulation of branched-chain amino acids and their respective α-keto-acids in body fluids. The main symptomatology presented by MSUD patients includes ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay and mental retardation but the neurological pathophysiologic mechanisms are poorly understood. Treatment consists of dietary restriction of the BCAA (low protein intake) supplemented by a BCAA-free amino acid mixture. It was verified that MSUD patients present L-carnitine (L-car) deficiency and this compound has demonstrated an antioxidant and anti-inflammatory role in metabolic diseases. Methods: The study evaluates the effect of L-car supplementation in MSUD patients on plasma inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-gamma (INF-g), and the correlation with malondialdehyde (MDA), as a marker of oxidative damage, and with free Lcar plasma levels. Seven classical MSUD patients (mean age 8.28 ± 2.87 years) under protein restrict diet protocol were supplemented with L-car capsules, at a dose of 50 mg/kg/day, not exceeding 1.5 g/day for 2 months and inflammatory cytokines, MDA and free L-car levels were analyzed in blood of MSUD patients before and after 1 and 2 months of Lcar supplementation. Results: Significant increases of IL-1β, IL-6, and INF-g were observed before the treatment with L-car. Moreover, a negative correlation between all cytokines tested and L-car concentrations and a positive correlation among the MDA content and IL-1β and IL-6 values were verified. Discussion: Our data show that L-car supplementation can improve cellular defense against inflammation and oxidative stress in MSUD patients and may represent an additional therapeutic approach to the patients affected by this disease.

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09. Urea cycle disorders

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Improved neurodevelopmental outcomes in patients with urea cycle disorders after liver transplantation Jun K 1, Shirou M 1, Ken M 1, Rieko S 1, Hiroshi M 1, Fumio E 1, Kimitoshi N 1 1

Department of Pediatrics, Graduate School, Kumamoto City, Japan

Background: Urea cycle disorders (UCDs) are among the most common inherited metabolic diseases in Japan. We investigated the clinical manifestations, treatment, and prognoses of 177 patients with UCDs who were evaluated and treated from January 1999 to March 2009 in Japan using a questionnaire survey. Among these 177 patients, 42 (7 with carbamoyl phosphate synthetase 1 deficiency, 27 with ornithine transcarbamylase deficiency, 7 with argininosuccinate synthetase deficiency, and, 1 with arginase 1 deficiency) underwent living-donor liver transplantation (LT). Methods: We evaluated whether LT could improve neurodevelopmental outcomes in patients with UCDs. Results: The neurodevelopmental outcomes of patients with a maximum ammonia concentration (MAC) of less than 300 μmol/L at the time of onset were not significantly different between the LT and non-LT groups (P = 0.222). However, the neurodevelopmental outcomes of patients who underwent LT were improved significantly compared to those of patients who did not undergo LT when the MAC at disease onset was 300 μmol/L or more (P = 0.008). Discussion: LT can improve control of the primary disease as well as neurodevelopmental outcomes in patients with severe UCDs and a MAC of 300 μmol/L or higher at disease onset. LT should be considered in patients with UCD and a MAC of 300 μmol/L or higher at the time of disease onset.

Inherited disorders of proline metabolism—update P-219 Rubio V 1 2 1 Inst Biomed Valencia CSIC, Valencia, Spain, 2CIBERER-ISCIII, Valencia, Spain

Neonatal intrahepatic cholestatis caused by citrin deficiency in a Southeast Asian hospital

Background: Proline is abundant in collagens and elastin and has antioxidant roles. Made in three steps from glutamate, the first two steps are catalyzed by the ALDH18A1-encoded bifunctional Δ1-pyrroline-5carboxylate synthetase (P5CS), yielding glutamate-5-semialdehyde, which is then converted to the urea cycle carrier and arginine precursor ornithine (intestine) or, after spontaneous cyclization, becomes Δ1pyrroline-5-carboxylate (P5C), which is reduced to proline by P5C reductases encoded by PYCR1 and PYCR2. Methods: Review of literature and of personal experience Results: Before 2011 P5CS deficiency was reported in only two families, but now several families have been identified, with newborns/ infants presenting cutis laxa, joint laxitude, cataracts, progeroid aspect, neurodevelopmental delay, gastric reflux, tortuous vessels and decreased preprandrial ornithine/proline levels (inconstant). Although reported as recessive, we found a patient with P5CS deficiency hosting a de novo ALDH18A1 mutation inferred as dominant. Very recently we identified ALDH18A1 mutations in dominant spastic paraplegia SPG9 families, and provided suggestive evidence for a dominant negative disease mechanism. Others have found more patients with spastic paraplegia and dominant or recessive inheritance depending on the mutation, as well as patients with cutis laxa phenotype associated with dominant mutations that, surprisingly, recurred de novo in the same residue in unrelated patients. PYCR1 deficiency was reported in 2009 with cutis laxa phenotypes and recessive inheritance, and in 2015 PYCR2 deficiency was reported with more drastic neurodevelopmental effects than PYCR1 deficiency. Discussion: There is unexplained phenotypic variability for ALDH18A1 loss of function mutations. Although proline biosynthesis defects are coming of age, a clear pathogenic chain from defective protein to pathological effect must be determined.

Ting T W 1, Phua K B 1, Lim J S C 1, Tan E S 1 1

KK Women’s and Children’s Hospital, Singapore, Singapore

Background: Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is an autosomal recessive condition due to underlying mutations of SLC25A13. SLC25A13 encodes aspartate-glutamate transporter which supplies aspartate from mitochondria to the cytoplasm for synthesis of argininosuccinate. Case report: We report six cases of NICCD in Singapore. Results: Four are Chinese, one is a Filipino-Chinese and one is an Indonesian. They presented between 1 and 2 months old for persistent conjugated jaundice. One had acute liver failure. All were tested positive for urine reducing substance with elevated total galactose in blood despite normal Gal-1-Puridyltransferase function. All showed high citrulline, high arginine and high threonine to serine ratio in plasma amino acids which are consistent with citrin deficiency. They all responded well to lactose free diet, with jaundice resolved and liver function normalised between 3 and 5 months after diet change. The oldest patient, currently 5.5 years old, is well and asymptomatic on unrestricted diet. Molecular genetic testing of SLC25A13 was done for 3 patients. Deletion and duplication analysis was done if sequencing was inconclusive. Two patients have compound heterozygote mutations of SLC25A13. One of them has a previously reported variant in one allele (c.851_854delGTAT, p.Met285Profs) and a novel variant on the other allele (deletion of 6674 bp involving entire exon 14). The other has two known pathogenic mutations. The last patient has only one novel variant (c.1478A > T, p.D493V) found and it is predicted to be pathogenic. Discussion: Urine reducing substance is a useful test to screen for citrin deficiency. Positive cases should be followed up with plasma amino acids and gal1-P-uridyltransferase function testing. Citrin deficiency is an important

S122 differential diagnosis in an infant with conjugated jaundice because it can treated by dietary intervention with good outcome.

P-220

Clinical and molecular investigations in five Turkish patients with citrin deficiency and identification of a novel mutation on SLC25A13 Kose M 1, Kagnici M 1, Erdur B 7, Erdemir G 7, Karakoyun M 6, Berksoy E 4, Bag O 5, Ceylaner S 3, Genel F 8, Unalp A 2 Div Metab Dis, Behcet Uz Child Hosp, IZMIR, Turkey, 2Div Ped Neurology, Behcet Uz Child Hosp, IZMIR, Turkey, 3Intergen, Genetic Diagnosis Centre, Ankara, Turkey, 4Div Emergency, Behcet Uz Child Hosp, IZMIR, Turkey, 5 Div Pediatrics, Behcet Uz Child Hosp, IZMIR, Turkey, 6Div Ped Gastroent, Tepecik Hosp, IZMIR, Turkey, 7Div Ped Gastro, Behcet Uz Child Hosp, IZMIR, Turkey, 8Div Ped Immunol, Behcet Uz Child Hosp, IZMIR, Turkey 1

Background: Citrin deficiency is an autosomal recessive genetic disorder caused by a defect in the mitochondrial aspartate/glutamate antiporter, citrin. The disorder manifests either as neonatal intra-hepatic cholestasis (NICCD) or occurs in adulthood with recurrent hyperammonemia and neuropsychiatric disturbances as adult onset type II citrullinemia (CTLN2). Case report: We report 5 patients with citrin deficiency. 4 patients were male and 1 patient was female. Two of them have NICCD, three of them have CTLN2. Results: Both NICCD patients showed typical clinical and biochemical changes with a diagnosis confirmed by the presence of previously reported mutations in the SLC25A13 gene. All of CTLN2 patients were siblings. Proband was a 15 year old mentally retarded male who had admitted to our emergency with disorientation. We learned that he had behavioural changes after 10 years old and resistant convulsions for 2 years. Laboratory data showed hyperammonemia and citrullinemia. Genetic analysis of the SLC25A13 gene identified a previously unreported novel c.478delC mutation. Genetic screening of siblings showed the same mutation. Discussion: This report of case series highlights the need to maintain a high index of suspicion in adolescents with an unexplained encephalopathy, with or without hyperammonemia, and to include ammonia analysis in the screening protocol because reaching an early diagnosis is of paramount importance for a good patient outcome. This is the first case series from Turkey being reported to be having NICCD and CTLN2, further emphasizing the fact that this disorder is truly pan-ethnic.

P-221

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 acid raised from 262 to 1300 mmol /creat (RR 0.0–1.9) in 36 total acute phases. Ammonia levels raised 12–30 h later after a close clinical follow up and laboratory tests. In 72 % of the total number of acute phases the boys were admitted to the hospital for IV treatment. Discussion: Our experience from the above 3 late onset OTC males emphasizes the great heterogeneity of this disorder which causes considerable diagnostic difficulties. To our knowledge so far only one similar case has been published (Burlina A. B. et al., J Inherit Metab Dis, 2006). He found raised orotic acid and uracil in the last urine sample after the child died. It is important to create inclusion criteria for late onset OTC diagnosis and follow up to avoid sad consequences.

P-222

Newborn screening may improve the neurological outcome in urea cycle disorders—data from the E-IMD registry Posset R 1, Garcia-Cazorla A 2, Valayannopoulos V 3, Chakrapani A 4 5, Teles E L 6, Dionisi-Vici C 7, Koelker S 1 1

Div Neuroped Metab Med, Univ Child Hosp, Heidelberg, Germany, Neurometab Unit, HSJD and CIBERER, Barcelona, Spain, 3Ref Centre IEM, Necker-Enf Malades Hosp, Paris, France, 4 Metab Med Depart, Children’s Hosp, Birmingham, United Kingdom, 5 Metab Unit, Great Ormond Street Hosp, London, United Kingdom, 6Metab Unit, Ped Serv, Hosp Sao Joao, Porto, Portugal, 7Bambino Gesu Children’s Hospital IRCCS, Rome, Italy 2

Background: Patients with urea cycle disorders (UCDs) have an increased risk of neurological diseases manifestation. Methods: Between 2011 and 2015, the EU-funded project “European registry and network for intoxication type metabolic diseases (E-IMD)” prospectively followed 456 patients with UCDs. Most patients were detected after clinical manifestation, whereas 23 patients were identified by newborn screening (NBS), mostly argininosuccinate synthetase (ASS-D; n = 6), argininosuccinate lyase (ASL-D; n = 9) and arginase 1 (ARG1-D; n = 4) deficiency. Results: NBS lowered the age at diagnosis for late onset ASL-D patients. Odds ratios showed a trend towards a decreased frequency of movement disorders for ASS-D, ASL-D and ARG1-D identified by NBS. In particular, initial peak ammonium level and age of disease onset are important predictors for the neurological outcome (except for N-acetylglutamate synthase deficiency, ARG1-D, and hyperornithinemia-hyperammonemia-homocitrullinuria syndrome). Discussion: Non-interventional variables should be included in analyses aiming to elucidate the benefit of NBS (if any) on the neurological outcome of UCDs. More outcome studies will be required to evaluate the long-term effect of NBS on UCDs.

Raised urinary orotic acid and uracil levels before hyperammonemia during repeated acute episodes in 3 late onset OTC males in Greece P-223 Drogari E 1, Paramera E 2 1 Metabolic Unit, Univ Child Hosp Ag Sophia, Athens, Greece, 2Neolab SA, Scientific Services, Athens, Greece

Clinical characteristics, mutation spectrum and outcomes of 32 patients with urea cycle disorders: a single center experience from Turkey

Background: Late onset ornithine transcarbamylase (OTC) deficiency is sometimes difficult to recognize due to the variety of its clinical and biochemical findings. Usually it presents with hyperammonemia and liver disease after acute episodes of vomiting which lead to lethargy and coma. Case report: We describe 3 late onset OTC males age 6, 17 and 22 years old today who present repeatedly with raised urinary orotic acid and uracil levels during deterioration, before they increase ammonia or glutamine in plasma (GC-MS). These findings repeat always and they follow one or more episodes of acute vomiting. 2/3 were diagnosed during the first year of life and the third during the second year of life. They have different genotypes. Their natural protein intake tolerance is 0,5–1,0 gr/kg/ day. All patients take sodium phenylbutyrate and amino acid supplements everyday. Results: We followed up the 3 males for 5–21 years. Uracil levels increased from 150 to 932 mmol/creat (RR 1.4–64.5) in 34 total acute phases and orotic

Aktuglu-Zeybek A C 2, Kiykim E 2, Zubarioglu T 2, Cansever M S 1 1

Central Lab, Cerr Med Fac, Ist Univ, Istanbul, Turkey, 2Div Metab Dis Nut, Cerr Med Fac, Ist Uni, Istanbul, Turkey Background: Urea cycle disorders (UCDs) include a variety of genetic defects, which lead to inefficient urea synthesis presenting usually with elevated blood ammonium level. Advances in the diagnosis and treatment of UCDs have led to a higher survival rate. The purpose of this study is to describe the characteristics of patients with UCDs in Turkey. Methods: Herein, we retrospectively investigated the clinical, biochemical and genetic data, treatment and prognosis of 32 patients with UCDs who were diagnosed and treated in Cerrahpasa Medical Faculty, Istanbul University, from January 2009 to March 2016.

S123

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Results: The most common UCDs were citrullinemia type 1 (9/32) and ornithine transcarbamylase deficiency (OTC) (9/32). 21/32 of the cases presented with acute intoxication type encephalopathy and 17/21 survived without mental retardation or with mild retardation. The genotypes of all patients are known, except one. 22/32 patients were following a protein restrictive diet supported with essential amino acid supplementation and 17/32 were being treated with sodium benzoate, sodium phenylbutyrate or carglumic acid depending on the UCD subtype. 8/32 of patients underwent with related living-donor liver transplant. 3/32 patients died on the follow-up (one after liver transplantation, one at initial admission and one due to sepsis). Discussion: Although expanded newborn screening with tandem mass spectrometry is not a part of the nationwide screening programme, the increased survival rate is most possibly due to early recognition and better treatments that are now available in Turkey. Studies like this can make it possible to analyze natural history and clinical practices in UCDs especially in countries where the diagnosis and prognosis are usually based on early diagnosis.

P-224

Spectrum and outcome of urea cycle defects in India Joshi M M 1, Shirke S M 1, Mahamunkar A P 1, Jalan A B 1, Kudalkar K V 1, Jalan R A 1, Shinde D H 1, Tawde R J 1, Borugale M A 1, Haeberle J 2 1 NIRMAN, Div of Biochemical Genetics, Navi Mumbai, India, 2University Children Hospital Zurich, Zurich, Switzerland

Background: Urea cycle disorders (UCD) are inborn errors of ammonia detoxification. The exact incidence of UCDs in India is not known due to absence of newborn screening or a registry. Most of the patients are detected symptomatically, adding to high morbidity and mortality. Aim was to evaluate the spectrum and outcome of patients with UCDs in India. Methods: This is a retrospective study of patients with UCD for last 16 years. We could biochemically diagnose a UCD in 57 patients (32 M, 25 F) presenting with hyperammonemia, vomiting, convulsions, failure to thrive and lethargy. Quantitative analysis of plasma ammonia, argininosuccinic acid and amino acids in plasma and urine and urinary orotic acid were done. Confirmation was done by molecular studies. Results: Out of 57 patients 22 expired in neonatal period and 2 expired in adolescence. 22 are lost for follow up and 11 are followed up regularly. 35/ 57 patients were confirmed to have a UCD by genetic means. Citrullinemia type 1 (25), argininosuccinic aciduria (6), ornithine transcarbamylase deficiency (OTCD) (2), CPS (1), argininemia (1) were found. No mutation could be identified in 22/57 patients. 11 patients with citrullinemia had a common mutation (ASS1 exon 15: c.1168G > A/p.G390R). No common mutation was identified in other groups of UCD. Prenatal diagnosis was carried out for 1 couple who had lost 3 babies with hyperammonemia. The couple was identified to be heterozygous carriers of c.991C > T mutation in exon 4 of NAGS gene. 3 children with citrullinemia type 1, 1 OTCD male child and 1 ASLD are doing very well. 1 child with citrullinemia received liver transplant. Discussion: Due to the absence of newborn screening, UCDs are detected only after development of symptoms in India, resulting in a high rate of morbidity and mortality (24/57). Citrullinemia is the commonest UCD (25/57) with a prevalent mutation seen in 12/25 patients. Early and aggressive treatment has resulted in good outcome in at least 6 patients.

P-225

A simple method for in vivo measurement of ureagenesis by GC-MS using stable isotopes and dried blood spots on filter paper Allegri G M 1, Deplazes S 1, Viecelli H M 1, Mathis D 2, Haeberle J 1, Thony B 12

1 Div Metabolism, Univ Child Hosp, Zurich, Switzerland, 2Div Clin Chem Biochem, Div Child Hosp, Zurich, Switzerland

Background: Management of inherited or acquired hyperammonemia depends mainly on the plasma ammonia level which is not a reliable indicator of urea cycle function, as its concentrations largely fluctuate. The gold standard to quantify urea flux in vivo is the use of stable isotopes to determine ureagenesis capacity. Here we developed and validated a simple in vivo method with [15N]ammonium chloride ([15N]H4Cl) as a tracer. Methods: Quantitative ureagenesis was assessed and validated in wild-type and mutant spfash mice, the latter with compromised urea cycle due to ornithine transcarbamylase deficiency, during fasted and non-fasted feeding states. Blood (5 μL) was collected through tail vein puncture before and after [15N]H4Cl intraperitoneal injections over a 2 h period. Non-labeled and [15N]urea were quantified by GC-MS after extraction and chemical derivatization. Comparable results were obtained for [15N]urea quantification from dried blood spots (DBS) collected on filter paper. Result: Blood, plasma and DBS can be used as matrices to quantify ureagenesis. Upon [15N]H4Cl challenge, labelled-urea production under fasting conditions in spfash compared to wild-type mice was reduced with the most significant difference at 30 min after injection (18.8 % of wild-type). Five consecutive injections over a period of 5 weeks had no effect on body weight or ureagenesis. Discussion: This approach requires a single intraperitoneal injection of tracer, followed by (5 μL) blood withdrawal from the tail vein after 30 min. The method is robust, simple and with no apparent risk for the animals, offering a sensitive, minimal-invasive, and fast measurement of ureagenesis capacity using DBS on filter paper. The stable isotope-based quantification of ureagenesis can be applied for the efficacy-testing of novel molecular therapies.

P-226

Towards gene therapy using non-viral minicircle-DNA vectors to treat hepatic ornithine transcarbamylase deficiency in the spf-ash mouse Deplazes S 1, Viecelli H M 1, Schlegel A 2, Cunningham S 3, Alexander I 3, Haeberle J 1, Thony B 1 1 Div Metabolism, Univ Child Hosp, Zurich, Switzerland, 2Swiss HBP TC, Dept Sx, Univ Hosp, Zurich, Switzerland, 3Child Med Research Inst, Westmead, Australia

Background: Ornithine transcarbamylase (OTC) deficiency is the most common inherited defect of the urea cycle, resulting in severe hyperammonemia and death if left untreated. As an alternative to viral vector-transduction for hepatic gene delivery and gene therapy, we have established gene transfer based on naked-DNA vectors devoid of any viral or bacterial sequences, so called minicircles (MC; abstract Viecelli et al., P-170). Here we aimed at experimental gene therapy in spf-ash mice, a model for OTC deficiency with 5–10 % residual enzyme activity. Methods/results: MC-vectors were generated containing expression cassettes with a short (0.3 kb) synthetic liver-specific promoter or the natural Otc promoter with a length of 1 kb, the murine Otc-cDNA or the codonoptimized murine Otc with or without truncated 5’-intron, and a polyA signal. OTC-MC vectors were delivered to mouse liver by hydrodynamic tail vein injection. Upon vector infusion and sacrifice of mice 10 days later, we found hepatic OTC enzyme activity in liver extracts between 30 and 70 % of wild-type; however, expression of transgenic OTC was confined to the perivenous area based on immunohistochemical analysis. Since the full active urea cycle is localized in the periportal area and thus delivery of therapeutic MC vectors needs to target the periportal hepatocytes we are currently establishing delivery via hydrodynamic portal vein injection. Thus far, we found transgene expression of MC-treated mice at a much lower levels than via hydrodynamic tail vein injection. Besides optimizing conditions for portal vein-delivery, injection via the biliary duct is also under evaluation. Analysis of in vivo ureagenesis to determine the therapeutic efficacy is done by stable-isotope-based quantification using [15N]ammonium as tracer (abstract Allegri et al., P-225). Discussion: Overall, non-viral MC-vectors have the potential to treat OTC deficiency in spf-ash mice but delivery to specifically target the periportal hepatocytes needs to be optimized.

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P-227

Results: We identified 43 patients from 33 families with NAGS mutations, of which 14 were novel. This adds to a total 36 NAGS mutations found globally in 56 patients from 42 families of which 76 % are homozygous. Of all mutations, 61 % are missense changes while lack or decrease of NAGS protein is predicted for ~1/3 of mutations. Missense mutation frequency is inhomogeneous along NAGS with a peak in exon 6 reflecting the paramount substrate binding/catalytic role of the C-terminal domain. Accordingly, phenotypes associated with missense mutations mapping in this domain are more severe than of missense mutations in the amino acid kinase domain. Enzyme activity and stability assays with the mutations recombinantly expressed and in silico structural analysis support the pathogenic role of most NAGSD-associated mutations found. Discussion: The diagnosis of NAGSD requires a high suspicion in order to arrive early enough at this diagnosis to start specific therapy before irreversible brain damage. Mutation analysis is straightforward and, in addition to confirming the diagnosis, has predictive value as missense mutations in the C-terminal domain in general seem to lead to a more severe phenotype. Grants: GV-Prometeo II/2014/029 and MEC-BFU2014-58229-P (VR); SNSF 310030_127184 and _153196 (JH).

A potential novel treatment for CPS1 deficiency based on pharmacological chaperones Diez-Fernandez C 1, Spodenkiewicz M 1, Underhaug J 2, Martinez A 2, Haeberle J 1 1 Univ Child Hosp, Div Metab, Zurich, Switzerland, 2Univ Bergen, Biorecogn Dept, Bergen, Norway

Background: Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step of the urea cycle, which converts neurotoxic ammonia into non-toxic urea. Mutations in the CPS1 gene (>200 mutations reported) can lead to CPS1 deficiency (CPS1D), a rare autosomal recessive inborn error of the urea cycle. Patients typically develop hyperammonemia, which can lead to encephalopathy, coma and death or mental retardation unless promptly treated. The only possible cure for CPS1D is liver transplantation and current management is in many cases insufficient for achieving long-term metabolic stability and survival. Therefore, searching for alternative therapeutic approaches is essential. Methods: We exploited the baculovirus/insect cell expression system to explore the disease-causing nature of 38 CPS1 mutations. To identify CPS1-stabilizing pharmacological chaperones, we purified large amounts of wild-type (wt) CPS1 and performed a high-throughput screening (HTS) with 11,000 compounds (including 1000 FAD approved drugs). The hits obtained were subjected to detailed characterization of their functional and molecular effects, using both wt CPS1 and unstable mutations. Results: Of the characterized CPS1 missense mutations, more than half (~60 %) trigger enzyme destabilization. In the HTS, eight compounds bound and stabilized wt CPS1. Of these, three did not substantially inhibit CPS1 activity, and one of them stabilized in addition unstable mutant forms. Discussion: Since most mutations studied here were destabilizing, a future treatment for CPS1D based on enzyme stabilization by pharmacological chaperones is meaningful. Of the 11,000 compounds screened, one bound, stabilized and did not inhibit CPS1 activity, both in wt and in unstable mutant forms. Although further in vivo studies are required, this compound is a promising therapeutic option for misfolding CPS1D mutations. This novel treatment can potentially have an enormous impact on the quality of life of CPS1D patients.

P-228

N-acetyl-L-glutamate synthase deficiency revisited: update on the mutational spectrum, impact of clinical mutations on enzyme functionality, and structural considerations Sancho-Vaello E 2, Marco-Marin C 2, Gougeard N 2, Fernandez-Murga L 2, Rufenacht V 1, Mustedanagic M 1, Rubio V 2, Haeberle J 1 1

Univ Child Hosp, Div Metab, Zurich, Switzerland, 2Inst Biomedicina IBVCSIC & CIBERER, Valencia, Spain Background: N-acetyl-L-glutamate synthase (NAGS) deficiency (NAGSD) is the rarest urea cycle defect and cannot be distinguished by clinical or biochemical means from carbamoyl phosphate synthetase 1 (CPS1) deficiency. This renders mutation analysis as method of choice for a clear diagnosis that is important for patients as NAGSD has substitutive therapy. Methods: Since 2003, we performed mutation analysis for NAGS and CPS1 deficiencies in > 400 families. Identified NAGS missense mutations were introduced into pure recombinant Pseudomonas aeruginosa NAGS to study their functional consequence. Structural analysis was added in silico to consolidate the expression findings.

P-229

Mild orotic aciduria in UMPS heterozygotes: a metabolic finding without clinical consequences Wortmann S B 1, Chen M 2, Wevers R A 4, Tiller G 3 1

Dept of Ped, SALK and PMU, Salzburg, Austria, 2Prevention Genetics, Marshfield, United States, 3Dept of Genetics, Kaiser Permanente, Los Angeles, United States, 4Dept of Lab Med, Radboud UMC, NIjmegen, Netherlands Background: Orotic aciduria is associated with disorders of the urea cycle and pyrimidine metabolism. Uridine monophosphate synthase (UMPS) catalyses the first step of the de novo pyrimidine synthesis. UMPS deficiency, due to biallelic mutations in UMPS, is an extremely rare condition with approximately twenty reported cases to date. Patients classically present with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, this disorder can lead to neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability. Case report and results: We identified mild and isolated orotic aciduria (2.5– 10.8 fold elevated) in nine unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/ developmental delay. Of note, none had blood count abnormalities, hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in the UMPS gene. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants as well as mild orotic aciduria in eight healthy family members. Discussion: We therefore conclude that heterozygous UMPS mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.

P-230

Linear growth is reduced in patients with urea cycle disorders Assatourian L 1, Trinh M 2, Macleod E 1, Simpson K 1, Park D 2, Ah Mew N 1, Members of the Urea Cycle Disorders Consortium3 1 Children’s National, Washington, United States, 2University of Maryland Baltimore County, Baltimore, United States, 3 Urea Cycle Disorders Consortium, Multiple institutions, United States

S125

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Urea cycle disorders (UCDs) result in reduced urea cycle flux and accumulation of toxic ammonia. Chronic therapy for urea cycle disorders includes dietary protein restriction and ammonia scavengers. Because adequate protein intake is required for normal growth, standard therapy may result in reduced growth in UCD patients. In fact, some smaller studies have demonstrated that linear growth may be impaired in UCD patients. The Longitudinal Study of Urea Cycle Disorders is the largest natural history study of UCD patients, conducted by the Urea Cycle Disorders Consortium (UCDC) of the National Institutes of Health Rare Disease Clinical Research Network. To investigate for growth deficiency, we evaluated linear growth, weight and head circumference in all enrolled UCD subjects. Methods: Complete data from at least two evaluations were available for 675 of 768 subjects, who contributed 9139 observations. Of these subjects, 456 were described as symptomatic, and 181 having neonatal-onset UCD. A linear mixed model was employed to test growth difference between groups. Results: Height/length was reduced in symptomatic patients by an average 1.5 cm (SD ± 0.62), as compared to untreated asymptomatic patients (p = 0.02). Additionally, in the most severely affected cohort, those with neonatal-onset disease, a marked reduction of 4.75 cm (SD ± 0.64) was observed in linear growth (p < 0.0001) as compared to the asymptomatic cohort. No differences were observed in weight. Head circumference was also reduced in the neonatal-onset cohort (p < 0.0001). Discussion: Symptomatic patients with UCDs have reduced height/ length, most evident in subjects with neonatal-onset disease. This is most likely secondary to dietary protein restriction. Additional analyses will include a comparison of UCD subject data with CDC/WHO growth curves, a breakdown of longitudinal growth by individual UCD, and correlation of growth with diet and biomarkers, including amino acids and pre-albumin.

P-231

Continuous renal replacement therapy for inherited metabolism disorders in infancy: report of 14 cases Aygun F 3, Zubarioglu T 1, Aygun F D 4, Cansever M S 2, Kiykim E 1, Aktuglu-Zeybek A C 1, Cam H 3 1 Div Nutr Metab Dis, Ist Univ Cer Med Fac, Istanbul, Turkey, 2Cent Lab, Ist Univ Cer Med Fac, Istanbul, Turkey, 3Div Ped Int Care, Ist Univ Cer Med Fac, Istanbul, Turkey, 4Div Ped Inf Dis, Ist Univ Cer Med Fac, Istanbul, Turkey

Background: Continuous renal replacement therapy (CRRT) is a well established treatment modality for patients with acute kidney insufficiency. As CRRT is a lifesaving blood purification method, it has started to be used in metabolic emergencies as hyperammonemic encephalopathy and leucine encephalopathy. In this study, we aimed to present clinical characteristics and outcomes of 14 patients with a diagnosis of inherited metabolic disorder that have been treated with CRRT. Methods: Patients who were diagnosed with hyperammonemic encephalopathy and leucine encephalopathy and received CRRT in PICU of Istanbul University Cerrahpasa Medical Faculty between November 2014 and December 2015 had been investigated retrospectively. Age, sex, diagnosis, intensive care duration, catheter location, complications during and after CRRT, serum levels of toxic metabolites, infection rates and mortalities of patients were recorded. Results: 14 patients were treated with CRRT for metabolic disorders. 10 patients had hyperammonemic encephalopathy due to urea cycle disorders and 4 maple syrup urine disease (MSUD) patients were hospitalized because of leucine encephalopathy. The age interval was between 2 days and 18 months, with a mean of 5,5 ± 7,4 months. The weight distribution was between 2,5 and 18 kg, with a mean of 7,3 ± 5 , 6 k g . 11 p a t i e n t s r e c e i v e d c o n t i n u o u s v e n o - v e n o u s hemodiafiltration, and 3 patients with MSUD received continuous veno-venous hemodialysis. All patients received high throughput

hemodialysis and hemofiltration. The average CRRT duration was 16,6 ± 15,6 h and two neonates died. Discussion: The aim of this study was to show that CRRT is an efficient method that can be used in toxic metabolic encephalopathies. However, more extensive newborn studies should be performed to determine the optimal conditions for CRRT in metabolic disorders’ treatment.

P-232

Unusual presentation of carbonic anhydrase VA deficiency in a 10-yearold male under medication with sultiam Harnacke K 1, Leiz S 2, Haack T B 3, Prokisch H 3, Haeberle J 4, Muntau A C 1, Santer R 1 1

Dept Pediatr, Univ Med Center Eppendorf, Hamburg, Germany, Dept Pediatr, Dritter Orden, Munich, Germany, 3Inst Hum Genet, Tech Univ, Munich, Germany, 4Div Metab Dis, Univ Child Hosp, Zurich, Switzerland 2

Background: The mitochondrial carbonic anhydrase VA (CAVA) plays a pivotal role in providing bicarbonate for carbamoyl phosphate synthetase 1 (CPS1), pyruvate carboxylase (PC), proprionyl-CoA carboxylase (PCC) and 3-methylcrotonyl-CoA carboxylase (3-MCC). In 2014, CAVA deficiency was first described as a differential diagnosis of hyperammonemia, as yet only described in the first years of life. Methods: Case study of a male child born to healthy non-consanguineous parents. Results: This boy had been treated with sultiam due to Rolando epilepsy since the age of 9. At 10 years of age, he suffered from a prolonged gastroenteritis and developed encephalopathy (GCS 6) with hyperammonemia (490 μmol/l) and lactic acidosis (9,1 mmol/l, BE −17,7 mmol/l) while being normoglycemic. Analysis of organic acids revealed increased excretion of ketones (acetoacetate, betahydroxybutyrate) and small amounts of 3-methylcrotonylglycine in urine. When Sanger sequencing of the CA5A gene was performed, an exon 1 deletion was suspected which was later confirmed on whole e x o m e s e q u e n c i n g . H o m o z y g o s i t y f o r a ~ 11 k b d e l e t i o n (NC_000016.9:87968105_87979792del) was detected in the patient and was conventionally confirmed when sequencing a junction fragment; both parents were shown to be heterozygous. Discussion: The fact that CAVA deficiency typically manifests in early life is thought to be the result of a postnatal maturational process of another mitochondrial carbonic anhydrase, CAVB, which, later in life, may compensate for reduced CAVA activity. A manifestation of CAVA deficiency in periods of increased metabolic demand beyond infancy would therefore require a second pathogenic factor besides biallelic pathogenic mutations of CA5A. Our observation suggests a role of carbonic anhydrase inhibitors, like sultiam, as possible triggering factors in the manifestation of this disorder.

P-233 Withdrawn P-234

Deciphering carbamoyl phosphate synthetase (CPS1) deficiency and urea cycle regulation by determining the structures of human CPS1 in the absence and in the presence of N-acetyl-L-glutamate De Cima S 1 2, Polo L M 1, Diez-Fernandez C 1 4, Martinez A I 4, Cervera J 2 4, Fita I 3, Rubio V 1 2 1 Inst Biomed Valencia CSIC, Valencia, Spain, 2CIBERER-ISCIII, Valencia, Spain, 3Ins Biol Mol Barcel, CSIC-Parc Cientific, Barcelona, Spain, 4Cent Invest P Felipe, Valencia, Spain

S126 Background: The 1500-residue multidomain enzyme CPS1 catalyzes the urea cycle first step and controls this cycle via allosteric regulation by its essential activator N-acetyl-L-glutamate (NAG). CPS1 deficiency (CPSD), a rare severe recessively inherited urea cycle disorder is largely due to missense CPS1 mutations (nearly 150 reported) that are unevenly distributed over the 38 CPS1 exons. CPS1 structure determination, in addition to shedding light on local mutation frequency, could provide vital information on whether a mutation is or is not disease-causing. Methods: We crystallized by vapor diffusion recombinant human CPS1 produced in high purity and elevated concentration using a baculovirus-insect cell system. CPS1 crystals without ligands or having bound ADP and NAG diffracted X-rays at ESRF and ALBA synchrotrons at respective 2.4 and 2.6-Å resolution. Phasing was achieved by molecular replacement using as model E. coli CPS. This enzyme, unlike CPS1, is NAG-insensitive, is always active and uses glutamine as substrate instead of ammonia. Results: Comparison of our crystal structures clarifies CPS1 on/offswitching by NAG. NAG binds at the C-terminal domain and triggers long-range changes that affect both distant active centers (one per phosphorylation step). These changes, in concert with nucleotide binding, dramatically remodel CPS1, stabilizing the catalytically competent conformation, and defining a long tunnel for passage of the reaction intermediate carbamate, from the first to the second phosphorylation site, where carbamoyl phosphate is produced. Discussion: These structures allow the rationalization of the effects of clinical mutations found in CPSD patients, clarifying why the central domain is a mutational hotspot and defining clinically important residues that are crucial for NAG binding or for NAG signal transmission to the catalytic sites. Support: GV-Prometeo II/2014/029 and MEC-BFU2014-58229-P (VR), BFU2012-36827 (IF) and SAF2010-17933 (JC).

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Targeted mass spectrometry-based metabolomics for the study of urea cycle enzymes and liver function Moedas M F 1 3, Farelo M A 1 3, Adam A A A 2, Van Cruchten A 3, IJlst L 3, Chamuleau R A F 2, Hoekstra R 2, Wanders R J A 3, Silva M F B 1 1 iMED-ULisboa, Fac Pharmacy, Univ Lisboa, Lisboa, Portugal, 2Tytgat Institute, AMC, Univ Amsterdam, Amsterdam, Netherlands, 3Lab Gen Met Dis, AMC, Univ Amsterdam, Amsterdam, Netherlands

Background: Hyperammonemia (HA) is a major biomarker of impaired nitrogen metabolism, characteristic for most Urea Cycle Disorders (UCDs). These inborn errors of metabolism have an estimated incidence of 1:8.000 and may result in severe disease. Also, a number of other more common conditions, namely specific drugs, may secondarily impair ammonia detoxification and cause adverse HA. Aims: (1) To develop sensitive methods using tandem mass spectrometry for the comprehensive determination of the activity of the core enzymes of UC. (2) To set-up bioassays, as valuable tools in drug discovery and development, for in vitro preclinical studies involving liver function and toxicity testing. Methods: The activities of carbamoyl-phosphate synthase (CPS) and ornithine transcarbamylase (OTC) were measured in homogenates of human liver and the human hepatoma cell line HepaRG by quantifying the amounts of citrulline produced with UPLC-MS/MS in the MRM acquisition mode using stable isotope labeled internal standards. Results: The results can be summarized as follows: (1) We developed optimized methods for the quantitative analysis of CPS and OTC in human liver and HepaRG cells and established linearity with protein up to 10 μg. (2) The obtained specific activities ranged from 80 % (OTC) [1] to 107 % (CPS) [2] of the assigned activities in liver homogenate. (3) We measured the activity of CPS and OTC in

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 HepaRG cells which revealed significantly lower activities compared to human liver. However, the activities of CPS and OTC in HepaRG cells demonstrated a viable and functional UC in this model. Discussion: This work reports novel analytical tools to study UC function, with a direct application for the diagnosis of UCDs in liver tissue. The developed bioassays will contribute to elucidate xenobiotics-induced effects on hepatic cells in vitro during preclinical drug development. Support: FCT (PT):SFRH/BD/69062/2010; [1] Pierson DL, J Biol Chem 253, 1978; [2] Kalousek F, J. Biol Chem 255, 1980.

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Studies on drug-induced modulation of urea cycle and nitrogen metabolism in human hepatoma HepaRG cells Farelo M A 1, Moedas M F 1 2, Ferreira J P 3, Micaelo E 1, Van Cruchten A 2, IJlst L 2, Wanders R J A 2, Silva M F B 1 1

iMED-ULisboa, Fac Pharmacy, Univ Lisboa, Lisboa, Portugal, 2Lab Gen Met Dis, AMC,Univ Amsterdam, Amsterdam, Netherlands, 3LCLEM, Fac Pharmacy, Univ Lisboa, Lisboa, Portugal Background: N-Carbamoyl glutamate (CG) is an analogue of N-acetyl glutamate (NAG), an activator of carbamoyl-phosphate synthase 1 (CPS1), which is used in the treatment of patients with NAG synthase (NAGS) deficiency [1]. Valproic acid (VPA) treatment may be potentially associated with hyperammonemia (HA) and hepatic encephalopathy, where inhibition of NAGS activity by the metabolite valproylCoA has been identified [2]. Aims: To elucidate the alterations on nitrogen metabolism and urea cycle (UC) induced by the pharmacological agents CG and VPA using the human hepatoma cell line HepaRG through analysis of respective metabolic profiles. Methods: Cells were cultured and incubated with the VPA, CG or a combination of both. UC intermediates (citrulline, arginine, ornithine, aspartate) and glutamate were analyzed and quantified by a novel method developed in LC-MS/MS. Extracellular metabolites and specific enzymes activities were quantified as biomarkers of UC function and hepatic cell metabolism. Results: Decreased consumption of ornithine and glutamate was observed in cells exposed to VPA, in a dose-dependent manner. The accumulation of ammonia in the extracellular medium was associated with the presence of VPA but, interestingly, urea was also produced in the same conditions. Coincubation with CG did not significantly revert VPA-associated increased ammonia levels. Discussion: This study shows that HepaRG cells in the presence of VPA are associated with HA and UC dysfunction. As HA was not reverted by co-incubation with CG, apparently the inhibition of NAGS by valproyl-CoA is most probably not the single mechanism causing UC dysfunction and HA, at least in these cells. Our data demonstrates that this cell model has a functional UC and that drug-induced changes on the exometabolome represent a valuable tool to study metabolic flux through this crucial pathway. Support: FCT(PT):SFRH/BD/69062/2010. [1] Caldovic, J Pediatr 145, 2004. [2] Aires, J Hepatol, 55,2011.

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Incidence of behavior and emotional problems in urea cycle disorders Simpson K 1, Ah Mew N 1, Members of the Urea Cycle Disorders Consortium 2 1 Children’s National, Washington, United States, 2Urea Cycle Disorders Consortium, Multiple Institutions, United States

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Urea cycle disorders (UCDs) are a group of rare inherited metabolic disorders caused by defects in the metabolic pathways necessary for the conversion of ammonia into urea. Hyperammonemia results in clinical or subclinical neurological sequelae, most often developmental delay or intellectual disability, but may manifest as psychiatric symptoms. While there have been descriptions of psychiatric manifestations during acute hyperammonemic episodes, there are few reports describing the incidence of chronic behavioral and emotional problems in this population. Methods: We reviewed behavioral and psychiatric data from subjects with confirmed UCD diagnoses enrolled in the Longitudinal Study of Urea Cycle Disorders, conducted by the Urea Cycle Disorders Consortium (UCDC) of the Rare Disease Clinical Network (RDCRN). Results: Of 705 subjects, 61 (8.6 %) reported self-injurious behavior, 46 (6.5 %) self-stimulation, and 22 (3.1 %) aggressive behaviors at one or more evaluations. 45 (6.3 %) reported depression, 57 (8.0 %) an anxiety disorder, 68 (9.6 %) ADHD, and 14 (1.9 %) an autistic spectrum disorder. Interestingly, self-stimulation was over-represented among subjects with ARG (7 of 27) and ASS (18 of 102). Discussion: The incidence of behavioral and emotional problems in our UCD cohort was in fact similar to the incidence in the general American population as reported by the Center for Disease Control and Prevention: depression (7.6 %), anxiety disorder (10.6 %), ADHD (11 %) and autism (1.5 %). However, stratification by disease type or disease severity may reveal differences in incidence between subgroups.

10. Organic acidurias: branched-chain

P-238 New in vitro model derived from brain conditional Mut−/− mice confirms cerebral ammonium accumulation in methylmalonic aciduria Remacle N 1, Forny P 3, Cudre-Cung H P 1, Do Vale Perreira S 1, Braissant O 2, Baumgartner M R 3, Ballhausen D 1 1 Cent Mol Dis, Lausanne Univ Hosp, Lausanne, Switzerland, 2Biomedicine, Lausanne Univ Hosp, Lausanne, Switzerland, 3Div Metab Dis, Univ Child Hosp, Zurich, Switzerland

Background: Methylmalonic aciduria (MMAuria) is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies are only partially effective in preventing neurological damage. We previously obtained interesting results on 3D organotypic brain cell cultures from wild-type (WT) rat embryos challenged with 2-MCA. Methods: In order to create an in vitro model which is closer to in vivo conditions, we now used 3D organotypic brain cell cultures derived from brain conditional knock-out (KO) Mut−/− mouse embryos. We tried to mimic an acute metabolic crisis by induction of a catabolic stress (fever). Biochemical analyses in culture medium and immunohistological studies of cell morphology and viability were performed. Results: In accordance with our previous findings in the WT rat model we found an important ammonium accumulation, an increase of the apoptosis rate and the suffering of neurons (cell bodies) and oligodendrocytes (signal loss). We found a substantial increase of glutaminase and a decrease of glutamate dehydrogenase protein expression by western-blot analysis. MMA was detected in the culture medium (~100 μM). Luminex analysis revealed altered expression levels of different chemokines. In cultures exposed to fever a secondary effect on oligodendrocytes (signal loss) was observed. Discussion: Our first results on this new model confirmed cerebral ammonium accumulation, increased apoptosis and suffering of different brain cell types. Further investigations on pathways of brain ammonium production are ongoing to understand the mechanisms leading

to intracerebral ammonium accumulation in MMAuria. Here we show for the first time that brain cells are capable of producing significant amounts of MMA. We further demonstrate that chemokines are activated thus indicating that neuroinflammation plays a role in the neuropathogenesis of MMAuria.

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Outcomes of patients with cobalamin C disease identified through newborn screening: a 16-year experience Ahrens-Nicklas R 1, Whitaker A 1, Aleman T S 1, Cuddapah S 1, Kaplan P 1, Yudkoff M 1, Ficicioglu C 1 1

The Children’s Hospital of Philadelphia, Philadelphia, United States

Background: Cobalamin C (cblC) disease is the most common inborn error of cobalamin metabolism. Newborn screening (NBS) for this disorder was implemented in our referral area 16 years ago. Over this time period, 12 patients have been identified through screening, they now range in age from 4 months to 16 years. Methods: We present a retrospective analysis of all cblC patients identified on NBS and followed at our center. Results: Average age at diagnosis was 8.1 days. Therapy was initiated immediately after diagnosis in each infant. 11/12 of the infants were symptomatic at the time of diagnosis. Symptoms included poor feeding, acidosis, and hypoglycemia. Mean initial C3 level was 9.17 umol/L (normal A). She has no mental impairment and has had chronic kidney injury (CKI) since 10 years of age. At 19 years, she developed bilateral vision loss. Visual acuity (VA) was 0.8 (right) and 0.1 (left). Left optic disc was minimally pale. Visual evoked potential (VEP) amplitudes were depressed with long latency. Electroretinogram (ERG) showed decreased potentials. Orbital MRI revealed T2 hyperintensity in left optic nerve. Pulse steroid was started. 15 months after onset, VA is bilaterally T), who also developed CKI after 10 years. At 11 years of age, she reported bilateral worsening of vision. She had bilateral disc atrophy, normal orbital MRI, normal ERG and absent VEP responses. Two years later, VA is 0.3 (right) and 0.1 (left). Vitamin B12, E and coenzyme Q were tried in both patients with no significant benefit. Discussion: Early diagnosis and treatment has improved survival in MMA, making chronic complications a major concern. Due to mental impairment and insidious onset, ON is probably underreported in MMA. Efficacy of potential treatments is not well described in the literature. It is not known if more stringent metabolic control can delay or prevent ON. CKI may have contributed to ON. Report of single cases may prove valuable in management of this rare complication. Routine screening of MMA patients for ON may provide early diagnosis of subclinical cases.

1 Children’s Research Institute, Washington, United States, 2Children’s National, Div Genet Metab Dis, Washington, United States

Background: Propionic acidemia (PA) and methylmalonic aciduria (MMA) are metabolic disorders that produce intoxication due to disruption of the propionate pathway. Individuals with these disorders can have long-term complications including stroke-like episodes, myopathy, renal failure and cardiomyopathy, similar to those found in individuals with dysfunctions in oxidative phosphorylation. Propionate and Krebs cycle pathways are metabolically linked in that both produce succinyl CoA, but no evidence of interactions between the enzymes in both pathways has been experimentally demonstrated. Methods: Mitochondria were collected from the livers of B6 wild-type mice using standard protocols and mitochondrial proteins were extracted under native conditions. Isolated proteins were then separated using size exclusion chromatography (SEC) on a Bio-Rad NGC Quest system and collected in 500 μl fractions. The proteins/complexes isolated in each fraction were then separated by SDS-PAGE and tested for the presence of the proteins of interest by immunoblot. Additionally, each fraction was probed for the presence of Krebs cycle enzymes’ activities. The Krebs enzymes that were probed include citrate synthase and oxo-glutarate dehydrogenase complex. Results: We have been able to identify a fraction from SEC analyses of protein complexes isolated from mitochondria with an estimated average size of over 670 KDa that contain members of both the propionate and the Krebs cycle pathways based on immunoblot analyses. Additionally, Krebs cycle enzyme activity was also detected in mouse liver mitochondrial proteins in correlation with the immunoblot results. Discussion: The results obtained in this project allow us to hypothesize a higher level of organization among the enzymes involved in the Krebs and propionate pathways in which they interact with each other in order to function correctly.

P-245

Optic neuropathy: a rare, late complication in methylmalonic acidemia Yildiz Y 1, Kalayci D 2, Bilginer Gurbuz B 1, Pektas E 1, Dursun A 1, Sivri H S 1 , Coskun T 1, Tokatli A 1 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 2Ophthalmol Clin, Numune Edu Res Hospital, Ankara, Turkey

Background: Optic neuropathy (ON) is a late complication in patients with methylmalonic acidemia (MMA). Eight MMA patients with ON are found in the literature. Presentation is variable with acute or insidious onset. Here, we present two MMA patients with ON. Methods: Records of MMA patients in a single centre with ON were reviewed. Metabolic and ophthalmologic findings were reported.

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Functional characterization of missense mutations identified in methylmalonic aciduria cblB type and rescue by pharmacological chaperone therapy Brasil S 1 2 3, Briso-Montiano A 1 2 3, Underhaug J 6, Merinero B Desviat R L 1 2 3 4, Ugarte M 1 3 4, Martinez A 6, Perez B 1 2 3 4 5

1 3 4 5

,

1 Centro Diag de Enferm Molec, Madrid, Spain, 2Centro de Bio Molec - SO (CBM-SO), Madrid, Spain, 3Univ Auton de Madrid (UAM) - CSIC, Madrid, Spain, 4CIBERER, Madrid, Spain, 5IDIPAZ, Madrid, Spain, 6Dept of Biomed, Univ of Bergen, Bergen, Norway

Background: Pharmacological chaperones (PCs) have emerged as a promising therapy in several disorders in which loss-of-function mutations affect protein folding. Our group described two possible PCs named Vand VI for ATP:cob(I) alamin adenosyltransferase (ATR), the enzyme responsible for the synthesis of adenosylcobalamin (AdoCbl) (Jorge-Finnigan A. et al., 2013). The aim of the present study was to evaluate the effect of five missense mutations affecting invariant ATR residues located in a key region for enzymatic activity (p.R186W, p.R186Q, p.R190H, p.R191Q and p.E193K) and their recovery with PCs. Methods: For this puropose a prokaryotic system was used and a specific eukaryotic disease cellular model overexpressing these mutations was generated to analyze the effect of these PCs and their combination with hydroxocobalamin (OHCbl) on stability, oligomerization and enzymatic activity. Results: The purification of these mutants showed that all present less trimers compared with wt ATR. For p.R191Q mutant no trimers were obtained. Differential scanning fluorimetry was performed and results showed the destabilizing effect of these mutations over ATR protein and the positive effect of both compounds enhancing thermal stability. The results for 14C-propionate incorporation assay, used as indirect enzymatic measure, showed the improvement of ATR activity. We observed that the combination of either one of the PCs with OHCbl or both with OHCbl represented the highest positive effect on ATR activity and stability. Molecular docking analysis was performed and the two compounds appear to bind to different locations in the protein supporting the synergetic effect observed. Discussion: Our results are promising and open the possibility for a combined therapy using PCs and OHCbl for cblB patients bearing destabilizing loss-of-function mutations and will be tested in a preclinical test in iPS-derived hepatocytes generated from a patient-derived fibroblast line. Work funded by ISCiii PI13/01239 and ERDF.

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progression of cell cycle and observed no difference. We also measured oxidative stress and observed increased mitochondrial superoxide and protein carbonylation levels in patients. Discussion: Our analysis revealed down-regulation of protein translation by eIF4B, indicating a downregulation of mTOR contrary to what is expected with accumulation of BCAAs. This is in line with studies of long-term treatments with BCAAs or mTOR activators. We observed up-regulation of markers for cardiac diseases, like DSP, which is in line with studies of BCAAs defect catabolism as a hallmark for cardiac diseases. These results correlate specific proteins to known regulatory effects of BCAAs, identifying protein markers for MSUD and also for cardiac diseases, cancer, and mitochondrial diseases.

Propionic acidemia: altered cellular and molecular pathways related to mitochondrial function in the animal model Alonso- Barroso E 1, Rivera-Barahona A 1, Perez B 1, Desviat L R 1, Richard E 1

1

Centro Biologia Molecular Severo Ochoa, Madrid, Spain

Background: The role of mitochondrial dysfunction and oxidative stress in the pathogenesis of many human diseases has become more evident in recent years. In the case of propionic acidemia (PA) caused by the deficiency of the mitochondrial propionyl-CoA carboxylase enzyme, the accumulation of toxic metabolites has been described to inhibit mitochondrial enzymes, thereby inducing oxidative stress. Our previous research on the hypomorphic PA mouse model has revealed important findings in line with these processes such as decreased levels and/or activity of several OXPHOS complexes, mtDNA depletion, alteration in antioxidant enzyme levels, lipid peroxidation and an increase in ROS production. Methods: Using the hypomorphic PA mouse model, we aimed to evaluate whether mitochondrial biogenesis is affected by analyzing gene expression related to this process by qRT-PCR, and whether apoptotic or necrotic cell death is induced by analyzing caspases and mitochondrial permeability transition pore (MPTP) component cyclophilin D levels by Western Blot. Results: We have evaluated at the mRNA level several transcriptional regulators of mitochondrial components, including nuclear hormone receptors such as PPARα/β/γ and ERRα; nuclear-encoded NRF1 and the mitochondrial transcription factor TFAM. We found that mRNA levels of PPARα, PPARβ, PPARγ and NRF1 were reduced in the hypomorphic PA model, specially in cardiac tissue. In addition, increased expression levels of cyclophilin D were observed mainly in PA mice liver compared to wildtype mice. Discussion: Our results suggest a downregulation of mitochondrial biogenesis pathway driven by PPAR family and NRF1 which play a major regulatory role in energy homeostasis, metabolic function and mitochondrial respiration, and also more sensitivity to MPTP opening resulting in necrotic cell death. These findings provide deeper knowledge of affected cellular pathways and molecular mechanisms of PA disease contributing to the pathophysiology.

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Regulation of proteins and cellular processes by branched-chain amino acids revealed by large-scale proteomics of fibroblasts from classic maple syrup urine disease patients Fernandez-Guerra P 1, Cheng L 3, Fenton R A 3, Bross P 1, Rodriguez-Pombo P 2, Palmfeldt J 1 1 Res Unit Mol Med, Aarhus Univ Hosp, Aarhus, Denmark, 2CEDEM, CBMSO-UAM, CIBERER, IDIPAZ, Madrid, Spain, 3Dept Biomedicine, Aarhus Univ, Aarhus, Denmark

Background: Branched-chain amino acids (BCAAs) are essential amino acids with multiple functions and are commonly used as dietary supplements in clinical procedures. However, their specific roles and cellular mechanisms are not clear. BCAAs levels are regulated by their catabolism where the rate limiting enzyme is branched chain a-ketoacid dehydrogenase (BCKDH). Aim: Analyse the cellular mechanisms of BCAAs using a single gene defect in BCKDH leading to maple syrup urine disease (MSUD). Methods: We performed large-scale proteomics on fibroblasts from healthy individuals and 5 unrelated classic MSUD patients with null mutations in E1 subunit of BCKDH. Results: We quantified more than 2200 proteins, with 70 proteins differentially regulated in patients, 63 % of them were up-regulated, including E2 subunit of BCKDH, and mitochondrial transporters: SLC25A11, SLC16A1. Pathway analysis revealed a profile associated with specific biological processes: downregulation of cell growth, protein translation, and anti-inflamatory response; upregulation of cellular adhesion, energy metabolism, intracellular and intercellular signalling, and structural and antioxidant proteins. To validate our findings we studied

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Effect of carglumic acid on hyperammonaemia in acute decompensation episodes of organic acidurias Valayannopoulos V 1, Garcia Segarra N 2, Del Toro M 3, Donati M A 7, Garcia-Cazorla A 4, Gonzales M J 4, Plisson C 5, Le Mouhaer J 5, Brachet E 5 , Chakrapani A 6 1 MaMEA IMAGINE, Necker-Enfants Malades, Paris, France, 2Ref Cent Inher Met Dis, Hop R Deb, Paris, France, 3Serv Neurol Infantil, Hosp Vall d’Hebron, Barcelona, Spain, 4Hosp St Joan Deu CIBER-ER, Barcelona, Spain, 5Med Aff, Orphan Europe, Paris, France, 6Metabol Med Dept, Great Ormond St Hosp, London, United Kingdom, 7Metab Unit AOU Meyer, Firenze, Italy

Background: Hyperammonaemia (HA), a key sign of decompensation in organic acidurias (OAs), can lead to severe neurological complications and should be treated rapidly. Methods: A post-hoc analysis of a retrospective study analysed efficacy of carglumic acid (CAR) ± ammonia (NH3) scavengers (SCAV) vs SCAV alone for reducing NH3 in patients (pts) with OAs and HA (plasma NH3 > 60 μmol/L) during decompensation episodes. NH3 was analysed in 12 h periods from 0 to 48 h and in 24 h periods from 48 to 120 h. Treatment-emergent adverse events (TEAEs) were recorded. Results: Of 98 episodes included, 38 were treated with CAR (34 pts), 33 with SCAV (22 pts) and 27 with both (COMBO [27 pts]). Episodes occurring in neonates: 45 % (CAR); 46 % (SCAV); 74 % (COMBO). Median episode duration (days): 6 (CAR/COMBO), 9 (SCAV). Median BL NH3 level (μmol/L): 199 (CAR); 122 (SCAV); 271 (COMBO). Episodes requiring extracorporeal detoxification (ED): 13 % (CAR); 30 % (SCAV); 11 % (COMBO). Data were censored at ED initiation. From treatment initiation to 120 h, mean reduction in NH3 from BL in COMBO and CAR groups was greater than in SCAV group despite both groups having higher BL NH3 levels; reductions were greatest in COMBO group. Time to achieve success/ improvement in 25 % of episodes (plasma NH3 ≤ 60 μmol/L): 34 h (CAR), 37 h (SCAV), 29 h (COMBO). Mean changes in plasma NH3 vs BL in the CAR, SCAV and COMBO groups, respectively, were: −13 %, +12 %, −27 % from 0 to 12 h (p < 0.05 SCAV vs COMBO); −47 %, −22 %, −52 % from 12 to 24 h (p = ns); −44 %, −5 %, −61 % from 24 to 48 h; and −66 %, −16 %, −76 % from 48 to 72 h (p < 0.05 CAR/COMBO vs SCAV for both timepoints). Number of TEAEs was similar in all groups and mainly related to the disease/condition. Discussion: CAR is well tolerated and efficacious for treatment of OA decompensation episodes. When given alone or combined with SCAV, reduction in NH3 was greater than SCAV alone in the first 72 h. Conflict of Interest declared.

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Disruption of 17β-hydroxysteroid dehydrogenase type 10 activity by amyloid-β peptide interaction in brain homogenates and in the recombinant protein Garcia-Villoria J 1, Pascual R 2, Ferrer A 2, Ribes A 1

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Hospital Clinic, IDIBAPS, CIBERER, Barcelona, Spain, 2Inst Biol Mol Cel, Univ Miguel Hernandez, Alicante, Spain Background: 17β-Hydroxysteroid dehydrogenase type 10 (HSD10) is a mitochondrial multifunctional enzyme mutation in the corresponding gene, HSD17B10, and is characterized by neurodegeneration in males. HSD10 has been associated w Alzheimer disease (AD), due to its ability to interact with the amyloid-β peptide (Aβ), suggesting that this interaction is one of the causes of mitochondrial dysfunction in AD. We studied HSD10 activity with the aim to screen therapeutic molecules that could disrupt the Aβ-HSD10 interaction and rescue the HSD10 activity. Methods: We measured HSD10 activity spectrophotometrically using 2methyl-3-hydroxybutiryl-CoA as substrate in 25 autopsied AD human brain samples and 8 age-matched controls. In addition, we obtained the recombinant protein of isoform 1 and measured its activity before and after inhibition by Aβ. Results: HSD10 activity decreases about 30–58 % in AD brain’s homogenates compared with controls. Similarly, the incubation of control brain homogenates with Aβ caused inhibition of HSD10 activity, but this inhibition decreased while increasing NAD+ concentration. This observation has also been corroborated in AD brain’s homogenates. Therefore, the measurement of HSD10 activity could reflect a change in the Aβ-HSD10 interaction. Consequently, we have established an in vitro system to measure the activity of the recombinant isoform 1 protein, which is predominantly expressed in brain. After testing more than 100 molecules, only NAD+ was able to rescue HSD10 activity. Discussion: We hypothesize that NAD+ precursors could be able to disrupt Aβ-HSD10 interaction and consequently, they could be a possible coadjuvant therapy for AD. In fact, it has been reported that treatment of AD mouse model with NAD+ precursors improves mitochondrial function and cognition.

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Incidence of pancreatitis in classical organic acidurias: single centre review Davison J E 1, Cleary M A 1, Dixon M 1, Skeath R 1, Petkovic D 2, McSweeney M 1, Grunewald S 1 1

Great Ormond Street Hosp, London, United Kingdom, 2Univ Zagreb, Zagreb, Croatia Background: Pancreatitis is a recognised complication in organic acidurias, causing significant morbidity. Accurate epidemiological data on the incidence is limited, with mostly anecdotal case reports in the extant literature. Methods: A single centre, retrospective notes review was undertaken to identify all patients with organic acidurias (vitamin B12 non-responsive methylmalonic aciduria (MMA), B12 responsive MMA (B12-MMA), propionic aciduria (PA) and isovaleric acidaemia (IVA)). Baseline demographics and genetic and biochemical diagnostics were collected. Episodes of pancreatitis were identified, including timing of event, clinical, biochemical and radiological features. Overall incidence of pancreatitis was determined. Kaplan-Meier (KM) survival analysis was deployed to analyse time to first episode of pancreatitis, with patients censored if deceased, lost to follow up or transitioned to adult services. Inter-group comparison was made by KM logrank test and hazard ratio (95 % confidence interval) calculation (MedCalc Statistical Software), to review time to first episode of pancreatitis and risk of pancreatitis in each cohort. Results: 63 patients were identified (MMA n = 22, B12-MMA n = 14, PA n = 12, IVA n = 15). Pancreatitis occurred in 13 % of patients: MMA 8/22 (36 %), median age first episode 4.15 years (range 1.59–7.45); B12-MMA 1/14 (7 %) at 7.14 years; PA 3/12 (25 %), median age 3.14 years (1.76–3.30); IVA 1/15 (6.7 %) at 9.43 years. KM analysis showed that groups differed significantly (p = 0.016), with MMA and PA having a significantly higher incidence and earlier onset of pancreatitis than IVA and B12-MMA patients. Discussion: In this cohort, the incidence of pancreatitis was higher than in previous studies, with a notable predilection in patients with classical B12 non responsive MMA. This complication should be monitored for closely,

and further work should explore potential aetiologies of pancreatitis, preventative strategies and optimal management of episodes.

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Developmental delay in a patient with mild isovaleric acidemia Pontoizeau C 1 2, Arnoux J B 1, Habarou F 1 2 3, Brassier A 1, Guemann A S 1, Chabli A 1 2, Vianey-Saban C 4, Chadefaux-Vekemans B 1 2, Acquaviva C 4, Read M H 5, De Lonlay P 1, Ottolenghi C 1 2 3 IEM Reference Centre, Necker Hosp, APHP, Paris, France, 2 Metab Biochemistry, Necker Hosp, APHP, Paris, France, 3 INSERM U1163, Imagine Ins, Paris, France, 4Metab Dis Dept, CHU Lyon, Bron, France, 5 Biochem Dept, CHU Caen, Caen, France 1

Background: Isovaleric aciduria (IVA) includes clinical forms of variable severity. Newborn screening (NBS) uncovered a potentially asymptomatic clinical form (“mild IVA”), recurrently associated with the mutation p.Ala311Val in the IVD gene and moderate accumulation of diagnostic metabolites (plasma C5-carnitine < 6 μmol/L and urinary isovalerylglycine, IVG, < 450 mmol/mol creatinine). Considering the lack of NBS for IVA in France, it is of interest that we identified a symptomatic patient with biochemical features of mild IVA. Methods: Between 2007 and 2015, we performed 277 urine organic acid analyses and 58 plasma acylcarnitine profiles for 34 patients suffering from IVA. We compared the values of these patients with the corresponding cohort of >25000 analyses from >15000 patients known or suspected for other metabolic diseases. Results: In all patients except one, IVG values were consistently above 200 mmol/mol of creatinine and plasma C5-carnitine above 3 μmol/L, with or without therapy, with important interindividual variation. The remaining patient had only moderate elevation of IVG values (25–155 mmol/mol) and C5-carnitine (2.6–3.5 μmol/L). This patient had been referred at 6 months for hypotonia and developmental delay. Genetic analysis of the IVD gene identified the c.932C > T (p.Ala311Val) mutation. Psychomotor delay persisted under nutritional management and glycine-carnitine supplementation. Discussion: We identified a symptomatic patient with biochemical features of mild IVA carrying a homozygous p.Ala311Val mutation in the IVD gene, raising the question of causality for the clinical findings. While we cannot rule out a second unidentified disease, psychomotor delay was previously reported in two patients with composite heterozygous mutations involving one p.Ala311Val allele. Although these findings may result from ascertainment bias, we suggest an additional degree of caution in the interpretation of NBS results and genetic counseling.

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Endocrinological aspects in propionic acidemia Stanescu S 1, Belanger-Quintana A 1, Arrieta F 1, Perez-Cerda C 2, Merinero B , Martinez-Pardo M 1

2

1 Un Enf Metab, Hosp Univ Ramon y Cajal, Madrid, Spain, 2CEDEM, Universidad Autonoma, Madrid, Spain

Background: Propionic acidemia (PA) is an inherited disorder caused by deficiency of propionil CoA carboxilasa. Most patients with this disorder present in the neonatal period with severe metabolic acidosis and hyperammonemia. However long-term complications present even in patients with good metabolic control, suggesting that the understanding of the disease course is still limited. Frequently the chronic complications involve the nervous system, the heart or the bone marrow. Little is known about the endocrinological aspects. Methods: We investigated 10 PA patients from a single metabolic center. Retrospective clinical information was collected by review of our medical records. Results: 4/10 patients (2 females and 2 males) presented with alteration of the thyroid function, all with subclinical hypothyroidism (high TSH, normal free

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T3/free T4). All had negative immunity markers for the thyroid disease. There was no relation with the acute decompensation (no metabolic acidosis or hyperammonemia) and we could not identify markers suggesting bad metabolic control. Three patients received treatment with oral levothyroxine, which normalized the thyroid function as expected. Discussion: There are no reports on the thyroid disease in the PA pathogenesis. Considering that the thyroid is involved in cardiac, nervous or hematological normal functions, we suggest periodical assessment in order to determine possible alterations.

P-254

Maple syrup urine disease in the Marmara region of turkey Balci M C 5, Karaca M 1, Zubarioglu T 1, Ozer I 6, Dorum S 2, Demirkol M 4, Gokcay G 3 1 Div Metab Dis, Istanbul Univ Child Hosp, Istanbul, Turkey, 2Div Metab Dis, Uludag Univ Child Hosp, Bursa, Turkey, 3Div Metab Dis, Istanbul Univ Child Hosp, Istanbul, Turkey, 4Div Metab Dis, Istanbul Univ Child Hosp, Istanbul, Turkey, 5Div Metab Dis, Tokat State Hosp, Tokat, Turkey, 6Div Metab Dis, Goztepe State Hosp, Istanbul, Turkey

Background: Maple syrup urine disease (MSUD) (OMIM #24860) is caused by branched-chain −keto acid dehydrogenase (BCKD) deficiency resulting in the accumulation of the branched-chain amino acids (BCAA) and their corresponding α-ketoacids. In Turkey MSUD is not included in the newborn screening program. In Marmara region of Turkey with a population of 20 million residents six metabolic centers with 18 pediatric metabolic physicians and 4 dietitians are responsible for the follow-up of MSUD patients. Objectives: The aim of the study was to characterize MSUD patients followed in the region with relevance to strategies such as dietary treatment and liver transplantation. Methods: Clinical features at referral and follow-up, treatment methods and current status of the patients were evaluated. Results: 85 patients (45 M, 40 F) diagnosed with MSUD, between 2001 and 2016, were studied. Classical MSUD (n = 80), intermediate MSUD (n = 4), and lipoamide dehydrogenase [E3] deficiency (n = 1) were diagnosed. Classical MSUD patients presented between first and 11 (mean: 5,1) days of life. Intermediate form MSUD patients, whose most common clinical manifestations were neurologic symptoms, were diagnosed between 8 and 24 months. Consanguinity was reported in 55 patients. 4 patients have familyhistory of MSUD. 44 late diagnosed patients who also had frequent metabolic decompensations had psychomotor and neurodevelopmental delay. 3 patients, 2 with poor dietary compliance and 1 with frequent metabolic decompensations, were liver transplanted. Discussion: The results of this study indicates that a better outcome in MSUD is associated with early diagnosis, good dietary compliance and timely intervention of elevated leucine levels.

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P-255

Renal impairment in methylmalonic aciduria: a review of six cases Seker Yilmaz B 1, Bulut F D 1, Kor D 1, Karabay Bayazit A 2, Yildizdas D 3, Anarat A 2, Onenli Mungan N 1 1 Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Div Ped Nephrol, Univ Cukurova, Adana, Turkey, 3Div Ped Intensive Care, Univ Cukurova, Adana, Turkey

Methods: Six patients from consanguineous Turkish parents, diagnosed as MMA were included in the study. These patients all have poor metabolic control. Renal functions were monitored during the follow-up. Creatinine clearance was evaluated with Schwartz equation, which is less dependent upon muscle mass, and estimated glomerular filtration rates (eGFR) were calculated. Results: The median age of the patients was 95 months (41–167) and the median follow-up period was 7 years (4–13). All of the patients presented with frequent episodes of dehydration and metabolic decompensation. While 5 of them were mut−/mut0, 1 of them was cblB (MMAB) variant. During the attacks progressive deterioration of renal functions were observed. Beyond that between the episodes, creatinine levels have begun to remain higher. Edema, hypertension and significant proteinuria were not detected. Hyperkalemia and elevated uric acid levels were remarkable. eGFR levels were between 28.4 and 59.4 mL/min per 1.73 m2. Despite the appropriate hydration and alkylation, renal impairment continued to increase. Currently none of our patients need renal replacement therapy. Discussion: Cardiomyopathy, pancreatitis, hepatic insufficiency, neurological impairment and progressive renal deterioration are frequent and serious complications of long-term survivors of MMA. As recurrent metabolic decompensations are compatible with poor renal prognosis, it is very important to monitor renal functions and prevent undesirable complications.

P-256

Maple syrup urine disease: consensus for nutritional treatment from the Marmara region of Turkey Saglam T 1, Balci M C 1, Karaca M 1, Zubarioglu T 2, Ozer I 3, Doruk S 4, Zeybek C A 2, Demirkol M 1, Gokcay G 1 1 Ped Nutr Metab Dis Ist Med Faculty, Istanbul, Turkey, 2Ped Nutr Metab Ist Univ Cer Med Facu, Istanbul, Turkey, 3Ped Metab Dis, Goztepe Edu Hospital, Istanbul, Turkey, 4Ped Metab Dis, Uludag Univ, Bursa, Turkey

Background: Maple syrup urine disease (MSUD) needs monitoring closely for a lifetime and nutritional treatment is critical for good prognosis. Different centers adopt different nutritional treatment strategies leading to confusion and misinterpretation among patients, especially among those living in the same region. This study aims at drawing conclusions about the treatment of MSUD from the experience of different centers and developing a regional consensus for treatment. Methods: The study is organized by six metabolic centers following MSUD (n = 86, 40 M, 46 F) patients in Marmara region of Turkey. Meetings were organized to discuss treatment and follow-up, food exchange lists, target plasma branched chain amino acid levels, family education, “sick-day” measures, elimination diet, liver transplantation and new treatment options in MSUD. Overall patient characteristics are gathered from centers. Results: Target protein and calorie amounts in the diets and plasma branched chain amino acid levels differed between centers. Exchange lists, “sick day” measures, and family education were similar. Center:s agreed that early and successful treatment could eliminate the need for liver transplantation. Number of hospitalization after diagnosis was 1,7 (mean: 2, range; 0–8) within the first year of life and only three patients were liver transplanted. Discussion: A similar approach to nutritional treatment of MSUD patients is essential to prevent controversies. Physicians and dietitians benefit from discussions and sharing experiences. Centers with more frequent monitoring, fast plasma amino acid analysis opportunities and easy patient communication methodologies are more successful in nutritional therapy and therefore in less need of transplantation.

P-257 Background: Methylmalonic acidemia (MMA) is an inborn error of metabolism caused by the deficiency of the mitochondrial methylmalonyl-CoA mutase (MUT). Chronic renal disease is a serious complication of MMA. Although the mechanisms of renal injury have not been elucidated, the few reported biopsies have consistently demonstrated tubular injury, interstitial fibrosis and glomerulosclerosis. The primary pathological mechanism may be mitochondrial dysfunction in the renal tubules.

Behavioral phenotype in a liver transgenic mouse model of methylmalonic acidemia Fraser J L 1, Arnold M L 1, Gagne J 1, Venditti C P 1 1 National Institutes of Health, Bethesda, United States

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Methylmalonic acidemia (MMA) causes acute metabolic crises and chronic end-organ damage, and requires lifelong protein restriction. Liver transplant improves metabolic outcome, yet patients remain at risk for metabolic stroke. The neurological phenotype of Mut−/− liver transgenic mice was employed as a model for neurological decompensation in MMA. The mice survive neonatal lethality by low-level expression of Mut, yet have massively elevated metabolites in the brain and decompensate with stress. Methods: Mut−/−;TgINS-Alb-Mut mice (“KO”, N = 9) and their heterozygous littermates (“Het”, N = 25) were fed high fat rodent chow (PicoLab Mouse Diet 20) to promote survival through the study period. Postnatal mice were assessed for weight, developmental milestones, and early reflexes. Testing for strength, motor, and coordination occurred weekly from 2 to 8 weeks of age and again at 8 months, using Rotarod, rope suspension, and ambulation. Twoway ANOVA repeated measures with Buonferroni post-test and Student’s twotailed t-test were performed. Results: Nine KO mice began early developmental testing, with 2 deaths between 2 and 3 weeks of life (survival = 77.8 %) with no subsequent deaths; the 2 dead pups demonstrated marked neurological decompensation 24–48 h prior to death, were not able to be tested and therefore were excluded from behavior analysis. No statistically significant differences existed between KO and Het in behavioral tests. Discussion: KO mice appear to be protected from neurological injury under low metabolic stress; unprovoked neurological injury does not appear to occur in this model. Mice were nutritionally supported on chow containing twice the fat content of standard rodent chow and 3 % less protein, and diet may have contributed to the outcomes of the KO mice, which were not under significant metabolic stress. These data serve as the benchmark for further studies on neurological injury in MMA mice.

11. Organic acidurias: others

P-258

Development and validation of a quality of life questionnaire for paediatric patients with intoxication-type inborn errors of metabolism Zeltner N A 1 2 3 4 5, Landolt M A 2 3 5, Baumgartner M R 1 3 4, Ensenauer R 8, Karall D 9, Koelker S 10, Muehlhausen C 7, Scholl-Buergi S 9, Thimm E 8, Quitmann J 6, Burgard P 10, Huemer M 1 4 1 Div Metab, Univ Child Hosp, Zurich, Switzerland, 2Dept Psychosom Psychiatr, Univ Child Hosp, Zurich, Switzerland, 3Child Res Center, Univ Child Hosp, Zurich, Switzerland, 4Radiz Rare Dis Initiative, Univ Zurich, Zurich, Switzerland, 5Dept Child Adolesc Psych, Univ Zurich, Zurich, Switzerland, 6Dept Med Psychol, Univ Med Center, Hamburg, Germany, 7 Dept Pediatr, Univ Med Center, Hamburg, Germany, 8Dept General Pediatr, Univ Child Hosp, Duesseldorf, Germany, 9Clinic for Pediatr, Medical Univ, Innsbruck, Austria, 10Center Paediatr Adolesc Med, Univ Hosp, Heidelberg, Germany

Background: Patients with intoxication-type inborn errors of metabolism (IT-IEM; e.g. organic acidurias) have to adhere to strict diet and medication and often face neurological sequelae and metabolic crises. Disease-specific health-related quality of life (HrQoL) assessment questionnaires are the method of choice to estimate the burden of a disease. To date, no such instrument is available for IT-IEM. Methods: A disease-specific patient- and parent-reported HrQoL questionnaire in German was constructed based on focus group interviews with patients and parents. Questionnaires were piloted in 21 subjects (n = 13 children and adolescents, n = 8 parents) and psychometrically tested in 122 subjects (n = 55 patients, n = 67 parents) for scale distribution, validity and reliability. Results: Items and scales of self- and proxy-questionnaires showed good concurrent validity with generic HrQoL questionnaires, internal consistency and split-half reliability. The established three main dimensions of physical, mental and social HrQoL were delineated using factor analysis. Acceptance of the questionnaire was good, but applicability was limited in patients < 8 years.

Discussion: The new IT-IEM-QOL questionnaire is a valid and reliable assessment tool with high content validity. It marks a promising step towards patientreported outcomes in research and clinical practice. Conflict of Interest declared.

P-259

Quinolinic acid provokes histopathological alterations associated with a neuroinflammatory response in striatum of Gcdh−/− mice: possible contribution of the kynurenine pathway in GA I neuropathology Amaral A U 1, Silva J C 1, Ribeiro R T 1, De Oliveira F H 2, Seminotti B 1, Leipnitz G 1, Colin-Gonzalez A L 3, Santamaria A 3, De Souza D O G 1, Wajner M 1 4 1 Departamento de Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil, 2Servico de Patologia, HCPA, Porto Alegre, Brazil, 3Lab Aminoacidos Excitadores, INNN, Cidade do Mexico, Mexico, 4Servico de Genetica Medica, HCPA, Porto Alegre, Brazil

Background: The pathogenesis of the striatum degeneration in glutaric aciduria type I (GA I) that usually occurs during inflammatory processes (infections) that are characterized by stimulation of the kynurenine pathway (KP) with high production of quinolinic acid (QUIN) is still a matter of debate. Methods: We investigated the effects of an acute intrastriatal administration of QUIN on the histopathology of striatum from 30-day-old wild type (WT) and glutaryl-CoA dehydrogenase knockout (Gcdh−/−) mice fed a high lysine (4.7 %) chow. We evaluated striatum morphology by hematoxylin and eosin, T lymphocytes (CD3), astrocyte activation (GFAP) and neuronal viability (NeuN) by immunohistochemistry and nitrotyrosine (YNO2) by immunofluorescence. Results: QUIN provoked vacuolation, edema and lymphocyte infiltration predominantly in the striatum of Gcdh−/− mice. Furthermore, QUIN induced a higher CD3 staining and increased number of YNO2 positive cells in the glutaryl-CoA dehydrogenase deficient mice, indicating, respectively, the presence of T lymphocytes and nitrosative stress. On the other hand, while QUINtreated WT mice showed an expected increase of GFAP staining, the level of this astrocytic protein was not changed in Gcdh−/− QUIN-injected mice, suggesting an abnormal response of Gcdh−/− astrocytes to QUIN toxicity. Finally, NeuN staining was reduced in both WT and Gcdh−/− mice treated with QUIN, indicating neuronal loss. Discussion: QUIN, whose brain production is increased during infectious/ inflammatory processes, provokes important histopathological alterations with the involvement of nitrosative stress that are associated with neurodegeneration in striatum of Gcdh−/− mice. It is presumed that activation of the KP during neuroinflammation may be implicated in the striatal damage of GA I patients during infections. Financial support: CNPq, PROPESq/UFRGS, FAPERGS, FINEP IBN-Net and INCT-EN.

P-260

Mevalonolactone disturbs mitochondrial homeostasis in brain of young rats: a potential mechanism of brain damage in mevalonic aciduria? Cecatto C 1, Silva J C 1, Amaral A U 1, Wajner A 1, Silva L H R 1, Nunes R C 1, Ribeiro R T 1, Godoy K S 1, Leipnitz G 1, Wajner M 1 2 1 Departamento de Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil, 2Servico de Genetica Medica, HCPA, Porto Alegre, Brazil Background: Mevalonic aciduria (MVA) is a multisystemic inflammatory disorder caused by severe deficiency of mevalonate kinase (MK) activity, leading to accumulation of mevalonic acid (MA) and mevalonolactone (ML) in tissues and biological fluids of affected patients. Developmental delay, cerebellar ataxia, psychomotor retardation and failure to thrive associated with hepatic alterations are usually observed. Considering that the pathogenesis of brain and liver alterations in this disease is poorly known, the present study evaluated

S134 the effects of MA and ML on important parameters of energy and Ca2+ homeostasis in rat brain and liver mitochondria. Methods: Mitochondrial membrane potential (ΔΨm), matrix NAD(P)H content, swelling and Ca2+ retention capacity, as well as respiratory parameters (state 3, state 4 and uncoupled respiration) and mitochondrial membrane fluidity were measured in mitochondrial preparations. Results: ML markedly decreased ΔΨm, NAD(P)H pool and Ca2+ retention capacity, and induced mitochondrial swelling in brain, with no alterations in liver mitochondria. These effects were completely prevented by cyclosporin A and ADP, as well as by ruthenium red, indicating the involvement of mitochondrial permeability transition (mPT) and of Ca2+. In contrast, ML did not alter the respiratory parameters and the mitochondrial membrane fluidity. Furthermore, MA was not able to alter any of the tested parameters, implying selective actions for ML. Discussion: The present data indicate that ML markedly impairs mitochondrial energy and Ca2+ homeostasis in brain with the involvement of mPT induction. Our data indicate that ML-induced disturbance of mitochondrial functions may contribute to the brain alterations characteristic of patients affected by MVA. Financial support: CNPq, PROPESq/ UFRGS, FAPERGS, FINEP IBN-Net and INCT-EN.

P-261

Unravelling 5-oxoprolinuria (pyroglutamic aciduria) due to bi-allelic OPLAH mutations: 20 new mutations in 14 families Sass J O 1, Gemperle-Britschgi C 2, Tarailo-Graovac M 3, Patel N 4, Walter M 6 , Jordanova A 7 17, Alfadhel M 5, Baric I 8, Coker M 9, Damli-Huber A 10, Faqeih E A 11, Garcia Segarra N 12, Geraghty M T 13, Jatun B M 14, Kalkan Ucar S 9, Kriewitz M 15, Rauchenzauner M 16, Bilic K 8, Tournev I 17 18, Till C 1 , Sayson B 3, Beumer D 3, Ye C X 3, Zhang L H 3, Vallance H 3, Alkuraya A S 4 , Van Karnebeek C D M 3 1 Bonn-Rhein-Sieg Univ Appl Sci, Rheinbach, Germany, 2Univ Child Hosp, Zurich, Switzerland, 3Univ British Columbia, Vancouver, Canada, 4King Faisal Spec Hosp Res Ctr, Riyadh, Saudi Arabia, 5King Saud bin Abdulaziz Univ Health Sci, Riyadh, Saudi Arabia, 6Univ Child Hosp, Freiburg, Germany, 7Univ Antwerp, Antwerp, Belgium, 8Univ Hosp Ctr, Zagreb, Croatia, 9Ege Univ Med Fac, Izmir, Turkey, 10kbo Child Ctr, Munich, Germany, 11Child Hosp King Fahad Medical City, Riyadh, Saudi Arabia, 12 Univ Hosp Ctr Vaudois, Lausanne, Switzerland, 13Child Hosp East Ont, Ottawa, Canada, 14Alesund Hosp, Alesund, Norway, 15Bernkastel-Wittlich Hosp, Wittlich, Germany, 16Hosp Ostallgau-Kaufbeuren, Kaufbeuren, Germany, 17Med Univ-Sofia, Sofia, Bulgaria, 18New Bulg Univ, Sofia, Bulgaria

Background: Primary 5-oxoprolinuria (pyroglutamic aciduria) is caused by a genetic defect in the γ-glutamyl cycle, affecting either glutathione synthetase or 5-oxoprolinase. While several dozens of patients with glutathione synthetase deficiency have been reported, with hemolytic anemia representing the clinical key feature, 5-oxoprolinase deficiency due to OPLAH mutations is less frequent and so far has not attracted much attention. This has prompted us to investigate the clinical phenotype as well as the underlying genotype in patients from 14 families of various ethnic backgrounds who underwent diagnostic mutation analysis following the detection of 5-oxoprolinuria. Methods: OPLAH was analysed by Sanger sequencing; two patients underwent whole exome or whole genome sequencing. The corresponding clinical phenotypes were obtained from medical records. Results: In all patients with 5-oxoprolinuria studied, bi-allelic mutations in OPLAH were indicated. An autosomal recessive mode of inheritance for 5oxoprolinase deficiency is further supported by the identification of a single mutation in all 9/14 parent sample sets investigated (except for the father of one patient whose result suggests homozygosity), and the absence of 5oxoprolinuria in all tested heterozygotes. It is remarkable that all 20 mutations identified were novel and private to the respective families. Clinical features were highly variable and in several sib pairs did not segregate with 5-oxoprolinuria. Discussion: Although a pathogenic role of 5-oxoprolinase deficiency remains possible, this is not supported by our findings. Additional patient

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 ascertainment and long-term follow-up is needed to establish the benign nature of this inborn error of metabolism. It is important that all symptomatic patients with persistently elevated levels of 5-oxoproline and no obvious explanation are investigated for the genetic etiology.

P-262

Impact of age at onset and newborn screening on outcome in organic acidurias Boy N 1, Heringer J 1, Valayannopoulos V 2, Garcia-Cazorla A 3, Lund A M 4, Chakrapani A 5, Wijburg F A 6 1 Div Inherit Metab Dis, Univ Child Hosp, Heidelberg, Germany, 2Centre Ref Mal Metab, Hop Univ Necker, Paris, France, 3Servicio Neurologica, Hosp San Joan deDeu, Barcelona, Spain, 4Centre for Inherit Metab Dis, Univ Hosp, Copenhagen, Denmark, 5Birmingham Child Hosp NHS Found Trust, Birmingham, United Kingdom, 6Dept Pediatrics, Academic Medical Center, Amsterdam, Netherlands

Background: To describe current diagnostic and therapeutic strategies in organic acidurias (OADs) and to evaluate their impact on the disease course. Methods: 567 OAD patients from the E-IMD registry were analysed including patients with methylmalonic (MMA, n = 164), propionic (PA, n = 144) and isovaleric aciduria (IVA, n = 83), and glutaric aciduria type 1 (GA1, n = 176). Statistical analysis included description and recursive partitioning of diagnostic and therapeutic strategies, and odds ratios (OR) for health outcome parameters. Symptomatic patients were divided into those presenting with first symptoms during (i.e. early onset, EO) or after the newborn period (i.e. late onset, LO). Results: Patients identified by newborn screening (NBS) had a significantly lower median age of diagnosis compared to the LO group, but not compared to the EO group. 71 % of all patients remained asymptomatic until day 8. Patients with cobalamin-nonresponsive MMA (MMA-Cbl−) and GA1 identified by NBS were less likely to have movement disorders than those diagnosed by selective screening. For other OADs, NBS effect was less clear. Variable drug combinations, ranging from 12 in MMA-Cbl to 2 in isovaleric aciduria, were used for maintenance treatment. The effects of specific metabolic treatment strategies on clinical outcome remained unclear because of the strong influences of age at onset (EO versus LO), diagnostic mode (NBS versus selective screening), and the various treatment combinations used. Discussion: NBS improves neurological outcome in GA1 and MMACbl−. Majority of symptomatic patients could have been identified earlier by NBS whilst still asymptomatic. Huge diversity of therapeutic interventions hampers the understanding of optimal treatment. Natural disease course and mode of diagnosis should be considered for evaluation of therapeutic effects.

P-263

Phenotype and genotype of a Spanish cohort with isovaleric acidaemia De Castro M J 1, Couce M L 1, Aldamiz-Echevarria L 2, Bueno M A 3, Barros P 4, Belanger A 5, Blasco J 6, Garcia Silva T 7, Marquez A 8, Vitoria I 11, Vives I 9, Fernandez-Marmiesse A 1, Perez B 10, Perez-Cerda C 10 1 Metab Unit Hosp Clin Univ, IDIS, CIBERER, Santiago de Compostela, Spain, 2Metab Unit Hosp Cruces Biocruces CIBERER, Bilbao, Spain, 3 Metab Dismorph Unit Hosp Univ Virg Rocio, Sevilla, Spain, 4Metab Bone Dis Resarch Gr, School Nurs, Extremadura, Spain, 5Nutr and Metab Unit, Hosp Ramon Cajal, Madrid, Spain, 6Gastr Hep Child Nutr Unit, Hosp Carlos H, Malaga, Spain, 7Pediat Rare Dis Unit, Hosp Univ 12 Oct, Madrid, Spain, 8Pediat Gastr Unit Metab Dis, Hosp Mat In, Badajoz, Spain, 9Metab Unit Pediat, Hosp Univ Virg Arrix, Murcia, Spain, 10Centr Diag Enf Biol Mol, IDIPAZ CIBERER, Madrid, Spain, 11Unit of Metab, Hosp Univ La Fe, Valencia, Spain

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Isovaleric acidaemia is a metabolic disorder of leucine metabolism due to a deficiency of isovaleryl-CoA dehydrogenase, resulting in the accumulation of isovaleric acid, isovalerylglycine (IVG) and isovalerylcarnitine (C5). The clinical presentation is highly variable ranging from severely affected to an asymptomatic state. Case report: This study analyzes clinical, biochemical and genetic data from a heterogeneous group of 16 IVA Spanish patients regarding phenotype (8 mild, 4 moderate and 4 severe) and disease detection (by newborn screening [NBS], familiar study [FS] or clinical symptoms [CS]). Results: Mean age at diagnosis from 8 NBS cases was 16 d, presenting blood spot C5 mean level of 4.2 μM and urine IVG mean of 562 mmol/molcreat, except one case that excreted 19069 mmol/molcreat and presented poor feeding and smell sweaty feet. The remaining cases (FS and CS) had plasma C5 mean level of 8.5 μM and urine IVG mean of 6405 mmol/molcreat. The percentages of IVA incorporation in fibroblasts in 3 NBS patients with mild disease were much higher (60–80 %) than in symptomatic patients (4.9–13 %). The average follow-up period, except one patient who died at 2 days of detection, was 9y,10m. Under treatment, three patients needed admission outside the detection period. Mean PDI/IQ in NBS patients had a value greater than for CS patients (96.8 vs 89). Genetic analysis revealed 15 different mutations, 6 previously described and 9 novel ones: p.Ile405Thr; p.Gly236Ser; p.Ala314Gly; p.Leu295Pro; p.Ser367Pro; p.Glu404Gly; c.111-112delGC; c.465 + 22_519delinsGTTG and a deletion comprising 1-2-3 exons. Discussion: 1) Urine IVG level at diagnosis seems to be better biochemical marker of prognosis than blood C5 levels. 2) Severe mutations correlate well with low incorporation of IVA in fibroblasts and worst clinical evolution. 3) Our results confirm the exceptionality of this classical organic aciduria regarding its milder neuropathological implications

P-264 3D organotypic Gcdh−/− brain cell cultures generate GA and 3-OHGA, and show histomorphological alterations under high lysine exposure Cudre-Cung H P 1, Remacle N 1, Do Vale Perreira S 1, Schmiesing J 3, Ivanisevic J 4, Muehlhausen C 3, Braissant O 2, Ballhausen D 1 1

Cent Mol Dis, Laus Univ Hosp, Lausanne, Switzerland, 2Biomedicine, Laus Univ Hosp, Lausanne, Switzerland, 3Dept of Bioch, Child Hosp, Univ Med Cent, Hamburg, Germany, 4Metabolomics Research Platform, Lausanne, Switzerland Background: Glutaric aciduria type I (GA-I) is caused by glutaryl-CoA dehydrogenase (Gcdh) deficiency and characterized by accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body fluids and tissues. Current treatment strategies are not in all cases effective to prevent brain damage despite early diagnosis by neonatal screening. We previously studied the cerebral toxicity of GA and 3-OHGA via their administration in 3D organotypic re-aggregated brain cell cultures issued from wild-type (WT) rat embryos. Methods: In order to be closer to in vivo conditions, we developed a 3D organotypic re-aggregated brain cell model issued from Gcdh−/− mouse embryos. We also investigated the effect of 10 mM L-lysine hydrochloride (Lys) added to this model at DIV 13. All cultures were harvested at DIV 14. Results: Accumulation of GA and 3-OHGA was observed in culture medium of Gcdh−/− aggregates while it was absent from WT cultures. Ammonium increased significantly in culture medium of Gcdh−/− aggregates similarly to rat aggregates exposed to 3-OHGA. No significant morphological brain cell alterations were observed in Gcdh−/− aggregates under normal culture conditions. Exposure to Lys led to reduction and shortening of astrocytic fibers and decreased signal intensity of oligodendrocytic markers in Gcdh−/− aggregates, and to a lesser extent also in WT aggregates. Discussion: This study provides evidence that Gcdh−/− developing mouse brain cells produce GA and 3-OHGA. As observed in Gcdh−/− mice, our new in vivo model displays the biochemical phenotype of GA-I, and shows histomorphological alterations when exposed to high Lys concentrations. We will now continue to investigate the mechanisms leading to brain cell suffering under Lys exposure in this model.

P-265

Persistent finding of suberic acid, azelaic acid and pimelic acid in organic acid profiles from a patient subsequently diagnosed with Wolman’s disease Hart C E 1, Wu H Y 3, Sharrard M 2, Tylee K 3, Church H 3, Jones S A 4 1 Clinical Chemistry, Sheffield Child Hosp, Sheffield, United Kingdom, 2Dept Paediatrics, Sheffield Child Hosp, Sheffield, United Kingdom, 3Willink Lab, Central Man Univ Hosp, Manchester, United Kingdom, 4Willink Unit, Central Man Univ Hosp, Manchester, United Kingdom

Background: Infantile onset lysosomal acid lipase (LAL) deficiency (Wolman’s disease) is characterised by diarrhoea, vomiting, failure to thrive, adrenal calcification and hepatosplenomegaly. It is diagnosed by measurement of the LAL enzyme. Case report: The patient presented with failure to thrive at 5 weeks of age. Amino acid, VLCFA and acylcarnitine results were unremarkable. The organic acid profile showed marked excretion of suberic, azelaic and pimelic acids. This finding was thought due to contamination and a fresh sample requested. Several subsequent samples showed the same pattern which was notable for the very consistent peak heights of the 3 compounds lending doubt to the idea that it was due to exogenous contamination but no conclusions could be reached about the significance. At 3 months the patient was noted to have hepatosplenomegaly and further tests were undertaken including white cell enzymes and chitotriosidase. Results: Chitotriosidase was elevated at 3431 μmol/L/h (NR = 4–120) and acid lipase deficient at 44 μmol/g/h (NR = 350–2000). A diagnosis of Wolman’s disease was made and the patient enrolled in a trial of enzyme replacement therapy with Sebelipase alfa. After treatment the organic acid profile showed near total resolution of the previous findings, lending weight to the idea that the findings were related to the underlying diagnosis. Discussion: There appears to be a link between suberic, azelaic and pimelic acids in a urine organic acid profile and a diagnosis of Wolman’s disease, although the mechanism remains obscure. A subsequent in depth study of the literature uncovered a case report of a similar finding in an abstract for a poster presented at the 2010 SSIEM. Since the symptoms of Wolman’s in the early stages may be non-specific and organic acid analysis is typically one of the first metabolic tests requested we feel wider knowledge of this link could speed up diagnosis significantly for some patients.

P-266

Early neurodevelopment in children with cblC defect Martinelli D 1, Gambardella M L 2, Ferrantini G 2, Lucibello S 2, Diodato D 3, Dionisi-Vici C 1, Mercuri E 2, Ricci D 2 1 Division of Metabolic Diseases, OPBG, Rome, Italy, 2Pediatric Neurology Unit, UCSC, Rome, Italy, 3Unit of Neuromuscular Disorders, OPBG, Rome, Italy

Background: A common clinical feature of infantile-onset methylmalonic acidemia with homocystinuria, cblC type, is psychomotor delay, presenting early in life, apparently irrespective of brain lesions and ocular signs. However, few studies have investigated the early neuropsychological phenotype in cblC defect. We aimed to study cognitive function development in our cohort of children with infantile-onset cblC defect and to verify a possible influence of clinical signs such as epilepsy. Methods: Patients (pts) were included if they had at least one neurodevelopmental assessment. We used the Griffiths Mental Development Scales. General quotient (GC) and the subquotients for each scale were calculated. The results were analyzed according to age at the assessment, age at the diagnosis (newborn screening versus symptomatic diagnosis), presence and age at onset of epilepsy.

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Results: 18 pts (age 17–74 months) were included in the study. All pts had 2 assessments at 9–12 months distance. We observed a progressive deterioration of GC and subquotients with major impairment of speech development after 24 months. 4 of 18 were diagnosed by newborn screening and had a GC within normal range between 24 and 36 months; 14 pts showed language and eye-hand coordination delay. 10 of 18 pts did not present epilepsy and showed the higher quotients; 5 pts presented seizures after 1 year of age and their quotients were lower than the previous group but close to borderline; 3 pts presented epilepsy in the first year and showed severe delay. Discussion: In our cohort of children with cblC defect, 15/18 (83 %) had a variable degree of psychomotor delay. Early impairment of speech development was a frequent finding between 24 and 36 months, that persisted after 36 months. Newborn screening was a positive predictor for early neuropsychological development, whereas presence and age at onset of epilepsy were the main predictors for a worse outcome.

P-267

Dysregulated proteins in a cellular model of methylmalonic acidemia Caterino M 1 2 3, Costanzo M 1 2 3, Minopoli G 1, Santorelli L Vecchio L 1 3, Raia M 1 3, Ruoppolo M 1 2 3

1 2 3

, Del

1 Dept Mol Med Med Biotechnol, Univ FedII, Naples, Italy, 2DiSciMuS RFC, Casoria, Italy, 3CEINGE Advanc Biotechnol, Naples, Italy

Background: Methylmalonic acidemias (MMAs) are severe autosomal recessive inborn errors of metabolism caused by the deficiency of the enzyme methylmalonyl - CoA mutase (MUT). MUT converts L methylmalonyl - CoA into succinyl - CoA, a Krebs cycle intermediate. The disorders are caused by mutations in the MUT apoenzyme or defective metabolism of the enzymatic cofactor, 5’deoxyadenosylcobalamin. MMA patients, classified as mut0, have no detectable residual enzyme activity. The brain damage is one of the hallmarks of disease. An in vitro silencing of MUT gene was performed using the RNA-interference strategy to produce a neuronal cellular system which could be used to study dysregulated pathway in the disease. Methods: The expression of MUT gene in neuronal SH-SY5Y cell line was reduced using siRNAs against MUT mRNA. The control SH-SY5Y cell line was transfected by using a random sequence. Cellular lysates were separated on a 10 % SDS gel. Each lane was cut in 3 mm slices and each slice processed by in situ hydrolysis protocol. Tryptic mixtures of peptides were analysed by tandem mass spectrometry. The relative abundance of each protein was determined by the spectral counting approach [1]. Results: The reduction of 70 % of mutase expression was observed after 48 h. The protein expression profile was analysed by quantitative proteomics. Dysregulated proteins were grouped according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Differentially expressed proteins were involved in metabolic processes, expecially Krebs cycle, apoptosis processe, cell cycle regulation and cellular response to stress. Discussion: The MUT silencing and the proteomic quantitative approach allow us to define the pathways directly perturbed by the metabolic disease. They represent targets to understand its molecular basis and for the design of therapies to alleviate clinical manifestations of MMA. [1] Caterino M. et al. Proteomics (2014) 14, 2286.

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Further evidence that D-glycerate kinase (GK) deficiency may be a nondisease Kalim A 1, Fitzsimons P E 2, Till C 3, Fernando M 4, Mayne P D 2, Sass J O 3, Crushell E 1

1

Met Temple Street, Child Univ Hosp, Dublin, Ireland, 2Bioch,Temple Street Child Univ Hosp, Dublin, Ireland, 3Bonn-Rhein-Sieg-Univ Appl Sci, Rheinbach, Germany, 4Univ Child Hosp, Freiburg, Germany Background: D-Glyceric aciduria is caused by deficiency in GK due to recessive mutations in the GLYCTK gene. GK catalyzes the conversion of Dglycerate to 2-phosphoglycerate which is an intermediary reaction in the catabolism of serine and fructose. Deficiency of GK leads to accumulation of Dglycerate which can be detected in urine organic acids. Debate exists as to whether this is a benign or disease-causing condition as the reported phenotypes vary significantly. Case report: We report two siblings from a consanguineous Pakistani family. The index case is a 5 year old boy with autism and global developmental delay. His urine organic acid analysis showed markedly incre ased excre tion of glycera te , dete rmined a s D-form by enantioselective gas chromatography. There was no oxalic aciduria. His younger sister (3 years old) is asymptomatic and developmentally normal (bilingual). Her urine showed similar amounts of D-glycerate. Results: Both children are homozygous for the novel mutation c.767C > G in exon 5 of the GLYCTK gene, predicted to affect the enzyme by replacing the evolutionary conserved proline with arginine (P256R). Both parents are heterozygous carriers. Discussion: Just 11 patients with D-glyceric aciduria have been described to date. Our cases are the 4th and 5th with genetic confirmation (cf. Hum Mutat 31:1280–1285, 2010). While GK is integral to distinct metabolic pathways, the disorder is reported to have a highly variable phenotype (ranging from asymptomatic cases to early death) and the question has already arisen as to whether GK deficiency is a benign disorder. The differing phenotype seen in these siblings suggests that this condition is likely to be an incidental finding and not the cause of the boy’s developmental delay and autism. Our two cases support the view that D-GK deficiency is a benign disorder. Long term follow-up studies with a greater number of individuals may be required for further confirmation.

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Two novel cases of Chiari malformation associated with glutaric aciduria type 1 Enright N 1, Glackin S 1, Caird J 2, Murphy N 3, King M D 4, Mayne P D 5, Monavari A A 1 1 Div Metab Dis, Univ Child Hosp, Temp St, Dublin, Ireland, 2Div Neuro Surg, Univ Child Hosp, Temp St, Dublin, Ireland, 3Div Endo Dis, Univ Child Hosp, Temp St, Dublin, Ireland, 4Div Neur Dis, Univ Child Hosp, Temp St, Dublin, Ireland, 5Div Clin Bioch, Univ Child Hosp, Temp St, Dublin, Ireland

Background: We report 2 unrelated cases of glutaric aciduria type 1 (GA1), who developed a Chiari malformation. We are not aware of any other reports of similar cases in the literature. Case report: Case 1 is a 23 year old man who was diagnosed on neonatal high risk screening. He was commenced on a lysine free and low tryptophan diet, with carnitine, from birth. He was subsequently diagnosed with growth hormone deficiency and hypothyroidism. During routine surveillance MRIs he was noted to have a Chiari 1 malformation with a syrinx. He has remained asymptomatic. Case 2 is a 14 year old girl also diagnosed on neonatal high risk screening. She was commenced on a lysine free and low tryptophan diet, with carnitine, from birth. She developed epilepsy at 11 years of age. At 14 she developed headaches, with deterioration in balance, and an MRI showed hydrocephalous, a Chiari 1 malformation and a syrinx. She had an endoscopic third ventriculostomy and her symptoms have resolved. Results: A Chiari 1 malformation and syrinx were discovered in 2 patients with GA1, one was symptomatic and required treatment, one was asymptomatic. One of these patients was also diagnosed with hypothyroidism and growth hormone deficiency. Discussion: The characteristic MRI findings in GA1 are widening of the Sylvian fissure, abnormalities of the basal ganglia, leukoencephalopathy and

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 brain atrophy. From our review of the literature we can find no other reports of Chiari malformation associated with GA1. Our patients began with the classical findings but then developed a Chiari malformation. These cases highlight the importance of MRI brain surveillance in these patients whether or not they are symptomatic. Acknowledgements: The authors would like to thank Dr E. Naughten for her great contribution to the care of the patients, and our colleagues Dr E. Crushell, Dr J. Hughes, Dr I. Knerr, Dr E.T. Treacy and all other members of the metabolic team who assisted in the care of these patients.

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3-Methylcrotonyl Co-A carboxylase deficiency detected by newborn screening as a cause of cardiomyopathy—case report Taybert J 4, Jablonska E 1, Polawski T 1, Kusmierska K 1, Kowalik A 1, Brzezinska M 2, Werner B 2, Oltarzewski M 1, Ploski R 3, Rydzanicz M 3, Sykut-Cegielska J 1 1 Screen Dept, Inst Mother and Child, Warsaw, Poland, 2Dept Ped Card General Ped, Warsaw Med Univ, Warsaw, Poland, 3Dept Med Genet, Warsaw Med Univ, Warsaw, Poland, 4Met Outpat Clin, Inst Mother and Child, Warsaw, Poland

Background: Since the newborn screening era 3-methylcrotonyl Co-A carboxylase (MCC) deficiency is regarded as a very mild disease with mostly asymptomatic cases. Therefore it is often not recommended to be included in the panel of diseases detected by MS/MS newborn screening (NBS). Case report: In the patient of Arabic origin of related young parents and normal pre- and perinatal period, abnormal acylcarnitine profile in NBS was identified; C5-OH-carnitine-6.24umol/l (ref.v. < 0.86), free carnitine–12.8umol/l. At age of 3.5 months C5-OH-carnitine was 65.80umol/l (ref.v. < 1.33), free carnitine–11.6umol/l. Organic acid profile confirmed 3-methylcrotonylglycinuria. Biotinidase deficiency was excluded. The child remained with no clinical signs until the age of 8 months when decreased appetite, small weight gain and dyspnea occurred; dilated cardiomyopathy was diagnosed which progressed rapidly to cardiac failure. Low-leucine diet and L-carnitine supplementation together with intensive pharmacological therapy were introduced with clinical improvement, but persistent echocardiographic picture. Further investigation relied on DNA diagnostics by whole exome sequencing aimed at finding genetic base for cardiomyopathy. Results: Analysis was performed with SureSelect enrichment kit and pairedend 2x100bp sequencing on IlluminaHiSeq1500. 88,998,954 reads were generated resulting in the 10x coverage of 95.2 % and 20x coverage of 89.9 % of the target. A homozygous mutation p.Ala524Thr/ c.1570G > A in the MCCC2 gene was found. The mutation (rs774241918) was previously associated with MCC deficiency. No other pathogenic variants were found including variants in cardiomyopathy genes. Discussion: To our knowledge this is the second case of MCC deficiency (previous one reported by Visser G et al. (Eur J Pediatr 2000) presenting as dilated cardiomyopathy. Our result suggests that such clinical manifestation may be added to the clinical picture of MCC deficiency, even in cases detected by NBS.

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15 years’ experience: diagnosis of organic acidemias at Quest Diagnostics Biochemical Genetics Laboratory Sharma R 1, Salazar D Z 1, Bonilla-Guerrero R 1, Davoodi-Semiromi A 1, Lobo R M 1, Lee J E 1, Neidich J A 1, Zhang K 1, Strom C M 1 1

Quest Diagnostics Nichols Institute, San Juan Capistrano, United States

Background: Organic acidemias are caused by defects in intermediate pathways of carbohydrate, amino acid, and fatty acid oxidation.

Methods: Organic acid analyis by GC/MS provides powerful tool for the diagnosis of many different disorders. Results: The result of 15 years experience in diagnosis of organic acidemias at the Biochemical Genetics laboratory at Quest Diagnostics is described. Of 2232 patients that were diagnosed primarily by organic acid testing in our laboratory, MCAD was the most common diagnosis (n = 543), followed by 3-methylcrotonylCoA-carboxylase deficiency (n = 273), methylmalonic acidemia (n = 233), glutaric aciduria type-1(n = 229), isovaleric acidemia (n = 208), propionic acidemia (n = 172), short-chain acyl-CoAdehydrogenase deficiency (n = 165), Ornithine Transcarbamaylase deficieny (n = 123), Tryosinemia type-1 (n = 61), 3-methyl glutaconic aciduria (n = 26), malonic acidura (n = 24), alkaptonuria (n = 23), betaketothiolase deficiency (n = 23), HMG-CoA-lyase deficiency (n = 23), 4hydroxybutyric aciduria (n = 16), Canavan disease (n = 16), 2-oxoadipic acidura (n = 14), ethylmalonic encephalopathy (n = 12), tyrosinemia type-2 (n = 12), multilple carboxylase deficiency (n = 11), 2-methylbutyryl-CoAdehydrogenase deficiency (n = 10), fumarase (n = 7), mevalonic aciduria (n = 7), dihydropyrimidine-dehydrogenase deficiency (n = 4), IsobutyrylCoA-dehydrogenase deficiency (n = 3). Discussion: Our results highlight the importance of biochemical genetic testing in a clinical setting for the diagnosis of inborn errors of metabolism such as urine organic acidemias and/or other inherited genetic diseases. Although individually rare, our experience suggests that the prevalence of these disorders may be much higher than historical data have shown. Early recognition of these disorders enables diagnosis and treatment before the onset of symptoms or before irreversible damage is sustained. It is important for clinicians to recognize that, as a group, these types of disorders are not uncommon Conflict of Interest declared.

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Ethylmalonic encephalopathy without ethylmalonic aciduria Yucel-Yilmaz D 1 2, Ozgul R K 1 2, Pektas E 1, Serdaroglu E 3, Yalnizoglu D 3, Dursun A 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 2Inst of Child Health, Hacettepe Univ, Ankara, Turkey, 3Div Pediatric Neurol, Hacettepe Univ, Ankara, Turkey 1

Background: Ethylmalonic encephalopathy (EE) is an infantile autosomal recessive metabolic disorder affecting the brain, gastrointestinal tract, and peripheral vessels. It is characterised by psychomotor regression and generalised hypotonia, later evolving into spastic tetraparesis, dystonia. The biochemical marker of the disease is markedly increased urinary ethylmalonic acid. Case Report: A 14-year-old female patient presented with ataxic, dysartric and dystonic symptoms. She was born from nonconsanguineous parents via C/S 3 weeks before term. Early developmental milestones were normal. Her gait and speech deteriorated after 6 and 10 years, respectively. Cranial neuroimaging showed bilateral minor hippocampal anomaly. Metabolic work-up including lactate, pyruvate, transferrin isoforms, blood and urine aminoacids, acylcarnitine profiles were normal. Urine organic acid analsyis performed two times at 8 month intervals were normal. Results: Whole exome sequencing performed due to undiagnosed genetic disease criteria revealed a known homozygous mutation (c. > T; p.Met1Ile) in the ETHE1 (ethylmalonic encephalopathy 1) gene. Discussion: The biochemical marker of the disease is markedly increased urinary ethylmalonic acid. The patient presented here has only dystonic symptoms without ethylmalonic aciduria. Whole exome sequencing is an important method not only for diagnosis of rare unknown diseases, but also for diseases showing an unexpected clinical and biochemical phenotype. This study was supported by TUBITAK (Project No:111S217)

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Clinical and molecular features of patients with glutaric aciduria type 1 in Malaysia

New symptomatic patients with glutaric aciduria type 3: further evidence of high prevalence of the c.1006C > T (p.Arg336Trp) mutation

Leong H Y 1, Abd Wahab S A 2, Yakob Y 2, Abd Azize N A 2, Mohd Khalid M K N 2, Ngu L H 1

Skaricic A 1, Zekusic M 1, Fumic K 1, Bilic K 1, Petkovic Ramadza D 2, Sarnavka V 2, Suman Simic A 2, Zschocke J 3, Baric I 2 4

1

Genetic Dept, Kuala Lumpur Hosp, Kuala Lumpur, Malaysia, 2Mol Diag Prot Unit, Inst Med Res, Kuala Lumpur, Malaysia Background: Glutaric aciduria type 1 (GA1) is an autosomal recessive disorder caused by deficiency of glutaryl-CoA dehydrogenase (GCDH) encoded by the GCDH gene. Methods: Clinical findings of seven GA1 patients in Malaysia were reviewed and bidirectional sequencing of the GCDH gene performed. Results: All the patients presented in infancy with macrocephaly (7/7), subdural haemorrhage/effusion (7/7), dystonia (2/7), seizure (3/7) and neuroregression (2/7). All had large urinary excretion of glutaric and 3-hydroxyglutaric acids. The patients on follow up are between 2 and 23 years old. 5 patients have severe motor disability, 1 has moderate disability and another mild motor disability. 1 patient passed away at 11 years due to pneumonia. Molecular analysis of the GCDH gene revealed 8 heterozygous mutations in 5 patients (c.382C > T, c.392A > T, c.892G > A, c.1060G > A, c.1063C > T, c.1168G > T, c.12442A > C, c.1261G > A). 2 patients exhibited homozygous mutations (c.227A > C, c.1157G > A). 3 of the missense mutations (c.1168G > T, c.227A > C, c.392A > T) were novel mutations located in highly conserved regions. Despite the small number of samples, c.1063C > T (R355H) and c.1244-2A > C were recurrent mutations, each detected heterozygous in two unrelated patients. Discussion: This study of Malaysian GA1 patients describes clinical features, novel and common mutations.

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1 Dept Lab Diagn, Univ Hosp Cen, Zagreb, Croatia, 2Dept Pediatr, Univ Hosp Cen, Zagreb, Croatia, 3Zen Med Gen, Med Univ Innsbruck, Innsbruck, Austria, 4School Med, Univ Zagreb, Zagreb, Croatia

Background: Glutaric aciduria type 3 (GA3) is an autosomal recessive disorder caused by mitochondrial succinate-hydroxymethylglutarate CoA-transferase deficiency. It is characterised by increased persistent, isolated excretion of glutaric acid. There is no distinctive phenotype associated with this disorder. The diagnosis is established by excluding other causes of glutaric aciduria and by gene analysis. Case Report: Patient 1 presented at the age of 15 months with recurrent vomiting, hypotonia, ataxia, left abducens paresis and symmetrical patchy white matter changes on brain MRI mimicking postvaccinal encephalopathy. She recovered completely. Patient 2 experienced episodes of agitation and unresponsiveness lasting 2 h during 3 consecutive nights at the age of 12 months. At the age of 3 years he had signs of attention deficit hyperactivity disorder. In both families parents were unrelated. Results: Organic acid analysis showed elevated urinary excretion of glutaric acid in both patients ranging from 39 to 98 mmol/mol of creatinine in patient 1 and from 90 to 133 mmol/mol of creatinine in patient 2 (N < 10). Glutaconic and 3-OH glutaric acid were found in traces. Acylcarnitine profiles were normal. Genetic analysis of the coding exons and adjacent intron regions of the succinyl-CoA:glutarate-CoA transferase (SUGCT) gene identified c.1006C > T (p.Arg336Trp) mutation in a homozygous state in both patients. Discussion: Our cases suggest that under certain circumstances GA3 may not be a benign condition, as indicated by encephalopathic crises experienced by the first reported patient more than 20 years ago after the first description. The c.1006C > T mutation is known to be causing GA3. Its appearance on four unrelated alleles in our patients points to its high frequency in different populations and possible hot spot of the SUGCT gene. More information and additional haplotype analysis are needed for a more accurate conclusion.

Clinical, neuroimaging, and genetic features of L-2-hydroxyglutaric aciduria: case series Canda E 1, Kose M 1, Eraslan C 2, Ucar S K 1, Habif S 3, Karaca E 4, Onay H 4, Ozkinay F 4, Coker M 1 Div Ped Metab Dis, Ege Univ Med Faculty, Izmir, Turkey, 2Div Radiology, Ege Univ Med Faculty, Izmir, Turkey, 3Div Biochemistry, Ege Univ Med Faculty, Izmir, Turkey, 4Div Genetics, Ege Univ Med Faculty, Izmir, Turkey 1

Background: L-2-hydroxyglutaric aciduria is a rare autosomal recessive encephalopathy caused by mutations in the L-2-hydroxyglutarate dehydrogenase (L2HGDH) gene. Methods: Here we discuss the clinical and molecular characteristics in patients with L-2-hydroxyglutaric aciduria. Results: There are seven patients with L-2-hydroxyglutaric aciduria. Median age is 18 (9.5–36) years. Six of them are female and one of them is male. The main symptoms of the patients were psychomotor retardation (5/7), cerebellar ataxia (5/7), extrapyramidal symptoms (6/7), and seizures (2/7). Characteristic magnetic resonance imaging findings include subcortical cerebral white matter abnormalities with T2 hyperintensities of the dentate nucleus, globus pallidus, putamen. Elevated urinary L-2 hydroxyglutaric acid was detected at the diagnosis median 150 (59–1460 nmol/mol creat). Two patients have homozygous R335X, two homozygous R282Q, one homozygous R302L, and one compound heterozygous P302L/A64T mutations in the L2HGDH gene. Discussion: Two of our patients have novel mutations. We want to mention that L-2-hydroxyglutaric must be considered in the differential diagnosis based on specific findings in cerebral MRI.

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N-acetylcysteine (NAC) therapy in ethylmalonic encephalopathy: the importance of changing the route of adminstration Burlina A B 1, Polo G 1, Cazzorla C 1, Giordano G 1, Zeviani M 2 1 Div Inherited Metab Dis, Univ Hosp, Padova, Italy, 2MRC-Mitochondrial Biology Unit, Cambridge, Italy

Background: Ethylmalonic encephalopathy (EE) is a rare autosomal recessive disorder due to loss of function mutations in ETHE1 gene characterized by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhea (Burlina et al., 1991). Despite dietary manipulations, oral supplementations with coenzyme Q10, metronidazole and NAC, progression of the clinical features occurred. Recently, liver transplant has been proposed as a new therapeutic option. Case report: A 8-year-old boy carrying a homozygous mutation in ETHE1 encoding an l55p substitution, was diagnosed at 6 months of age. At 29 months of age, we added NAC (100 mg/kg/bw) to metronidazole orally and an improvement was observed. Last year, he had a sudden episode of metabolic lactic acidosis requiring ventilation treated only with NAC ev and in few hours he showed a normalization of lactic acidosis. The patient was put only on NAC ev daily and normal diet. Results: After 9 months, the weight is at 50th percentile, no recurrent petechiae, acrocyanosis and chronic diarrhea are present. Serial evaluation of metabolites concentrations (plasma lactate, ethylmalonate, thiosulfate and C4 acylcarnitine) before and after changing route of administration showed

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 reduced concentrations, but still above the reference limit, indicating a specific and considerable effect of the ev therapy. We measured NAC (HPLC fluorimetric assay) in the same plasma samples: the concentration were 10 and 56 umol/L respectively (nv 0,23-1,12 umol/l). The levels remained stable for 48 hrs after infusion. No side effects were observed during the change of route of administration. Discussion: The results presented here warrant NAC ev treatment alone normalizes the main symptoms of the disease. Early diagnosed patients by neonatal screening could prompt to presymptomatic treatment with the aim of preventing irreversible brain damage and allows alternative experimental therapies such as liver transplantation or gene cell therapy.

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Some cases of elevation of 3-hydroxy-isovaleryl carnitine are caused by a defect in biotin transport

presented with lactic acidosis, hypoglycaemia, liver dysfunction or seizures and most died without other investigations. Targeted DNA sequencing was applied for variants in TAZ, TMEM70 and SERAC1. The whole exome sequencing was performed in six neonates. Results: Up to now, the molecular testing was completed in 14 neonates from 11 families. Surprisingly, the cause of the disease was established in all studied probands, including: SERAC1 in five, CLPB in four and TMEM70 in one. “NOS 3-MGA-uria” was recognized in four neonates bearing mutations of CPS1. Recurrent variants were identified in SERAC1 (p.Ser608Thrfs*5) and CLPB (p.Arg417*) (Wortmann et al. 2012, 2015). It should be mentioned that out of the project, we identified the TAZ mutations in two neonates with increased lactate excretion but not 3-MGA which appeared only after 3 months. Discussion: 3-MGA-uria in a neonate provides strong indication for DNA analysis. The whole exome sequencing is preferable due to unspecific symptomatology. Prior screening for recurrent SERAC1 and CLPB variants may be justified in Polish population. The authors thank Doctor Saskia Wortmann for molecular study of CLPB and SERAC1. The study was partly supported by CMHI S136/13 project.

Bobrinina V 2, Vitsyna O 1, Baydakova G 1, Zakharova E 1 1 FSBI Res Cent for Med Genet, Moscow, Russian Federation, 2Clos JointStock Comp Genoanalytica, Moscow, Russian Federation

Background: Biotin, water-soluble vitamin, is coenzyme of five mammalian carboxylases. Two diseases with biotin deficiency are known: biotinidase and holocarboxylase synthetase deficiencies. Both of them are characterized by the elevation of 3-hydroxy-isovaleryl carnitine (C5OH) in biological fluids. In some cases the increase of C5OH level is unexplainable and can be the result of a defect in others metabolic pathways Materials and methods: Blood spots from 6 patients with isolated increased level of C5OH were analyzed (range from 1,159 to 15,371; cut off >0,82 mcmol/l). The normal activity of biotinidase in plasma and no mutations in holocarboxylase synthetase gene excluded the biotinidase and holocarboxylase synthetase deficiencies. >Results: We observed one family with two affected children with a similar clinical picture. After the biotinidase and holocarboxylase synthetase deficiencies were excluded, whole exome sequencing (WES) was performed. Two sequences variants in the sodium dependent multivitamin transporter gene (SMVT) were found: c.838C > T (p.Arg280Trp) and c.1310C > A (p.Pro437Gln). Both have high predicted scores of pathogenicity and low (less than 0,5 %) frequency of occurrence in populations. Another 5 patients with unexplainable C5OH elevation in blood were screened for mutations in SMVT gene by direct DNA sequencing. No mutations were found. Discussion: In some cases the elevation of C5OH level may be explained by mutations in SMVT gene, but additional functional studies are necessary for understanding the role of mutations in SMVT gene.

12. Carbohydrate disorders

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Successful treatment of an adolescent with glycogen storage disease type Ib and severe Crohn-like colitis with elemental nutrition and an antiTNFα-agent Spenger J 1, Sperl W 1, Mayr J A 1, Wortmann S B 1 1

Div Metab Dis, Univ Child Clinic, Salzburg, Austria

3-Methylglutaconic aciduria in neonates—molecular study

Background: Glycogen storage disease type (GSD) Ib is a congenital disorder of glycogen metabolism that is associated with neutropenia and neutrophil dysfunction. An inflammatory bowel disease (Crohn like colitis) occurs in a subset of patients. Treatment options are limited and include granulocyte colony-stimulating factor, antibiotics and local aminosalicylates. In classic Crohn’s disease elemental nutrition and anti-TNFα-agents have been proven successful. Until now in the literature only one GSD Ib patient has been reported who received (and responded to) an anti-TNFα-agent. No reports on elemental nutrition are available. Case report: Here, we present the successful treatment of an adolescent with GSD Ib and severe Crohn-like colitis with elemental nutrition and an antiTNFα-agent. Results: Under elemental nutrition, anti-TNFα-treatment and stable metabolic control gastrointestinal symptoms ameliorated, calprotectin in stool nearly normalised and the patient showed impressive weight gain and growth spurt. Discussion: Elemental nutrition and anti-TNFα-agents are successful and well tolerated in the treatment of Crohn-like colitis in GSD Ib.

Wojtylo M 1, Trubicka J 2, Pajdowska M 3, Rokicki D 1, PiekutowskaAbramczuk D 2, Oltarzewski M 4, Jablonska E 4, Pronicka E 1 2

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1 Dept Ped Nutr Metab Dis, Ch Mem Health Ins, Warsaw, Poland, 2Dept Genet, Ch Mem Health Ins, Warsaw, Poland, 3Dept Bioch and Exp Med, Ch Mem Health Ins, Warsaw, Poland, 4Screening Dept, Ins Mother and Child, Warsaw, Poland

Background: 3-Methylglutaconic aciduria (3-MGA) is a marker for a heterogeneous group of metabolic disorders, including primary, secondary and “not otherwise specified (NOS)” 3-MGA. The secondary 3-MGA is related to the genes responsible for phospholipid remodelling (TAZ, SERAC1), mitochondrial membrane (OPA3, DNAJC19, TMEM70) and a newly discovered CLPB. The aim of the study was to assess a molecular basis of 3-MGA in Polish neonates who had undergone selective GC-MS screening during the period of 1995– 2015. Patients and methods: Increased 3-MGA excretion was found in 37 neonates with a disorder of unknown cause out of 356 positive tests (10.4 %). They

Glycogen storage disease type IX in a boy with 3-methylglutaconic aciduria previously suspected of Barth syndrome Szymanska E 1, Rokicki D 1, Ciara E 4, Pronicki M 3, Pajdowska M 2, Trubicka J 4, Pronicka-Iwanicka K 5, Szymanska S 3, Pasnicka M 1, Tylki-Szymanska A 1 , Pronicka E 1 4 1 Dept Ped Nutr Met Dis, Ch Mem Health Ins, Warsaw, Poland, 2Ch Mem Health Ins, Warsaw, Poland, 3Dept Pathol, Ch Mem Health Inst, Warsaw, Poland, 4Dept Genet, Ch Mem Health Ins, Warsaw, Poland, 5Dept Audiol, Ch Healt Mem Ins, Warsaw, Poland

Background: 3-Methylglutaconic aciduria (3-MGA) is a nonspecific metabolic marker associated with mitochondrial dysfunction, including Barth

S140 syndrome. However, 3-MGA may also be present in the urine of patients with other metabolic disorders such as glycogen storage diseases. Case report: A boy born in 2012 as a former 2.6 kg preterm infant at 35th week gestation completed through caesarian section due to mother’s diabetes and oligohydramnios, Apgar score of 8 points. Perinatal period was complicated with severe, transient hypoglycemia of 2 mg/dl and there was cardiomegaly found in the ultrasound examination. At 6 months of age hypertransaminasemia of 180–200 IU/l without cholestasis was observed. The boy presented with mildly delayed psychomotor development, hypotonia, enlarged liver, and characteristic facial features with chubby cheeks. Metabolic screening revealed lactic acidosis of 33.6 mg/dl, elevated triglycerides (200 U/l) with decreased HDL level of 31 mg/dl, mild transient neutropenia, hypoglycemia of 56 mg/dl, and 3-MGA-uria. VGT test showed lactate increase following glucose load. BTHS was taken under consideration but TAZ gene sequencing did not identify any pathogenic mutation. Muscle and liver biopsies were performed. Histopathologic examination of the muscle was normal, while there was glycogen storage found in hepatocytes on electron microscope scanning. Results: Massive parallel sequencing technology including clinical exome was performed which has revealed a novel pathogenic variant c.749C > T p.(Ser250Leu) in PHKA2 gene associated with GSD type IX-alpha. Nutritional management with high-protein diet was commenced. Discussion: 3-MGA-uria as a biochemical finding is not specific enough to establish a diagnosis, and it may be associated with more inborn errors of metabolism than it used to be considered. The reported case demonstrates that unspecific excretion of 3-MGA in the urine can be a cause of misdiagnosis.

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Novel SLC37A4 mutations and molecular characterization in Korean patients with glycogen storage disease Ib Park H D 1, Choi R 1, Ko J M 2, Lee J 3, Lee D H 3, Hong S J 4, Choe Y H 5 1 Dept Lab Med, Samsung Med Cent, Seoul, Republic of Korea, 2Dept Pediatr, Seoul Nat Univ Child Hosp, Seoul, Republic of Korea, 3Dept Pediatr, Soonchunhyang Univ Hosp, Seoul, Republic of Korea, 4Dept Pediatr, Catholic Univ Daegu, Daegu, Republic of Korea, 5Dept Pediatr, Samsung Med Cent, Seoul, Republic of Korea

Background: Molecular techniques are fundamental in the accurate diagnosis and therapeutic approach to glycogen storage diseases (GSDs). The aim of this study was to evaluate the SLC37A4 mutation spectrum in Korean GSD Ib patients and to report novel mutations. Methods: Nine Korean patients with GSD Ib were included in this study. The median age of onset was 14.5 months (range 3 months to 10.9 years). All patients have SLC37A4 mutations that were detected with PCR-direct sequencing and/or whole exome sequencing (WES). A comprehensive review of the literature on previously reported SLC37A4 mutations was also conducted. The Human Gene Mutation Database (HGMD, http://www.hgmd.org/) was checked for previously reported sequence variants. Results: Nine different pathogenic mutations were identified in the nine patients with GSD Ib. Among them, four novel mutations were identified: c.148G > A (pGly50Arg), c.320G > A (p.Trp107*), c.412 T > C (p.Trp138Arg), and c.818G > A (p.Gly273Asp). The most common mutation type was missense mutations (66.7 %, 6/9), followed by nonsense mutations (22.2 %, 2/9). Notably, the most common mutation identified in the Korean population was c.443C > T (p.Ala148Val), which comprises 39.9 % (7/18 tested alleles). This mutation has not been reported in GSD Ib patients among other ethnic populations. The c.1042_1043del (p.Leu348Valfs*53) was the second most frequent mutation (16.7 %) and this has also been frequently reported in mixed Caucasian (27–31 %) and German (32 %) populations. Other mutations such as c.352 T > C (p.Trp118Arg) and c.1015G > T (p.Gly339Cys) that have been reported frequently in other ethnic populations have not been reported in Korean population. Discussion: Considering the SLC37A4 mutation spectrum in Korean patients with GSD Ib, molecular diagnostic methods with direct sequencing and/or WES are needed to provide accurate diagnoses. This study expands the knowledge of the SLC37A4 mutation spectrum in Korean patients with GSD Ib.

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Intracerebroventricular D-galactose injection provokes motor coordination impairment and cerebellar damage in Wistar rats Rodrigues A F 1, Biasibetti H 1, Pierozan P 1, Schmitz F 1, Zanotto B S 1, Sanches E F 1, Dal Magro D D 2, Netto C A 1, Wyse A T S 1 1 UFRGS, Porto Alegre, Brazil, 2Universidade Regional de Blumenau, Blumenau, Brazil

Background: Classical galactosemia is characterised by tissue accumulation of galactose (GAL). However, GAL dietary restriction is not able to prevent patients from symptoms like impaired motor coordination, which starts to develop early in life. Furthermore, GAL is endogenously produced, leading to the believe that this metabolite participates in the toxicity of classical galactosemia. The cerebellum is well known to play a key role in movement control, and another common feature of classical galactosemia is cerebellar atrophy. Methods: GAL (4 mM) was injected intracerebroventricularly in 60-days-old Wistar rats, control animals received saline. The volume of the solution (saline or GAL) administered was 5 μl. Motor coordination was assessed 1 h and 24 h after injection by using the beam walking test. In the cerebellum, histopathological (hematoxylin/eosin and immunohistochemistry) and biochemical (active caspase-3 and BDNF levels) parameters were performed 24 h after injections. Results: Motor coordination impairment was found 24 h after GAL injection. GAL also decreased the number of cells in the molecular and granular layer of the spinocerebellum, while GAL reduced the number of cells in the molecular layer of the cerebrocerebellum. Immunohistochemistry analyses suggested that the cell types lost were neurons and astrocytes in the spinocerebellum and neurons in the cerebrocerebellum. Furthermore, the results showed that galactose increased the immunocontent of active caspase-3 and decreased total BDNF immunocontent. Discussion: The results support that intracerebroventricular GAL administration causes motor impairment due to cell loss in the cerebellum which is, at least in part, caused by apoptosis. The findings also contribute to understand better the neurotoxic mechanisms behind the neuropathology of classical galactosemia.

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International clinical guidelines for the management of classical galactosemia: diagnosis, treatment and follow-up Welling L 1, Bernstein L E 2, Berry G T 3, Burlina A B 4, Eyskens F 5, Gautschi M 6, Grunewald S 7, Gubbels C S 3, Knerr I 8, Labrune P 9, Van der Lee J H 11, MacDonald A 10, Murphy E 12, Portnoi P A 13, Ounap K 14, Potter N L 15, Rubio-Gozalbo M E 16, Spencer J B 17, Timmers I 18, Treacy E P 19, Van Calcar S C 20, Waisbren S E 21, Bosch A M 1 1

Dept of Pediatrics, Academic Med Center, Amsterdam, Netherlands, 2Nutr Dept, The Child Hosp Colorado, Aurora, United States, 3Boston Child Hosp, Harvard Med Broad Ins, Boston, United States, 4Dept of Pediatrics, Univ Hospital, Padova, Italy, 5Dept Metab Dis, Antwerp Univ Hosp UZA, Antwerp, Belgium, 6 Pediatric Endocrinol, Univ Child Hosp, Bern, Switzerland, 7Metab Unit, Great Ormond Street Hosp, London, United Kingdom, 8Inh Metab Dis, Temple St Child Univ Hosp, Dublin, Ireland, 9 Dept Pediatrics, Hopital Antoine Beclere, Clamart, France, 10Birmingham Child Hosp, Birmingham, United Kingdom, 11 Ped Clin Res Office, Academic Med Center, Amsterdam, Netherlands, 12Charles Dent Metabolic Unit, London, United Kingdom, 13Galactosemia Support Group, West Midlands, United Kingdom, 14Dept of Genetics, Tartu Univ Hosp, Tartu, Estonia, 15Washington State University, Spokane, United States, 16Dept of Ped, Maastricht Univ Med Centre, Maastricht, Netherlands, 17Emory Univ School of Medicine, Atlanta, United States, 18 Dept Cogn Neurosc, Maastrich Univ, Maastricht, Netherlands, 19NCIMD, TSCUH and MMUH Hospitals, Dublin, Ireland, 20 Oregon Health and Science University, Portland, United States, 21Boston Child Hosp, Harvard Med School, Boston, United States

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Classical galactosemia (CG) is an inborn error of galactose metabolism. Despite early dietary treatment (a galactose-restricted diet), many patients suffer from long-term complications. Treatment and follow-up of CG patients vary significantly worldwide and international guidelines based on systematic review of the literature are lacking. To provide patients around the world the same state-of-the-art care, members of The Galactosemia Network (GalNet) developed evidence-based and internationally applicable clinical guidelines. Methods: Twenty-one experts from eight European countries and the USA participated. We used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. Key questions were formulated by experts and patients during an initial GalNet meeting. A systematic review of the literature was performed and two independent investigators extracted evidence from included articles. Recommendations were formulated by experts, based on evidence (if available) or expert opinion. We evaluated quality of the body of evidence and assigned each recommendation a strength of recommendation. Consensus for each statement was reached by conference calls, e-mail rounds and a final consensus meeting. Results: A total of 41 recommendations were formulated, addressing diagnostic methods, duration and composition of dietary treatment, biochemical monitoring and clinical follow-up of 1) cognitive and speech and language development, 2) neurological complications, 3) psychosocial development, 4) endocrine complications and fertility outcome, 5) bone health and 6) cataracts. For all recommendations over 90 % consensus was reached. Discussion: These are international, evidence-based practice guidelines for the diagnosis, treatment and follow-up of CG.

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Systematic review and meta-analysis of intelligence quotient in earlytreated individuals with classical galactosemia Welling L 1, Antshel K M 9, Colhoun H 7, Gautschi M 2, Grunewald S 3, Holman R 8, Van der Lee J H 4, Treacy E P 5, Waisbren S E 6, Bosch A M 1 1

Dept of Pediatrics, Academic Med Center, Amsterdam, Netherlands, Pediatric Endocrinol, Univ Child Hosp, Bern, Switzerland, 3Metab Unit, Great Ormond Street Hosp, London, United Kingdom, 4Ped Clin Res Office, Academic Med Center, Amsterdam, Netherlands, 5NCIMD, TSCUH and MMUH Hospitals, Dublin, Ireland, 6Boston Child Hosp, Harvard Med School, Boston, United States, 7Dept of Pediatrics, Trinity College, Dublin, Ireland, 8 Clin Research Unit, Academic Med Center, Amsterdam, Netherlands, 9Dept of Psychology, Syracuse University, Syracuse, United States 2

Background: Despite adherence to a galactose-restricted diet, individuals with classical galactosemia (CG) are at risk for developing long-term complications such as cognitive impairment. Different patient populations and test methods have been used in published studies assessing cognitive outcome, which hampers conclusions regarding the prevalence of intellectual disabilities in this disorder. The primary aim of this systematic review was to review the literature and conduct a meta-analysis on intellectual performance in early-treated (≤4 weeks of life) individuals with CG, assessed with comparable test instruments. The Full Scale Intelligence Quotient (FSIQ) was the primary variable of interest. Verbal IQ, Performance IQ and correlation between age and IQ were also examined. Methods: We developed computerized search strategies for MEDLINE, EMBASE and PsychInfo. Two independent investigators performed the article selection process, risk of bias assessment and data-extraction. Individual patient case data were pooled for analysis using linear mixed model analysis. Results: Five articles were included in this review, of which four provided data for the meta-analysis. Data on 87 individuals (median age 13 years, range 3 to 38 years) were used to assess mean FSIQ in all CG individuals. The FSIQ ranged from 47 to 122, with a mean score of 87 (95 % CI; 81 to 94). 40% of all individuals performed more than a standard deviation below the general population mean (FSIQ < 85), almost half attained scores within the low-average range (FSIQ 85–100), and a minority (15 %) attained scores above 100. There was no significant correlation between FSIQ and age. Data on verbal and performance IQ were rarely reported.

Discussion: Early-treated CG patients are at risk for having impaired cognitive abilities, with a mean FSIQ of 87 in this study. However, IQ varies significantly between individuals with CG. There is no indication that older patients have lower FSIQ.

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Bone health in classic galactosemia: systematic review and meta-analysis Welling L 1, Van Erven B 3, Van Calcar S C 5, Doulgeraki A 7, Eyskens F 2, Gribben J 8, Treacy E P 4, Vos R 9, Waisbren S E 6, Rubio-Gozalbo M E 3, Bosch A M 1 1 Dept of Pediatrics, Academic Med Center, Amsterdam, Netherlands, 2Dept Metab Dis, Antwerp Univ Hosp UZA, Antwerp, Belgium, 3Dept of Ped, Maastricht Univ Med Centre, Maastricht, Netherlands, 4NCIMD, TSCUH and MMUH Hospitals, Dublin, Ireland, 5 Oregon Health and Science University, Portland, United States, 6Boston Child Hosp, Harvard Med School, Boston, United States, 7Institute of Child Health, Athens, Greece, 8 Guy’s and St Thomas’ NHS Found Trust, London, United Kingdom, 9Dept Methodol and Stat, Maastricht Univ, Maastricht, Netherlands

Background: Individuals with classic galactosemia (CG) are at risk for longterm complications, despite adherence to a galactose-restricted diet. Previous studies have reported an association between CG and decreased bone mass. The primary objective of this systematic review with meta-analysis was to determine the extent of bone mineral density (BMD) reduction in CG patients of all ages. The secondary objective was to assess other indicators of bone status. Methods: Systematic search strategies were developed by a trained clinical librarian. Selection of relevant manuscripts, risk of bias assessment and dataextraction were performed independently by two investigators. Aggregate data (mean BMD Z-scores per study) were pooled for analysis. Results: A total of ten studies were included in the review, four of these in the meta-analysis. Meta-analysis of BMD Z-scores in children and adults measured at the lumbar spine (LBMD; 4 studies; n = 112), total hip (HBMD; 2 studies; n = 58), and femoral neck (FBMD; 2 studies; n = 73) were performed. Overall effect sizes were: LBMD −0.70 (95 % CI: −0.88, −0.52); HBMD −0.89 (95 % CI: −1.14, −0.64); FBMD −0.63 (95 % CI −1.29, 0.02). The mean BMD Z-score was lower in adults than in children. Vitamin D levels are frequently in the low reference range in adult individuals with CG. Few studies assessed minerals, other hormones and bone turnover markers (BTM). Discussion: The mean BMD Z-score in individuals with CG is −0.7 compared to 0 in the normal population, but still is in the normal range. This indicates that bone health in general is affected in CG and that more patients, compared to the general population, will have a BMD Z-score ≤ −2 SD. Therefore, regular evaluation of bone health seems warranted.

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Infertility and low natural pregnancy rates in female patients with classical galactosaemia in the Republic of Ireland Losty E 1, Crushell E 1, Hughes J 1, Monavari A A 1, Treacy E P 1, Connolly G 1 , Knerr I 1 1

National Centre for IMD, Dublin, Ireland

Background: Classical galactosemia (Gal) is a metabolic disorder caused by mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. It has been part of the Irish Newborn Bloodspot Screening Program since 1972. Gal causes primary ovarian insufficiency (POI) in the majority of female patients, starting at an early age with subsequent infertility. POI is a diet-independent long-term complication with a variable course; only a low spontaneous conception rate has been described. Methods: Ethical approval was sought and obtained for an extensive chart review of female Gal patients at child bearing age. Patients were coded to

S142 allow the data to be anonymised. Clinical data obtained included menstrual/ pregnancy history, usage of hormonal replacement therapy (HRT), genotype and hormonal profiles. We examined the chances of spontaneous pregnancies in our female Gal patients, currently being treated at the National Centre for Inherited Metabolic Disorders TSCUH in Dublin. Results: The majority of female patients did require HRT commencing at pubertal age. Spontaneous pregnancies did occur in two different GALT mutations despite POI diagnosis. Discussion: The focus of this study was to display pregnancy chance despite POI diagnosis in the context of Gal in the Republic of Ireland. Counselling with information on inheritance, HRT and options for reproduction should be offered involving the expertise of a paediatric metabolic consultant and gynaecologist and other members of a multidisciplinary team.

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Atypical manifestation of classic galactosemia with succinylacetone excretion Shkurko T A 1, Doronina Y 1, Kormoz S 1, Trofimova N 1, Baydakova G 2, Pichkur N A 1, Olkhovych N V 1, Gorovenko N 1 1 Cen Orph Dis Nat Child Hosp Ohmatdyt, Kyiv, Ukraine, 2Lab Inh Met Dis, Res Cen Med Gen, Moskow, Russian Federation

Background: Galactosemia is a rare, hereditary disorder of carbohydrate metabolism that affects the body’s ability to convert galactose to glucose. The disorder is caused by a deficiency of an enzyme galactose-1-phosphate uridyl transferase (GALT) which is vital to this process. Early diagnosis and treatment with a lactose-free diet is absolutely essential to avoid profound intellectual disability, liver failure and death in the newborn period. Classic galactosemia and clinical variant galactosemia can both result in life-threatening health problems unless treatment is started shortly after birth. Case report: A 7-day old boy presented with feeding problems, vomiting, weight loss, muscle hypotonic,hematuria. Jaundice was not observed. He developed liver failure and coagulopathy, with the rapid deterioration of the condition. Investigations revealed increasing levels of tyrosine, succinylacetone and α-fetoprotein in blood. The treatment of tyrosinemia was started immediately, but it was not effective. Further investigation revealed he had GALT deficiency with homozygous GALT mutations being a heterozygous carrier of a FAH mutation. Results: Thus he was diagnosed with galactosemia and started a correct treatment with good responsiveness. Discussion: Over 95 % of patients with galactosemia have manifestation of jaundice and hyperbilirubinemia, which was not observed in this case. Raising of succinylacetone and α-fetoprotein may have been caused by heterozygous carrier of FAH gene and increased plasma tyrosine often indicates liver involvement in the pathological process. Metabolic diseases are characterized by multisymptomatic picture, so it is very important to start early treatment and use diagnostic protocols that allow to select right therapeutic measures.

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Hepcidin, interleukin-6 levels and iron metabolism parameters in patients with hepatic glycogen storage diseases Nalin T 1 2, Sperb-Ludwig F 1 2, Siebert M 2, Weinstein D A 3, Derks T G J 4, De Souza C F M 2, Schwartz I V D 1 2 1 Univ Federal do Rio Grande do Sul, Porto Alegre, Brazil, 2Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil, 3University of Florida, Gainesville, United States, 4University Medical Center of Groningen, Groningen, Netherlands

Background: Hepatic glycogen storage diseases (GSD) are genetic diseases characterized by recurrent episodes of hypoglycemia. Anemia has been recognized as a frequent manifestation of these disorders.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Objective: To evaluate hepcidin and IL-6 concentrations in patients with hepatic glycogen storage diseases (GSD) and their association with anemia and other parameters of iron metabolism. Methods This was a cross-sectional study with a convenient sampling strategy. Levels of hepcidin, IL-6, and markers of iron metabolism (hemoglobin, iron, ferritin, transferrin, and transferrin saturation) were measured in 32 patients receiving uncooked cornstarch therapy for GSD (GSDIa = 18; GSDIb = 7; GSDIII = 3; GSDIXa = 3; GSDIXb = 1; median age 9.5 years). Additional data were obtained by means of chart review. Nonparametric methods were used for data analysis. Results: Nine patients (GSDIa = 3/17; GSDIb = 6/7) were anemic (mild = 4; moderate = 5). Five patients had hepatic adenomas. Eight patients had hyperferritinemia, and one had elevated transferrin saturation as well. Hepcidin correlated positively with ferritin levels (r = 0.375; p = 0.034). IL-6 correlated with hemoglobin (r = −0.572; p = 0.001), iron (r = −0.538; p = 0.001), transferrin (r = −0.550; p = 0.001), and transferrin saturation (r = −0.425; p = 0.015). There was no correlation between hepcidin and IL-6 levels (p = 0.057). Patients with GSD Ib had the highest IL-6 levels. There was no difference between patients with and without anemia (median = 69.4 ng/ mL, IQR = 42.2–81.6 and median = 55.4 ng/mL, IQR = 42.1–67.7, respectively) (p = 0.182), and with and without liver adenoma (median = 67.1 ng/mL, IQR = 39.4–75.9 and median = 53.6 ng/mL, IQR = 37.4–69.5, respectively) (p = 0.453), regarding hepcidin levels. Discussion: Anemia is a common finding in hepatic GSD, especially in GSD Ib, the type of GSD associated with the highest IL-6 levels. These findings suggest that inflammation is strongly associated with development of anemia in hepatic GSD, particularly in GSD Ib.

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Complete reversal of glycogen storage disease type 1a complications after liver transplantation Gomes D C 1, Gaibino N 1, Guerra A 1, Oliveira A 1 1

North Lisbon Hosp - Metab Dis Ref Center, Lisbon, Portugal

Background: Glycogen storage disease type Ia (GSD1A) is a liver glycogenosis that typically manifests in the first months of life with hypoglycemia and lactic acidosis. Treated patients can progress into adulthood with a good quality of life but multiorgan complications may arise when metabolic control is suboptimal. Case report: 32 years old male patient diagnosed with GSD1A in the first months of life, treated with overnight feeding during infancy and uncooked cornstarch only thereafter. He progressed into adulthood without any admissions but without glycemic control. At the age of 28 years, when he was first evaluated at our adult clinic, he had an enlarged irregular liver. An abdominal MRI showed 16 adenomas, the largest measuring 5 cm. He also had microalbuminuria, severe hypertriglyceridemia, hyperuricemia and normochromic normocytic anaemia. He was lost to follow up until the age of 32 when he started having multiple admissions for acute metabolic decompensations, despite increasing dosage of uncooked cornstarch and progressively reduced fasting intervals. He refused continuous overnight feeding. Severe hypertriglyceridemia was unresponsive to fibrates or statins. Abdominal CT showed persistent multiple hepatic adenomas. A year later he was submitted to orthotopic liver transplantation (OLT) without any immediate complication. Results: His metabolic profile became normal just after surgery. On an 8 months follow-up visit his lipid profile, 24 h urine albumin, uric acid levels and hemoglobin were normal. His quality of life was significantly improved. Discussion: This case is an unusual presentation of a GSD1A patient who progressed well during infancy but had multiple disease complications in adulthood requiring OLT. Previous studies have shown that despite correcting the fasting abnormalities, extra-hepatic complications of GSD1A may persist. In this particular case, on a short-term follow-up, all were successfully reversed.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 P-290

The use of indirect calorimetry for energy requirement measurements in children with hepatic glycogen storage disease type I

Results: Genetic analyses of the siblings revealed homozygosity for mutation c.736C > T on the GYS2 gene confirming the diagnosis. The third patient was found to be homozygous for c.1145G > A. Discussion: GSD0 is more common than previously assumed. Recognition of the variable phenotype spectrum of GSD0 and routine analysis of GYS2 are essential for the correct diagnosis.

Szymanska E 1, Ehmke vel emczynka Seliga E 1, Rokicki D 1, TylkiSzymanska A 1, Ksiazyk J 1 P-292 1

Dept Ped Nutr Met Dis, Ch Mem Health Ins, Warsaw, Poland

Background: Patients with glycogen storage disease (GSD) type I have predisposition to obesity due to high calorie diet with cornstarch. Objective energy requirement measurements in GSD are crucial. The aim of this study was to measure resting energy expenditure (REE) in children with GSD type I using indirect calorimetry. Methods: Nine patients with GSD type I (Ia:Ib; 5:4), aged 9.2 (5.5–15.8) years were included into the study. The average fasting time preceding IC was 2.7 h (SD ± 0.9). REE was measured using an indirect calorimeter. The results were compared with predicted values using Schofield’s equation and calculated from weight and height. Results: Median body mass [median (interquartile range)] was 36.3 kg (19.7– 60.5) and median height was 141 cm (105–157). Median uncooked cornstarch intake (g/24 h) was 180.0 (55.0–335.0), which was 872.3 kcal/24 h (SD ± 425.6) and median diet energy (kcal/24 h) was 1443 (SD ± 356), median total calories intake was 2189.6 kcal/24 h (SD ± 423). Average measured REE was 1677.6 (SD ± 336.6), median predicted REE was 1272.0 (SD ± 206.8) and median REE (Scho) was 1178.8 (SD ± 238.2). Measured REE was increased in GSD I when compared to the predicted values calculated from weight and height (p = 0.001) and from Schofield’s equation (p = 0.00004). Discussion: An increased energy requirement and obesity found in children with GSD I might be due to a high calorie diet/excessive cornstarch intake which should be taken into account for their dietary management.

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The variable clinical phenotype of hepatic glycogen synthase deficiency Kasapkara C 1, Aycan Z 4, Acoglu E 3, Senel S 3, Oguz M M 3, Ceylaner S 2 1 Div Metab Dis, Dr Sami Ulus Child Hosp, Ankara, Turkey, 2Intergen Genetic Center, Ankara, Turkey, 3Div Pediatr, Dr Sami Ulus Child Hosp, Ankara, Turkey, 4Div Ped Endocr, Dr Sami Ulus Child Hosp, Ankara, Turkey

Background: Glycogen synthase deficiency, also known as glycogenosis (GSD) type 0 is an inborn error of glycogen metabolism caused by mutations in the GYS2 gene, which is transmitted in an autosomal recessive trait. It is a rare form of hepatic glycogen storage disease with less than 30 cases reported in the literature so far. The disorder is characterized by fasting hyperketotic hypoglycemia without hyperalaninemia or hyperlactacidemia. It is a glycogenosis with lack of liver glycogen synthesis, therefore hepatomegaly is not observed in patients with glycogen synthetase deficiency. Symptoms of fasting hypoglycemia in patients with GSD 0 usually appear for the first time in late infancy when weaning from overnight feeds. Seizures associated with low blood glucose may also occur, but they are rare. Clinical management is therefore based on frequent meals composed of high protein intake during the day and addition of uncooked cornstarch in the evening. Case report: Herein we report three new cases of liver glycogen synthase deficiency (GSD 0). The first patient presented at the age of 4 years with recurrent hypoglycemic seizures. The second patient who is the brother of the first patient presented at 15 months with asymptomatic incidental hypoglycemia. Glucose monitoring in both patients revealed daily fluctuations from fasting hypoglycemia to postprandial hyperglycemia and lactic acidemia. A third patient was consulted for ketotic hypoglycemia and postprandial hyperglycemia at the age of 5 years.

A case of a rapidly progressive renal impairment in glycogen storage disease type 1a—the management and preparation for the renal transplant. Stepien K M 1, Solomon L 2, Duncan M 2, Nair B 3, Hendriksz C J 1 1 Mark Holland Metab Unit, SRFT, Salford, United Kingdom, 2Renal Unit, Preston, Preston, United Kingdom, 3Histopathology Dept, Preston, Preston, United Kingdom

Background: Glycogen storage disease (GSD) type 1a is a rare autosomal recessive condition. Renal manifestations start in childhood and progress silently. They include proteinuria, haematuria, nephrocalcinosis, distal renal tubular acidosis, hypercalciuria, hypocitraturia and gradual renal failure. The progression of renal complications can be decelerated by improvement of the metabolic control with diet and treatment with allopurinol and ACE Inhbitors. Case report: A 19-year old Asian female was diagnosed with GSD 1a in childhood. Plasma glucose control was difficult to control. She had a PEG tube to prevent overnight hypoglycaemia because her fasting tolerance was less than 4 h. Her caloric intake was 3000 kcal per day that resulted in gradual weight gain (BMI 30 kg/m2). She reached early adult life with no major physical or mental impairment, when she was noted to have evidence of kidney disease. She still needed overnight feeding, had polycystic ovaries syndrome and suffered from anxiety. Results: Her eGFR fell from 51 to 25 ml/min and urine protein: creatinine ratio increased from 203 mg/mmol to 770 mg/mmol between June 2015 and April 2016. Other results included serum albumin 40 g/L, urate 1005 μmol/L, total cholesterol 19.8 mmol/L and lactate 7.51 mmol/L. Ultrasound and plain x-ray showed normal renal anatomy with no stones. Renal biopsy showed global glomerular sclerosis and mild mesangial expansion of surviving glomeruli, moderate tubular atrophy and interstitial fibrosis. The proximal tubules were full of glycogen. Discussion: Modern management enables patients with GSD 1a to reach adult life. Kidney failure develops as a result of the enzyme defect, and may progress inevitably as a result of secondary processes such as hyperfiltration. Management is challenging and multidisciplinary. Possible approaches include single kidney or combined kidney–liver transplantation. Conflict of Interest declared.

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Plasma acylcanitines and urine organic acids profiles provide evidence for possible mitochondrial dysfunction in glycogen storage disease type Ia Rossi A 1, Ruoppolo M 2, Formisano P 3, Villani G 2, Albano L 2, Gallo G 2, Moccia A 3, Parenti G 1, Strisciuglio P 1, Melis D 1 1 Dept Trans Med Sci Sect Peds, Fed II Univ, Naples, Italy, 2Dept Mol Med Biotech, Fed II Univ, Naples, Italy, 3Dept Trans Med Sci Sect Path, Fed II Univ, Naples, Italy

Background: Glycogen storage disease type I (GSDI) is an inborn error of carbohydrate metabolism caused by mutations of either the G6PC gene (GSDIa) or the SLC37A4 gene (GSDIb). It has been recently shown that GSDIa patients are at higher risk for developing metabolic syndrome and insulin-resistance (IR). A possible role of mitochondrial dysfunction in developing IR has been proposed. Mitochondrial disorders are traditionally investigated by plasma acylcanitines (ACs) and urine organic acids (UOA) profiles.

S144 The aim of the present study was to analyze the molecules of intermediary metabolism to determine whether an alteration of mitochondrial function exists in GSDI patients and its possible role in IR. Methods: 13 GSDIa and 7 GSDIb patients and 26 and 14 age and sex-matched controls, were enrolled. Plasma ACs, UOA and surrogate markers of IR (HOMA-IR, QUICKI, ISI, VAI) were measured. Results: GSDIa patients showed higher short-chain ACs (C0, C2, C3,C4, C6DC) and long-chain ACs (C14, C16, C16:1, C16OH, C18, C18OH) levels and urinary excretion of lactate, pyruvate, ethylmalonate, fumarate, malate, 3methylglutaconate, 3-methylglutarate, suberate, aconitate, sebacate, 2-ketoglutarate, 4-octenedioate than controls (p < 0.05). GSDIb patients showed higher C0 and C4 levels than controls (p < 0.05). In GSDIa patients C12 and C16 levels correlated with insulin serum levels, HOMA-IR, QUICKI and ISI (p < 0.05); long-chain ACs levels correlated with cholesterol, triglycerides and VAI (p < 0.05). Discussion: Increased plasma long-chain ACs and abnormal UOA profile suggest overload of mitochondrial lipid oxidation and Krebs cycle/respiratory chain inefficiency leading to mitochondrial stress. Correlation data are consistent with the reported hypothesis that IR is associated to mitochondrial dysfunction. It can be hypothesized that inefficient beta-oxidation generates chain-shortened ACs that may affect insulin signaling pathway, leading to IR.

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Alglucosidase alfa enzyme replacement therapy as a therapeutic approach for glycogen storage disease type IV

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Some authors state that growth hormone (GH) therapy is strictly contraindicated in GSD because near-normal growth occurs with optimal metabolic control and, because there is no GH deficiency, GH therapy increases glycogenolysis and may increase the rate of complications. But there is a subset of GSD Ib patients with poor growth despite adequate dietary treatment and dietary compliance. Case report: A 12-year-old male patient was first presented to our unit at the age of 6 years because of hepatomegaly and poor growth. The bioquemical findings were consistent with GSD Ib including mild hypoglycemia (56 mg/ dl), neutropenia (987/mm3), hyperlactacidemia (3 mmol/L), hyperuricemia (6 mg/dl) and hyperlipidemia (triglycerides 253 mg/dL); genotype c.1042_1043delCT (p.L348fsX400) /c.1062 C > A (AAC > AAA; p.Asn354Lys, exon 8), gene SLC37A4. Results: Despite a course of uncooked cornstarch (1 gr/kg/4 h) with good metabolic control, mild complications (occasionally cold and epistaxis), and no inflammatory bowel disease, growth rate did not improve. At 10 years, height was 122 cm (< P 1; SDS −3.15); weight was 22 kg (P3; SDS −1.86); BMI: 14.78 (P12; SDS −1.17); growth velocity: 3.5 cm/year. GH secretion was evaluated with propranolol plus exercise stimulation test (HG 30 min 3 ng/ml; normal values > 7 ng/ml) and with clonidine stimulation test (GH maximum peak 5.45 ng/ml; normal values > 10 ng/ml). IGF-1 levels were persistently low: 12.3 ng/ml, and normal thyroid function. GH therapy was started and the growth rate improved to 5 cm/year. There was no metabolic decompensation prior to GH therapy. Liver funtion did not worsen either. Discussion: Although GH therapy response was suboptimal in our case, we did not detect any metabolic or clinical decompensation. There is a need to consider the usefulness of GH treatment in certain cases of GSD Ib.

Sun B 1, Yi H 1, Gao F 1, Yang C 1, Austin S 1, Kishnani P S 1 1

Div Med Genet, Duke Univ Med Ctr, Durham, United States

Background: Glycogen storage disease type IV (GSD IV) is characterized by deposition of less-branched and poorly soluble glycogen in various tissues. The progressive hepatic type is the most common form of GSD IVand patients develop irreversible cirrhosis and often die of liver failure in early childhood. Liver transplantation is the only treatment option for GSD IV. We investigated the feasibility of using recombinant human acid-α glucosidase (rhGAA, Alglucosidase alfa), an FDA approved therapy for Pompe disease, as a treatment approach for GSD IV. Methods: Male GSD IV mice were used to test 3 dosages of rhGAA treatment: 20 mg/kg (human equivalent dose), 40 mg/kg, and 100 mg/kg. Weekly intravenous injections of rhGAAwere administrated for 4 weeks starting at 9 weeks of age. A group of age-matched untreated mice were used as controls. All mice were sacrificed 48 h after the last injection following overnight fasting. Fresh tissues were immediately frozen and stored at −80 °C until testing for GAA activity and glycogen content. Results: Significant increases in GAA activity were observed in tissues from GAA-treated mice in a dose dependent manner. The greatest increase was found in liver, which had 29- 48- and 67- fold increases over untreated controls at the 3 doses from low to high, respectively. Enzyme uptake was less efficient in skeletal muscles as the GAA activity was increased by only 1.6- fold at 100 mg/kg. Glycogen contents were significantly reduced in the livers from the 40 mg/kg (−21 %) and 100 mg/kg (−25 %) treatment groups. Discussion: Our data suggest that rhGAA is feasible for treating hepatic glycogen storage in GSD IV. Greatly increased rhGAA activity may enhance glycogen clearance in lysosomes, promote glycogen transport to lysosomes, and thus reduce cytoplasmic glycogen levels in GSD IV. Another possibility is that excess rhGAA leaks into the cytoplasm from the lysosomes and helps degrade glycogen deposits. Conflict of Interest declared.

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Growth hormone therapy in glycogen storage disease Ib (GSD Ib) Ruiz Pons M 1, Rial Rodriguez J M 2, Valerio E 1 1

Div Metab Dis, Univ Ntra Sra Cand Hosp, Santa Cruz de Tenerife, Spain, Div Endocrin Dis, Univ Ntra Sra Cand Hop, Santa Cruz de Tenerife, Spain

2

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Study of gonadal, brain and bone damage in a zebrafish model for classic galactosemia Van Erven B 1, Vanoevelen J M 2, Bierau J 3, Coelho A I 1, RubioGozalbo M E 1 1

Dept Pediatr and Dept Clin Genet, MUMC+, Maastricht, Netherlands, 2Dept Clin Genet, Unit Clin Genom, MUMC+, Maastricht, Netherlands, 3Dept Clin Genet, MUMC+, Maastricht, Netherlands Background: Classic galactosemia is a genetic disorder of galactose metabolism, resulting from profound galactose-1-phosphate uridylyltransferase (GALT) deficiency. The current standard of therapy—galactose-restricted diet—is life-saving but cannot prevent the development of ovarian, brain and, to a lesser extent, bone damage. Aims: We generated a GALT knock-out zebrafish, aiming to gain new insights on galactosemia chronic complications. Biochemical analysis showed essentially null enzyme activity; developmental-specific phenotypic characterization is now in progress. Methods: We crossed our GALT knock-out transgenic zebrafish line to two reporter lines: i) fluorescent primordial germ cells (PGCs) (vasa:GFP) and ii) fluorescent myelin (mbp:GFP) transgenic lines. These GALT knock-out lines with labeled PGCs and fluorescent myelin provide two excellent models to study the damage to female gonads and brain damage in embryonic, juvenile and adult stages. In addition, bone development will be studied using tissue staining. Results: The GALT knock-out and heterozygous vasa:GFP fish are currently being studied by fluorescence analysis regarding PGC number, organization and migration. The GALT knock-out and heterozygous mbp:GFP fish will be studied by fluorescence analysis to evaluate the myelination process and myelin microstructure. Furthermore, a bone and cartilage staining will be performed in GALT knock-out and heterozygous fish, in order to evaluate bone development. The GALT wild-type fish will be evaluated in parallel with all analyses and used as a control. Discussion: These studies will increase knowledge on the developmental stage of the organ-specific onset of damage, as well as on the time-specific extent of damage, thereby providing crucial information for the establishment of a new therapeutic strategy.

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Cytosolic phosphoenoylpyruvate carboxykinase deficiency (cPEPCK) presenting with gastrointestinal hemorrhage

Discussion: This finding indicates that Duarte allele D314 might be far more common in the Pakistani population than in European and North American ones. Unexpectedly, galactitol was found in the urine of all patients receiving a galactose-restricted diet, and it was postulated that this sugar alcohol has its origin in part from endogenously produced galactose.

Tangeraas T 1, Tveten K 2, Astrup H 3, Rootwelt T 1, Backe P H 4, Woldseth B 1 1 The Norwegian Unit for NBS and diagn IEM, Oslo, Norway, 2Dept of Med Genetics and laboratory, Skien, Norway, 3Dept of Paed Sorlandet Hosp Trust, Kristiansand, Norway, 4Inst for Clinical Medicine Univ of Oslo, Oslo, Norway

Background: cPEPCK catalyzes the formation of phosphoenolpyruvate from oxaloacetate, and deficiency of the enzyme affects gluconenogenesis and causes reduced fasting tolerance. Case report: A previously healthy girl (16 months) presented with severe gastrointestinal hemorrhage (Hb 6.7 g/dl, p-glucose 5.6 mmol/l) and affected liver function following a gastroenteritis episode (ALAT 888U/l, ASAT 1290U/l, INR 1.4 and albumin 29 g/l. Multiple ventricular ulcers were ascertained but no cause identified. Later she was admitted twice during intercurrent infections with ketotic hypoglycemia, metabolic acidosis and elevated lactate levels. A similar biochemical pattern was confirmed during a controlled fasting test provoking hypoglycemia after 17 h fast. The girl shows normal development on regular feeds and glucose polymer emergency regimen. Urine analysis of methylated organic acids by GC-MS. Whole Exome Sequencing (WES, trio testing) was performed using Nextera Rapid Capture Exome (Illumina) and NextSeq 500. Results: Urine after 12 h fast showed increased excretion of 3-hydroxybutyrate as well as fumaric and glutaric acids. WES revealed compound heterozygosity in the PCK1 gene NM_002591.3 c.925G > A (p.Gly309Arg) and c.1415del (p.Gly472Alafs*84). The first variant is predicted to be pathogenic (PolyPhen, SIFT, MutationTaster, Align GVGD). The second deletion results in a frameshift at amino acid 472 followed by the substitution of the next 83 amino acids and ends with a premature stop codon. Discussion: Only a few cases of cPEPCK deficiency have been published, but cPEPCK deficiency was recently revived in a case presenting with liver failure after gastroenteritis. Our case shares some of the same clinical and biochemical findings such as liver affection after gastroenteritis and secretion of TCA metabolites in urine. Our case further expands the clinical, biochemical and genetic findings of cPEPCK deficiency.

P-298

Endogenous synthesis of galactose in galactosemics with galactose restricted diet Mahmood U 1, Imran M 1, Cheema H A 2, Suleman H 2, Mahmood S 1

P-299

Images from F-DOPA PET scan in congenital hyperinsulinism: not always a clue for diagnosis Maines E 1, Giacomello L 2, Gaudino R 3, D’Onofrio M 4, Salgarello M 5, Gugelmo G 1, Bordugo A 1 1 Inher Metab Dis Unit, NBS Center, AOUI, Verona, Italy, 2Pediatric Surgery Unit, AOUI, Verona, Italy, 3Div Pediatric Endocrinology, AOUI, Verona, Italy, 4 Dept of Radiology, AOUI, Verona, Italy, 5Dept Nucl Med, Sacro Cuore Hospital, Negar, Verona, Italy

Background: Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycaemia in the neonatal period and early infancy. [18F]dihydroxyphenylalanine positron emission tomography (18F-DOPA PET) or integrated 18F-DOPA PET computed tomography (18F-DOPA PET/ CT) are useful to distinguish focal from diffuse CHI forms by detecting hyperfunctional pancreatic areas. The differentiation between focal and diffuse forms is very important because the surgical management is radically different. Case report: A 13 month-old girl was referred to our clinic because of diazoxide unresponsive CHI due to a paternally inherited mutation in the ABCC8 gene. A partial pancreatectomy had been performed when she was 3 months old in her developing country of origin (Kosovo). After surgery, despite medical therapy with octreotide (40 μg/Kg/d), she continued to experience recurrent episodes of severe hypoglycaemia. To evaluate the opportunity of another surgical intervention, we performed 18F-DOPA PET/CT, which showed a possible focal lesion in the anatomical region of the pancreatic tail. On the other side abdominal magnetic resonance imaging (MRI), performed to elucidate 18F-DOPA PET/CT images, showed that only the pancreatic head was preserved with pancreatic parenchyma terminating at the isthmus level. Abdominal MRI was necessary to elucidate that 18F-DOPA uptake was related to a loop of bowel occupying the empty pancreatic bed. Oral nifedipine was added (0.5 mg/Kg/d) to the treatment with good results and no severe hypoglycaemic episodes. Results: Our case suggests that bowel uptake can be a possible pitfall in the interpretation of 18F-DOPA PET/CT in children affected by CHI. Discussion: When 18F-DOPA PET/CT results do not fit with clinical data, abdominal MRI may provide valuable information to ensure accurate identification of uptake origin, allowing an accurate correlation of the radiotracer activity with the underlying anatomic or pathologic equivalent.

1 University of Health Sciences, Lahore, Pakistan, 2The Childrens Hospital and ICH, Lahore, Pakistan

P-300 Background: Type I galactosemia is an inborn error resulting from mutations on both alleles of GALT gene which leads to absence or deficiency of galactose-1-phosphate uridyltransferase (GALT). Galactose-1-phosphate accumulates within cells and surplus galactose is reduced to galactitol or oxidized to galactonate. Patients with this condition have substantial motor, cognitive, and psychiatric impairments despite dietary treatment. Classical galactosemia is frequently associated with Q188R, S135L and K285N mutations and N314D is associated with Duarte galactosemia and is wide spread among various worldwide populations. Methods: The present study aims at detecting Q188R, S135L, K285N mutations and N314D variant in the GALT gene in 8 galactosemia patients and 190 unrelated normal subjects all of Pakistani origin through ARMS and urinary galacitol by quantitative Benedict’s test. Results: S135L and K285N mutations were present neither in galactosemia patients nor in normal subjects. Only one galactosemia patient carried Q188R mutation that was in homozygous state. However, N314D variant was frequently found in affected (7out of 16 alleles) and normal subjects (55 out of 380 alleles).

Phenotypic and genotypic features and long term follow-up 36 Turkish galactosemia patients Kor D 1, Seker Yilmaz B 1, Bulut F D 1, Oktem M 2, Kolasin P 2, Onenli Mungan N 1 Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Duzen Lab, Ankara, Turkey

1

Background: Classic galactosemia is a rare autosomal recessive disorder due to galactose-l-phosphate uridyl transferase (GALT) deficiency, leading to the accumulation of galactose and metabolites. The treatment is dietary restriction of galactose. Even with dietary adherence, neurocognitive problems and ovarian failure are frequently seen complications. Galactosaemia is not a part of Turkish newborn screening programme. We report the geno-phenotypic features and long term follow-up of 36 galactosemia patients from the south part of Turkey.

S146 Methods: This study was a retrospective analysis of 36 galactosemia patients. Results: 36 patients from 34 families were enrolled in the study. Diagnosis was confirmed with the level of GALT and/or mutation analysis. Median age of diagnosis was 23,6 ± 18,9 days (prenatal 175 days). Genetic analyses of the GALT gene revealed 2 novel mutations out of 11 different mutations. The previously reported p.Q188R (c.563A > G) mutation was also the most frequent mutation in our study. Symptoms/signs were feeding difficulties, vomiting, diarrhea, cholestasis, and hepatomegaly. 12 patients were operated for cataract and recurrence did not occur. All patients received a galactose-restricted diet, calcium and vitamin-D supplementation. Median age at last visit was 59 months (2–190). Among the long-term complications growth retardation was observed in 6 patients. Latent hypocalcaemia and vitamin-D insufficiency were detected in 4 and 20 patients, respectively. A gallstone was observed in one patient. No patients had chronic liver disease. 4 cases had mild mental-motor retardation due to hypoglycemia. Linguistic skills were affected in 7 patients (19,4 %). Except 2 female patients, one with ovarian insufficiency and one with precocious puberty, all of them were prepubertal. Discussion: Here we present a large group of Turkish galactosemia patients and long term follow-up results. 2 new mutations and a patient with precoccus puberty are the new outcomes of this study.

P-301 Type Ib glycogen storage disease presenting as severe hypertrygliceridemia Buonuomo P S 1, Ponzi E 2 3, Lepri F R 4, Maiorana A 2, Macchiaiolo M 1, Ceccarelli S 5, Scalzone M 1, Rana I 1, Saura F 6, Calandra S 7, Novelli A 4, Bartuli A 1 1

Rare Diseases, Bambino Gesu’, Rome, Italy, 2Metabolic Unit, Bambino Gesu’, Rome, Italy, 3Department of Genomic Medicine, UCSC, Rome, Italy, 4 Laboratory of Medical Genetics, Bambino, Rome, Italy, 5Haematology, Bambino Gesu’, Rome, Italy, 6Laboratory Medicine, Bambino Gesu’, Rome, Italy, 7Biomedical, Metabolic and Neural Science, Modena, Italy Background: Lipid disorders can occur either as primary event or secondary to an underlying disease. Defining and treating the underlying cause of hypertriglyceridemia is the essential step toward restoring the lipids and lipoproteins to normal, especially in severe cases who are at risk for acute pancreatitis. Case report: A 10-month-old female infant referred for hepatosplenomegaly and failure to thrive presented milky serum due to extreme hypertriglyceridemia (11.000 mg/dl) promptly treated with plasmapheresis. A genetic defect of the intravascular lipolysis of TGrich plasma lipoproteins was suspected, and candidate genes (LPL, APOC2, APOA5, GPIHBP1, and LMF1) were initially sequenced, After TG normalization, asymptomatic hypoglycemia (29 mg/dl) became evident. The combination of fasting hypoglycemia, hepatomegaly with increased echogenicity, hyperlipidemia, lactic acidosis and 3methylglutaconic aciduria raised the suspicion of glycogen storage disorder type I (GSD1). Results: Sequencing of the candidate genes for familial chylomicronemia revealed a common heterozygous mutation for the LPL gene [(Ex6) c.953A > G, AAT > AAG, p.Asn318Ser (Asn291Ser)]. Remarkably, DNA was screened with ‘hypoglycemia gene custom panel’ by Nextera Rapid Capture Enrichment (Illumina) assay and the patient was found to be homozygous for the mutation c.1015 G > T (p.Gly339Cys) in SLC37A4 gene, causing GSD1b. The baby started a high carbohydrate-low lipid diet with normalization of blood glucose and lipid profile. Discussion: In GSD1 hypertriglyceridemia results from increased synthesis and decreased serum lipid clearance. The presence of a concomitant LPL mutation is consistent with severe hypertriglyceridemia found in our patient, which in turn delayed the diagnosis by interfering with routine blood tests. Although there are various causes of milky serum in infants, a strong suspicion of GSD should be kept in mind when hepatomegaly, hypoglycemia and hypertriglyceridemia are detected.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 P-302 New cases of PRKAG2 mutations presenting in infancy: a possible therapeutic approach using alglucosidase alfa (Myozyme) enzyme replacement therapy Austin S L 1 , Torok R 1 , Phornphutkul C 2 , Buckley A 3 , Sun B 1 , Govendrageloo K 4, Perrin H 5, Kishnani P S 1 1 Dept of Peds, Duke Univ, Durham, United States, 2Dept of Peds, Brown Univ, Providence, United States, 3Dept of Pathology, Duke Univ, Durham, United States, 4Peds Cardio, Netcare Sunninghill Hosp, Johannesburg, South Africa, 5Peds Neuro, Netcare Waterfall City Hosp, Johannesburg, South Africa

Background: PRKAG2 encodes the γ2 subunit of AMP-activated protein kinase (AMPK), which is an important regulator of cardiac metabolism. Mutations in PRKAG2 cause a cardiac syndrome comprised of ventricular hypertrophy, preexcitation, and progressive conduction system disease. Significant variability exists in the presentation and outcomes of patients with PRKAG2 mutations. The features often resemble the cardiac manifestations of Pompe disease. Case report: Here, we add three cases to the five previously described where patients with PRKAG2 mutations presented with symptoms in infancy. Results: In all three of our cases, Pompe disease was the initial suspected diagnosis, with two patients going on to receive enzyme replacement therapy (ERT) (Myozyme / Lumizyme). However, Pompe disease was eventually ruled out, and a disease causing PRKAG2 mutation was identified subsequently in each case. In one case, hypotonia and muscle weakness were the presenting symptoms, which improved on ERT. ERT was stopped after the PRKAG2 mutation was identified, and his myopathy worsened. When ERT was restarted, a clinical benefit was again noted. Discussion: We highlight the potential for PRKAG2 mutations to mimic Pompe disease in infancy and the need for confirmatory testing via sequencing when diagnosing Pompe disease. Also, we outline the benefit a patient showed while on ERT treatment, the decline in his condition when the infusions were discontinued, and the significant positive response when ERT was reinitiated. This further supports the role of ERT in clearing cytoplasmic glycogen. Conflict of Interest declared.

P-303 Hypoglycemia in children: uncovering the genetic basis of related metabolic disorders by custom gene panel in an Italian cohort Ponzi E 1, Maiorana A 1, Lepri F R 2, Novelli A 2, Dionisi-Vici C 1 1 Metabolic Unit, Bambino Gesu Child Hosp, Rome, Italy, 2Med Genet Unit, Bambino Gesu Child Hosp, Rome, Italy Background: Hypoglycemia is a common finding of many IEM, as GSDs, galactosemia, HFI, CHI, defects of gluconeogenesis, FAO, ketogenesis, ketolysis, and mitochondrial disorders. Early diagnosis is essential to start specific dietary and pharmacological strategies. Although clinical presentation and metabolic workout can orient the diagnosis to a specific group of IEM, an extensive genetic analysis is necessary to rapidly identify critical genes and pathways to confirm the diagnosis. The widely used Sanger gene-by-gene sequencing is time consuming and cost-expensive, because of high number and large size of the involved genes. Methods: We simultaneously analyzed 72 genes involved in IEM presenting with hypoglycemia with next generation sequencing (NGS) Nextera Rapide Capture protocol-Illumina. Gene cluster analysis and interpretation were oriented by patient phenotype. All mutations were confirmed with Sanger sequencing. New variants were validated with the SIFT, Align GVGD, Polyphen2 and Mutation Taster software. Results: Pathogenic mutations were detected in 22/45 patients with different clinical phenotypes. We identified 30 mutations in genes involved in CHI (6 ABCC8, 1 AKT2, 1 GLUD1, 1 HNF1A, 1 INSR), FAO (4 ETFDH, 1 ETFA, 2 HADHB), GSDs (2 GYS2, 1 SLC37A4, 1 PHKA2), gluconeogenesis defects (1 FBP1), galactosemia (2 GALT), HFI (1 ALDOB), ketogenetic defects (3 HMGCS2) and mitochondrial disorders (2 POLG). Mutation types: 22

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 missense mutations, 2 intronic variants, 3 out of frame deletions and insertions, 1 stop loss, 2 stop gain; 22/30 were new pathogenetic variants. Discussion: The use of a custom gene panel allowed a rapid targeted diagnosis in 50 % of patients. Discrepancy between biochemical and clinical hallmarks in some patients might be explained by involvement of multiple genes sharing similar pathways in carbohydrates metabolism. In conclusion, NGS provides an accurate high-throughput technology for rapid genetic diagnosis of metabolic hypoglycemias.

P-304

The association of the c.-119_-116delGTCA mutation and the c.940A > G variant of GALT gene in Taiwanese newborns Chiu Y H 2, Chen J H 3, Liu Y N 2, Liu M Y 4, Chiang S H 5, Chiang C C 1, Ho H C 6, Liu T T 4, Hsiao K J 5 1 Chinese Found Health, Taipei, Taiwan, 2Dept Edu Res, Taipei City Hosp, Taipei, Taiwan, 3Dept Life Sciences, Natl YM Univ, Taipei, Taiwan, 4VYM Genome Res Center, Natl YM Univ, Taipei, Taiwan, 5Prev Med Found, Taipei, Taiwan, 6Taipei Inst Pathol, Taipei, Taiwan

Background: Infants with Duarte variant (D2) galactosemia remain 14– 25 % of control GALT activity in their erythrocytes and typically, but not always, asymptomatic. Besides, asymptomatic children with Duarte variant (D2) galactosemia showed relatively increased risk of developmental difficulties by mid-elementary. Therefore, the correct diagnosis, close follow-up and regular check for the ability of galactose metabolism is suggested to prevent developmental delay and other unknown phenotypes in individuals with Duarte variant galactosemia. Although the c.119_-116delGTCA mutation was the true disease causing mutation, the detection for the c.940A > G variation was widely used for the diagnosis of Duarte variant (D2) galactosemia in many populations. In this study, we evaluate the association of the c.940A > G variant and the c.-119_116delGTCA mutation in Taiwanese population. Methods: The c.940A > G variant and the c.-119_-116delGTCA mutation were analyzed in 2,817 infants who were negative for galactosemia in newborn screening and 93 infants with transient neonatal gelactosemia in Taiwan. Results: In 2,817 non-galactosemia newborns, 59 newborns carried c.940A > G variants, among which only 83.1 % (49/59) newborns carried both the c.940A > G variants and the c.-119_-116del mutation. In 93 newborns with transient neonatal galactosemia, 10 newborns carried c.-119_-116del mutations, but one of which is not associated with c.940A > G variant. Discussion: Our data demonstrated that the c.-119_-116delGTCA mutation was not always associated with the c.940A > G variant. We therefore recommended direct analysis of the c.-119_-116delGTCA mutation for determining Duarte variant (D2) galactosemia.

P-305

Unveiling the mutational spectrum of classic galactosemia in Croatia Rivera I 1, Angelo S 1, Pavlu-Pereira H 1, Silva M J 1, Tavares de Almeida I 1, Petkovic Ramadza D 2, Sarnavka V 2, Vukovic J 2 3, Fumic K 4, Krzelj V 5, Lozic B 5, Puseljic S 6, Baric I 2 3 1 iMedUL, Fac Phar, Univ Lisboa, Portugal, Lisboa, Portugal, 2Dept Ped, Univ Hosp Cen Zagreb, Croatia, Zagreb, Croatia, 3School Medicine, Univ Zagreb, Croatia, Zagreb, Croatia, 4Dept Lab Diag, Univ Hosp Cen Zagreb, Croatia, Zagreb, Croatia, 5Dept Pediat, Univ Hosp Cen Split, Croatia, Split, Croatia, 6 Dept Pediat, Univ Hosp Cen Osijek, Croatia, Osijek, Croatia

Background: Classic galactosemia (MIM #606999) is an autosomal recessive disorder caused by deficient activity of galactose-1-phosphate uridyltransferase (GALT). Patients usually develop symptoms in the neonatal

period, the most dangerous being liver failure which can be successfully treated by dietary galactose restriction. Nevertheless, many patients develop longterm complications, mostly neurologic, whose pathophysiology is still poorly understood. This disorder is characterized by high allelic heterogeneity of the GALT gene, which is thought to play a determinant role in biochemical and clinical phenotypes. The objective of this study was the molecular characterization of Croatian galactosemic patients. Patients and methods: A cohort of 15 unrelated patients, corresponding to 30 independent mutant alleles, was studied. After PCR amplification of individual exons and related intronic boundaries, GALT gene (GenBank accession NG_009029.1) was scanned for mutations by direct sequence analysis. Segregation analyses were performed when appropriate. Results: Although remaining four alleles to identify, the mutational spectrum observed in Croatian patients revealed to be quite restricted since only three mutations, 2 missense and 1 nonsense, were identified. As expected, the most frequent mutation is p.Q188R (15 alleles) followed by p.K285N (8 alleles). Interestingly, a novel mutation (p.R123X) was identified in homozygosity in a patient and in compound heterozygosity with p.Q188R in another patient. Furthermore, the number of patients homozygous for the p.Q188R mutation (less than 25 %) is considerable lower than in most galactosemic populations. Discussion: The present work started unveiling the mutational spectrum of classic galactosemia in Croatia, and it showed that most Croatian patients may benefit from the novel therapeutic approaches that are being developed, namely the use of chaperones like arginine.

P-306

Safety and acute complications of dietary management in patients with hepatic glycogen storage disease Peeks F 1, Steunenberg T A H 1, Mitchell J J 2, Lubout C M A 4, Mundy H 3, De Boer F 1, De Souza C F 5, Weinstein D A 6, Derks T G J 1 1 Div Metab Dis, Univ of Groningen, Groningen, Netherlands, 2Div Ped Endocrin, Montreal Child Hosp, Montreal, Canada, 3Met Unit, Evelina Child Hosp, London, United Kingdom, 4Emma's Child Hosp, Amsterdam, Netherlands, 5Med Gen Service, Hosp de Clinicas, Porto Alegre, Brazil, 6 GSD Program, Univ of Florida, Gainesville, United States

Background: Hepatic glycogen storage diseases (GSDs) are a group of very rare inherited disorders affecting carbohydrate metabolism, for which diet is the cornerstone of management. Safety and acute complications associated with dietary management have been poorly documented. Methods: To study acute complications associated with dietary management in GSD patients, a questionnaire was distributed via 7 GSD patient organizations representing patients from at least 10 countries. Results: In total, 191 patients with median age of 14.1 years (range: 0.5–66.1) responded. Most prevalent GSD subtypes were: 95x GSDIa, 27x GSDIb, 15x GSDIIIa and 18x GSDIX. Management was considered safe by 69 % (131/ 191) of the patients, but 47 % (89/191) reported at least one acute complication associated with dietary management: 41 hypoglycemias, 19 % seizures/epilepsy, and 6 % coma. Patients with GSDIb reported highest prevalence of complications: 70 %. Among patients who reported complications, most frequently reported causes were: not waking up by alarm clock (58 %), forgetting a meal (45 %), infections (37 %), gastric tube occlusion (35 %), and connector leakage (34 %). Most complications occured before the age of 12 years and during night time. As a consequence of complications, 4 % were managed at home, 62 % experienced hospital admittance and 33 % were admitted to the intensive care unit. At the time of the severest complication (i.e. coma, seizures, death), there was no statistical difference between either the number of patients with or without complex carbohydates (33 vs 27), or with or without continuous drip-feeding (23–37). Most commonly reported safety precautions were glucose meters (92 %), alarm clocks (61 %), and written emergency protocols (60 %). Discussion: Dietary management for hepatic GSDs is associated with considerable acute complications, of which the causes can be prevented, theoretically. There are no published guidelines concerning safety precautions.

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C20:3n9. The average of triglyceride levels was 302 ± 184 mg/dL, total cholesterol was 197 ± 50 mg/dL, high-density lipoprotein cholesterol was 31 ± 11 and low-density lipoprotein was 105 ± 36 mg/dL. Discussion: GSD1A fatty acid composition had been described showing a substantial elevation in C16:0, C16:1n7 and C18:1n9. We also found increased levels of the monounsaturated C16:1n7 and C18:1n9, but decreased levels of C16:0, most likely due to an up-regulation of de novo hepatic lipogenesis also by the enhanced SCD-1 activity. No patient had a biochemical evidence of essential fatty acid deficiency.

Adaptive and maladaptive behavior in hyperinsulinism Caviglia S 2, Bazzu P 2, Maiorana A 1, Dionisi-Vici C 1 1 Div Metab Dis, Bambino Gesu Hospital, Rome, Italy, 2Clinical Psychology Unit, Bambino Gesu Ch, Rome, Italy

Background: Congenital hyperinsulinism (CHI) is the most frequent cause of persistent hypoglycemia in children, due to an inappropriate secretion of insulin. In our clinical experience we have investigated CHI patients as they are forced to follow a strict pharmacological and dietary treatment to avoid neurological symptoms and sequelae. Methods: The Vineland Adaptive Behavior Scales (VABS) questionnaire was administered to parents of 48 CHI patients aged between 1–25y (mean 10,0y ± SD6,47) aiming to investigate specific domains (Communication, Daily Living Skills, Socialization and Motor Skills) and assess the Maladaptive Behavior Index score QA. Results: We herein focus on adaptive behavior considering >85AQ average score and < 85AQ maladaptive behavior index function. An adaptive behavior score was found in 54,17 % of children (mean 84,7 ± SD19,9). Patients with maladaptive behavior score 45,83 % fall especially in Daily Living Skills and Communication in spite of socialization domain. Patients with adaptive behavior, also, fall in Daily Living Skills and Communication domains. In particular a statistically significant difference emerges especially between Daily living Skills and Socialization domains. In adaptive behavior group 19,2 % patients manifested a compromised QI, while 36,3 % of maladaptive group presented normal QI. Female group 56,25 % falls especially in Daily Living Skills (mean 106.59 ± SD153,97) in spite of preserved communication domain (mean 86,74 ± SD25,98). Male group falls in Daily Living Skills (mean 82,48 ± SD23,27) and communication maintaining Socialization and Motor Skills domains. Discussion: Our patients with CHI present an AQ score compromised due to the frequent monitoring of glycemia, strict adherence to therapy and dietary regimens. Parents’ perception identified Daily Living Skills domain as the most compromised permitting us to handle and improve their lives.

P-308

Free fatty acid composition in Brazilian patients with type IA glycogen storage disease Vallejo D L 1, Grunert S 2, Tucci S 2, De Souza C F M 3, Nalin T 1, Schwartz I D 3 1 Uni Federal do Rio Grande do Sul, Porto Alegre, Brazil, 2University Hospital Freiburg, Freiburg, Germany, 3Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil

P-309

Clinical utility of a next generation sequencing panel in the genetic diagnosis of glycogen storage diseases Kyriss M N 1, Bliven K 1 1

PreventionGenetics, Marshfield, United States

Background: The glycogen storage diseases (GSDs) are a group of inherited metabolic disorders that result from a defect in a glycogen metabolic enzyme(s). At least 14 genes are reported to be involved in these disorders. While the GSDs are broadly divided into two categories based on primary symptoms (hepatic or neuromuscular), patients can also present with clinical features that involve both liver and muscle, making clinical diagnosis and identification of a probable causative gene difficult. To increase detection rates, reduce time to diagnosis, and improve cost-effectiveness, we released a NextGen sequencing panel that covers 14 genes known to be causative for various GSDs. Methods: A custom-designed oligo capture probe set was used to capture patient DNA for the 14 known GSD genes. For each gene, the full coding region of every exon plus 20 base pairs of flanking non-coding DNA on either side of each exon were captured and sequenced on a NextGen platform. Sanger sequencing was performed on low coverage (130/85 mmHg was recorded in 3/8 GSD I, 6/12 GSD III and 3/8 GSD IX. HDL was < 1.03 mmol/l in males or < 1.29 mmol/l in females, in 4/7 GSD I, 3/12 GSD III and 2/7 GSD IX. Triglycerides were >1.7 mmol/l in 5/6 GSD I, 1/3 GSD III and 1/3 GSD IX. Discussion: Patients with GSD I and III, versus GSD IX, had higher BMI and tendency to diabetes suggesting greater insulin resistance in severe liver GSD and overlap with metabolic syndrome. Therapeutic feeding regimens and necessary avoidance of fasting in those severely affected are likely contributory. Potential roles of altered body composition, skeletal muscle metabolism, and therapeutic modifications, warrant further exploration.

P-312 P-314 Polyol levels in the diagnosis of transaldolase deficiency Stradomska T J 1, Tylki- Szymanska A 2, Pawlowska J 3, Ryciak E 4, Dobrzanska A 4, Socha P 3

Unusual evolution in glycogen storage disease type VI due to growth hormone deficiency

1 Dept Biochem, Child Mem Health Inst, Warsaw, Poland, 2Div Metab Dis, Child Mem Health Inst, Warsaw, Poland, 3Div Gastr Hepat, Child Mem Health Inst, Warsaw, Poland, 4Div Neonat, Child Mem Health Inst, Warsaw, Poland

Ben Chehida A 1 2, Ben Massoued S 1, Ben Abdelaziz R 1 2, Hajji H 1 2, Boudabous H 1 2, Ben Turkia H 1 2, Abdelmoula M S 1, Kaabachi N 1, Azzouz H 1, Tebib N 1

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1

La Rabta Hospital, Tunis, Tunisia, 2Medical School, Universite Tunis Elmanar, Tunis, Tunisia

P-316

Background: Glycogen storage disease type 6 (GSD 6) is characterized by fasting hypoglycemia, hepatomegaly and growth retardation. Usually, it is a benign disorder that does not require any treatment. Clinical and biochemical abnormalities may resolve with age; most adults are asymptomatic. Case report: We report an unusual case of GSD 6 that had persistent hypoglycemia and retarded growth until teenage, due to growth hormone deficiency. Results: A 16-month-old Tunisian boy was investigated for hepatomegaly. History found seizures preceded by pallor and sweating, frequent nocturnal awakenings claiming feeding every 4–5 h. Psychomotor development was normal. He had short stature (−3 SD), hepatomegaly (13 cm). Laboratory investigations found cytolysis (8 x normal) and hypertriglyceridemia (2.78 g/l). CK, uric acid and tubulopathy balance were normal. His fasting tolerance was max. 4 h with ketosis but no lactic acidosis in hypoglycemia. Echocardiography and EMG were normal. The diagnosis of GSD 6 was confirmed by liver biopsy showing elevated glycogen content and decreased hepatic phosphorylase enzyme activity. The evolution was remarkable because of the persistence of a fasting tolerance of max. 4 h until the age of 12 years with worsening of growth retardation (−4 SD). Associated GH deficiency was confirmed by low GH in hypoglycemia. MRI showed pituitary adenoma. Discussion: Although GSD 6 causes fasting hypoglycemia and retarded growth that tend to disappear with age, comorbidity must be suspected in cases with an unusual clinical course.

Monocarboxylate transporter 1 deficiency: a novel heterozygous mutation resulting in acute ketoacidosis

13. Disorders of fatty acid oxidation and ketone body metabolism

P-315

Morbidity and mortality among exclusively breastfed neonates with medium-chain acyl-CoA dehydrogenase deficiency Ficicioglu C 1 2, Ahrens-Nicklas R 1, Pyle L 1

Paquay S 1, Sass J O 2, Finckh U 3, Melchior M 5, Gobert C 1, Sznajer Y 4, Nassogne M C 1 1 Div Pediatr Neurol and Metab Dis, UCL, Brussels, Belgium, 2Div Natural Sci, Univ Applied Sci, Rheinbach, Germany, 3Bereich Labor and Human Genet, MVZ, Dortmund, Germany, 4Div Human Genet, UCL, Brussels, Belgium, 5Div Pediatr, Namur, Belgium

Background: Monocarboxylate transporter 1 (MCT1) deficiency has been recently described as a cause for recurrent ketoacidosis due to defect on ketone bodies utilization. This newly characterized disorder was revealed in ketoacidotic patients when succinyl-CoA:oxoacid-CoA transferase (SCOT) and mitochondrial acetoacetyl-CoA thiolase (MAT) deficiencies were ruled out. Homozygous as well as single heterozygous mutations in the SLC16A1 gene encoding MCT1 were found to be related to clinical symptoms of MCT1 deficiency. Pathogenic heterozygous mutations have also been reported in asymptomatic siblings, suggesting that additional genetic and environmental triggers might be needed to develop ketoacidotic crisis. Moreover, the same mutation was described in symptomatic patients, whether homozygous or heterozygous. Case report and results : A 17-months-old boy born from unrelated parents presented with a severe ketoacidotic episode following infection. Biochemical analyses did not reveal metabolic markers for MAT deficiency. SCOT deficiency was excluded by sequencing of OXCT1 gene. Molecular analysis of the SLC16A1 gene found a heterozygous mutation c.442G > A for p.Gly148Arg, not described in the literature so far but affecting highly conserved amino acids. Computer based analyses (PolyPhen, SIFT, Mutation Taster) indicate that the mutation could influence the protein function and be pathogenically relevant. Discussion: MCT1 deficiency is probably underestimated and should be kept in mind in the differential diagnosis of ketoacidotic events when diabetes and ketolysis defects have been investigated. Recognition of the defect will be of significant benefit for the patient since preventive measures will avoid damaging effects of severe ketoacidosis. Clinical manifestations can occur both with homozygous or heterozygous mutations. Additional data will have to better define genotype/phenotype correlation.

1

The Children’s Hospital of Philadelphia, Philadephia, United States, Perelman School of Medicine at UPENN, Philadelphia, United States

2

Background: Despite greatly improved morbidity and mortality among infants with medium-chain acyl-CoA dehydrogenase deficiency (MCAD) since implementation of universal newborn screening (NBS), a population of neonates still becomes ill before their positive screen results are available. Exclusive breastfeeding is a proposed risk factor in this group. Since initial studies of MCAD NBS, breastfeeding rates have increased substantially. In this study we quantify the current risk of early decompensation in MCAD neonates and identify factors associated with poor outcomes. Methods: We completed a retrospective analysis of neonates with MCAD referred to our center between 2010 and 2015. Results: 46 infants were diagnosed with MCAD during the study period. 11 of 46 (23.9 %) were symptomatic prior to or at the time of the abnormal NBS report. Of these patients 4 presented with death or cardiac arrest, and 7 had hypoglycemia and/or lethargy. 100 % of symptomatic patients were exclusively breastfed, while only 40.6 % of asymptomatic patients were exclusively breastfed (p = 0.0008). During the study period, there was an upward trend in breastfeeding rates among patients without a known family history of MCAD. In 2010–2011 45.5 % of patients were exclusively breastfed; this increased to 64.7 % in 2012–2013, and 87.5 % in 2014–2015. Over this same time period rates of decompensation prior to NBS results significantly increased from 9.09 % in 2010–2011, to 23.5 % in 2012–2013, and 75 % in 2014–2015 (p = 0.003). Discussion: Exclusively breastfed neonates with MCAD are at risk for early metabolic decompensation. As breastfeeding rates rise, close management of feeding difficulties is essential for all neonates awaiting NBS results.

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The effect of riboflavin is limited in Japanese patients with multiple acylCoA dehydrogenase deficiency (MADD) Yamada K 1, Kobayashi H 1, Hasegawa Y 1, Yamaguchi S 1 1 Dept of Ped, Shimane Univ, Izumo, Japan Background: madd, also called glutaric acidemia type II, is caused by a defect in electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH). MADD is traditionally classified into the neonatal- and late-onset types. It was reported that riboflavin was effective for almost all patients with late-onset type of MADD. However, patients who do not respond to riboflavin exist in Japan. Methods: Effect of riboflavin is retrospectively investigated in 33 patients with GA2 identified by genetic analyses in our laboratory as follows: (1) excellent response, in which both biochemical abnormalities and clinical symptoms were improved; (2) good response, in which either biochemical or clinical findings were improved; (3) partial response, in which limited biochemical or clinical improvement was observed; and (4) no response. Results: There were 4, 5, and 24 patients with mutations in ETFA, ETFB, and ETFDH genes, respectively. Only one patient with ETFA deficiency displayed excellent response, although other patients with ETFA or ETFB deficiency died in early infancy. Patients with ETFDH deficiency included 7 neonatalonset and 17 late-onset types that were divided to 11 infantile-onset, 4 myopathic, and 2 pre-symptomatic forms. All patients with the neonatal-onset type showed no response. The response in the late-onset form was excellent in 2 cases, good in 2 patients, or partial in 2 subjects. Seven patients did not

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 respond. One patient was not treated with riboflavin, and data of 3 patients were not available. In infantile-onset form, all 3 patients who did not respond to riboflavin died in early childhood, whereas all patients with excellent, good, or partial response survived. Discussion: Riboflavin was less effective for Japanese MADD patients compared to the previous reports. The cause of discrepancy between Japanese and Caucasian patients in response to riboflavin is unknown, but may be associated with low frequency of myopathic form in Japan.

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Evaluation of natural or synthetic AMPK activators for correction of inborn fatty acid oxidation defects in patient cells

respectively). After metabolic emergency treatment CK decreased to near normal and stable levels around 3,500 U/l, respectively, when acute cardiac failure occurred. Results: Endomyocardial biopsy confirmed presence of parvovirus B19 in myocardium in both patients. After initial stabilisation patient 1 had recurrent episodes with thoracal pain, disturbance in conduction and repolarisation. She survived an episode of pulseless ventricular tachycardia and ventricular fibrillation requiring prolonged resuscitation. In patient 2, continuous deterioration of cardiac function and progressive multiorgan failure necessitated initiation of ECMO treatment. Subsequently, the patient had to be converted to a biventricular assist device, where she died while waiting for heart transplantation. Discussion: Acute cardiac failure in previously stable LCHADD patients may be promoted by parvovirus B19-associated myocarditis. The outcome is poor and may be improved by early diagnosis and intravenous immune globulin (IVIG) therapy.

Bastin J 1, Le Bachelier C 1, Boutron A 1, Djouadi F 1 1

INSERM U1124, Paris, France

Background: Mitochondrial fatty acid oxidation (FAO) disorders form a large group of inborn diseases without pharmacological treatment to date. Targeted strategies based on pharmacological stimulation of residual FAO capacities were shown to potentially correct FAO in patient fibroblasts with mild carnitine palmitoyl transferase 2 (CPT2), or very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. Here, we tested the effects of activators of AMP-activated kinase (AMPK), a Ser/Thr kinase considered as a key cellular energy sensor. Methods: 8 CPT2-deficient-, 9 VLCAD-deficient-, and control fibroblasts were treated for 2 days with synthetic (AICAR, Abbott A769662, Metformin) or natural (berberine, quercetine) compounds reported to act as direct or indirect AMPK activators. Possible changes in 3H-palmitate oxidation, FAO and respiratory chain (RC) enzymes mRNAs, and CPT2 and VLCAD proteins’ levels were then analyzed, together with the phosphorylation of AMPK. Results: Unexpectedly, and at odds with literature data obtained from immortalized cells, neither berberine, nor quercetine, nor metformin, were able to induce AMPK phosphorylation, and neither did any of these compounds improve FAO in CPT2 or VLCAD-deficient fibroblasts. Exposure to AICAR, presently considered as a poorly specific AMPK activator, resulted in highly variable results among the different cell lines. The only treatment leading to parallel increases in AMPK phosphorylation and in FAO capacities was that using A769662 (300 μM, 48 h). Discussion: These results suggest that the data on pharmacological activation of AMPK obtained in rodent cell lines are not always transferable to human fibroblasts. Overall, only the direct AMPK activator A769662 could induce significant FAO stimulation (+30 to +120 %) in CPT2- and in VLCADdeficient fibroblasts after a 48 h treatment. Experiments using siRNA against AMPK are ongoing to analyze the role of AMPK in mediating FAO changes in response to A769662.

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Acute cardiac failure in LCHADD patients caused by parvovirus B19 infection Haas D 1, Koelker S 1, Opladen T 1, Gorenflo M 4, Gottschalk U 3, Santer R 2, Muehlhausen C 2

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Experimental evidence that long-chain 3-hydroxylated fatty acids accumulating in LCHAD deficiency disrupt bioenergetics without altering redox homeostasis in heart, liver and skeletal muscle of adolescent rats Silva J C 1, Cecatto C 1, Amaral A U 1, Hickmann F H 1, Wajner A 1, Godoy K S 1, Goncalves A M 1, Milnitsky B 1, Leipnitz G 1, Wajner M 1 2 1 Departamento de Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil, 2Servico de Genetica Medica, HCPA, Porto Alegre, Brazil

Background: Long-chain 3-hydroxylated fatty acids (LCHFA) are accumulated in LCHAD deficiency, an inherited disorder characterized by heart, liver and skeletal muscle alterations whose pathogenesis is poorly understood. Since hyperlactic acidemia is usually found in this disease, suggesting mitochondrial dysfunction, we investigated the role of the accumulating LCHFA on energy and redox homeostasis. Methods: Mitochondrial membrane potential (ΔΨm), matrix NAD(P)H content, Ca2+ retention capacity and respiratory parameters were measured in heart, liver and skeletal muscle mitochondria of adolescent rats. Levels of thiobarbituric acid-reactive substances, carbonyl formation, sulfhydryl oxidation and reduced glutathione were also determined in homogenates from these tissues. Results: 3-Hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids significantly increased state 4 (resting) and markedly decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respiration, as well as ΔΨm, NAD(P)H content and Ca2+ retention capacity in all examined tissues. Furthermore, these effects were totally prevented by cyclosporine A and ADP, as well as by ruthenium red, indicating the involvement of mitochondrial permeability transition (mPT) and of Ca2+. Noteworthy, the dicarboxylic analogue of 3HTA did not alter any of the tested parameters, pointing out to a selective effect of the monocarboxylic LCHFA. However, 3HPA and 3HTA were unable to alter all tested parameters of redox homeostasis. Discussion: Our data strongly indicate that 3HTA and 3HPA behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and mPT inducers. It is therefore presumed that disruption of energy homeostasis elicited by the major accumulating metabolites in LCHAD deficiency may contribute to the tissue alterations and symptoms presented by patients affected by this disease. Financial support: CNPq, PROPESq/UFRGS, FAPERGS, FINEP IBN-Net and INCT-EN.

1

Div Metab Dis, Univ Child Hosp, Heidelberg, Germany, 2Div Metab Dis, Univ Child Hosp, Hamburg, Germany, 3Cardiology, Univ Child Hosp, Hamburg, Germany, 4Cardiology, Univ Child Hosp, Heidelberg, Germany Background: Cardiomyopathy is a frequent complication of LCHADD but cardiac function remains stable in the majority of early treated patients diagnosed by newborn screening and aggressive management of metabolic decompensations. Case report: We report on two female LCHADD patients: patient 1 age 3 and patient 2 age 15 y, homozygous for the common c.1528G > C mutation in HADHA. Both had normal echocardiography prior to a minor infection with moderate metabolic decompensation (CK max. 30,000 and 23,000 U/l,

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Interim results from an open-label phase 2 study assessing the safety and clinical effects of investigational UX007 (triheptanoin) in subjects with long-chain fatty acid oxidation disorders (LC-FAOD) Vockley J 1, Burton B 2, Berry G T 3, Longo N 4, Phillips J 5, Sanchez-Valle A 6 , Tanpaiboon P 7, Grunewald S 8, Murphy E 9, Bowden A 10, Zhang L 10, Cataldo J 10, Marsden D 10, Kakkis E 10

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1

Univ of Pittsburgh, Pittsburgh, United States, 2Ann Robert H Lurie Child Hosp Chicago, Chicago, United States, 3Boston Children Hospital, Boston, United States, 4University of Utah, Salt Lake City, United States, 5Vanderbilt Univ Medical Center, Nashville, United States, 6USF Health, Morsani College of Medicine, Tampa, United States, 7Children’s National Medical Center, Washington, DC, United States, 8Great Ormond St UCL Instit Child Health, London, United Kingdom, 9National Hosp for Neuro and Neurosurgery, London, United Kingdom, 10Ultragenyx Pharmaceutical Inc, Novato, United States

contrast to 2/19 NBS patients. Neurological examination during regular clinical visits was abnormal in 4/17 pre-NBS patients and in 1 NBS patient. Discussion: The clinical outcome in patients diagnosed through NBS appears to be favourable compared to the outcome in those diagnosed pre-NBS. This could be an effect of early start of treatment, but may also be due to detection of mutations with a milder natural disease course. To better understand the clinical spectrum of patients picked up through NBS and to determine the effect of different treatment strategies, long-term follow-up is needed.

Background: LC-FAOD are a group of autosomal recessive disorders that lead to an inability to convert long-chain fatty acids into energy, causing serious liver, muscle, and heart disease. Methods: This single-arm, open-label phase 2 study evaluated 29 patients (10 months to 58 years) with LC-FAOD demonstrating severe ongoing disease despite standard-of-care. 25 patients completed the study. Most patients qualified based on severe musculoskeletal disease. After a 4-week run-in on current regimen (27 of 29 patients on MCT oil), UX007 (triheptanoin), a medium oddchain fatty acid, was titrated to a target dose of 25–35 % of total daily caloric intake. Patients were evaluated over 24 weeks on several endpoints, including cycle ergometry, 12-min walk test (12MWT), and quality of life (QoL). Patients only performed assessments appropriate for their age and clinical status. Results: At week 24 cycle ergometry testing, 7 age-and condition-eligible patients showed a mean 60 % (+446.8 watts) increase (min, max: −388, +2438) over baseline (+744.6 watts). At week 18 for 12MWT, eight eligible patients showed a mean 28 % (+188 meters) increase (min, max: −80, +880) from baseline (+673.4 meters). Adult self-reported, health-related QoL (SF12v2) physical component summary scores were significantly improved, mean increase 8.9 points, supporting clinical meaningfulness of the changes in exercise tolerance tests. No difference was seen in pediatric parent-reported scores (SF-10). 18 patients (62 %) had treatment-related adverse events, predominantly gastrointestinal (55 %) and mild-to-moderate in severity. Discussion: In patients with severe LC-FAOD, treatment with UX007 showed improvements in exercise tolerance and muscle function, and were associated with positive changes in self-reported QoL. Conflict of Interest declared.

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The effect of newborn screening on clinical outcome in very-long-chain acyl-CoA dehydrogenase deficiency Bleeker J C 1 2 4, Ferdinandusse S 2, Houtkooper R H 2, Wanders R J A 2, Van der Pol W L 3, Visser G 1 1 Dept Metab Dis, Wilhelmina Child Hosp, Utrecht, Netherlands, 2Lab Genet Metab Dis, AMC, Amsterdam, Netherlands, 3 Dept Neurology and Neurosurgery, UMCU, Utrecht, Netherlands, 4Dept Metab Dis, Emma Child Hosp, AMC, Amsterdam, Netherlands

Background: With the introduction of very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD) in newborn screening (NBS) programs, the number of diagnosed patients has rapidly increased. Many infants are asymptomatic at time of diagnosis. We determined the effect of NBS on clinical outcome in VLCADD. Methods: Long term follow-up study. Dutch VLCADD patients diagnosed before (pre-NBS) and through NBS were evaluated by a standardized protocol in the Dutch Expertise Center for Fatty Acid Oxidation Disorders. Data were collected on signs and symptoms, treatment, course of the disease and motor and cognitive development. Results: Data on outcome after 9 years of NBS are presented. Of 57 registered patients (26 pre-NBS, 31 NBS), 6 died (4 pre-NBS, 2 NBS). Of the living patients, 36 were seen during regular follow-up (17 pre-NBS, 19 NBS). Hypoglycemia was detected in 10/17 pre-NBS patients with a median age of 10 months (0–2 years). One NBS patient developed hypoglycemia shortly after birth. Cardiomyopathy was present in 3/17 pre-NBS patients and in none of the NBS patients. Myopathy was present in 15/17 pre-NBS patients, in

Expanded newborn screening for VLCAD deficiency (VLCADD): four years’ experience Merinero B 1, Alcaide P 1, Morais A 2, Garcia-Silva M T 3, Martin Hernandez E 3, Quijada P 3, Pedron C 4, Dulin E 6, Yahyaoui R 7, Egea J M 9, Belanger A 5 , Blasco J 8, Ferrer-Lopez I 1, Leal F 1, Ugarte M 1, Ruiz-Sala P 1, Perez B 1, Perez-Cerda C 1 1 CEDEM, Univ Auton Madrid, CIBERER,Idipaz, Madrid, Spain, 2U Nutric Inf Enfer Metab, Hosp La Paz, Madrid, Spain, 3U Pediatr Enfer Raras, Hosp Doce Oct, Madrid, Spain, 4Sec Gastroenterol Nutric,Hosp Nino Jesus, Madrid, Spain, 5Serv Pediatr, Hosp Ramon y Cajal, Madrid, Spain, 6Lab crib neonat, Hosp Gregorio Maranon, Madrid, Spain, 7Lab Metabolop, Hosp Regional Malaga, Malaga, Spain, 8Sec Gastroenterol Nutric,Hosp Reg Malaga, Malaga, Spain, 9Lab Metabolop, CBGC, Hosp V Arrixaca, Murcia, Spain

Background: Identification of VLCADD is possible in the expanded newborn screening (NBS) due to the increase of tetradecenoylcarnitine (C14:1) and the ratios C14:1/C2, C14:1/C16, C14:1/C12:1 in dried blood spots. Although the detection of this disease is not mandatory in Spain, it is included in some regions. Methods: Revision of the clinical, biochemical and/or molecular findings of 52 cases with suspected VLCADD detected in 619.906 screened newborns in the last 4 years. Results: Confirmatory testing included analysis of plasma acylcarnitines in all cases, determination of dehydrogenase activity toward palmitoyl-CoA in lymphocytes by MS/MS and/or ACADVL gene analysis in selected cases. Twelve true-positive cases were identified by increased C14:1 and ratios, a deficient VLCAD activity ( A mutation) and method of diagnosis (NBS versus clinical) using student’s t-test. Results. MCAD deficiency in Utah was more frequent than the national average (1:7,738 versus 1:17,759). Children with MCAD deficiency had normal growth. c.985G > A homozygotes had higher NBS C8-carnitine (23.4 ± 19.6 versus 6.6 ± 3.0 μmol/L) and lifetime plasma C8-carnitine levels (6.2 ± 5 versus 3.6 ± 1.9 μmol/L) compared to compound heterozygotes (p < 0.001 for both). C8-carnitine did not change significantly with age while C2-carnitine decreased (p < 0.001), possibly reflecting reduced carnitine supplementation typically seen with age. Homozygotes had significantly higher liver transaminases than compound heterozygotes (ALT 41.9 ± 6.2 versus 31.5 ± 3.7 U/L, AST 63.9 ± 5.8 versus 45.7 ± 1.8 U/L, p < 0.05 for both). On average, homozygotes had more hypoglycemia events than compound heterozygotes (1.4 versus 0.5 events/patient) as did patients diagnosed clinically compared to those diagnosed by NBS (1.9 versus 0.8 events/ patient), though not statistically significant. Neonatal death was observed in one patient homozygous for the common mutation, but severe neonatal complications (hypoglycemia, cardiac arrhythmia) were also seen in patients with other mutations. Discussion. Homozygosity for the common ACADM mutation was associated with increased levels of C8-carnitine and transaminases. NBS provides opportunity to reduce morbidity in all patients with MCAD deficiency.

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Compensatory mechanisms in OCTN2 deficient mice, a murine model of primary carnitine deficiency (PCD)

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Hypoparathyroidism in mitochondrial trifunctional protein deficiency Diodato D 2, Taurisano R 2, Maiorana A 2, Boenzi S 2, Rizzo C 2, Bellacchio E , Semeraro M 2, Carducci C A 1, Leuzzi V 1, Bertini E 2, Dionisi-Vici C 2

2

1 Child Neurol Div, Univ La Sapienza, Rome, Italy, 2Div Metab Dis, Bambino Gesu Child Hosp, Rome, Italy

Background: Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed by 4 alfa subunits, encoded by HADHA and retaining the LCEH and LCHAD activities, and four beta subunits encoded by HADHB, retaining LKAT activity. Among FAO defects, MTP presents with unique clinical features which include peripheral neuropathy, pigmentary retinopathy and, more rarely, hypoparathyroidism (HT). To date HT has been reported only in 5 MTP patients. Case report: Here we report on two MTP patients with HT. P1, the first reported MTP patient with HT (Dionisi-Vici et al., 1996), is now a 24 year-old woman with intermittent myoglobinuria and severe sensory-motor peripheral neuropathy; P2 is a 1 year old baby diagnosed through NBS presenting with recurrent episodes of rhabdomyolysis. Both showed hypocalcemia, hypophosphatemia, low PTH levels and were treated with vitamin D3 and Ca supplement. Results: Genetic analysis of HADHB gene revealed the homozygous change c.1165A > G (p.N389D) in P1, and compound heterozygous c.206C > T (p.T69I)/c.1280G > A (p.G427E) mutations in P2. Discussion: The homozygous p.N389D change, present in P1, was already reported in another MTP patient with HT (Labarthe et al., 2006). This mutation seems to alter the formation of HADHA-HADHB complexes, since it is located close to the HADHA-HADHB interface; a reduced capacity to form the hetero-complexes probably interfere also with the HADHB catalytic activity. A similar effect may also occur with the p.A392V change, described by Naiki et al. (2013) in other two MTP patients with HT. The p.T69I and p.G427E mutations present in P2, are both predicted to be deleterious, possibly affecting either the formation of HADHA-HADHB complexes or HADHB catalytic activity. All these findings might suggest a possible genotype/phenotype correlation. Although rare, HT should be carefully screened in MTP deficiency.

Mingirulli N 1, Tucci S 1, Melchionda L 1, Spiekerkoetter U 1 P-327 1

Lab Metab Dis, Univ Child Hosp, Freiburg, Germany

Background: Homeostasis of carnitine is maintained by an efficient renal reuptake via the organic cation transporter 2 (SLC22A5). Hereditary OCTN2 deficiency (PCD) leads to a severe renal loss of carnitine with subsequently impaired mitochondrial fatty acid ß-oxidation (FAO). The clinical spectrum of PCD is very wide even within siblings carrying the same mutations. We aim to investigate metabolic adaptation mechanisms that might explain the heterogeneity of the clinical phenotype of PCD in OCTN2 deficient mice. Methods: OCTN2−/− mice and wildtype controls (n = 10) were treated with carnitine and sacrificed at 12 weeks after terminating therapy for 2 weeks. Endogenous carnitine biosynthesis and alternative carnitine transporters were quantified in kidney and liver by qPCR. Mitochondrial biogenesis was investigated in kidney and skeletal muscle at mRNA level and by westernblot analysis of OXPHOS. In skeletal muscle the expression of troponine genes encoding for glycolytic and oxidative muscle fiber-types was examined. Results: Our data show a stimulated mitochondrial biogenesis (i.e. upregulation of PPARα, PGC1α) at RNA level associated with an increase in OXPHOS in skeletal muscle and kidney. The latter presented with an upregulation of all enzymes involved in carnitine biosynthesis (i.e. GBBD) and an enhanced expression of alternative carnitine transporters (i.e. SLC22A4). In skeletal muscle an increased expression of troponine genes encoding for oxidative muscle fibers (i.e. TnniA1, TnncA1) was detected. Discussion: Carnitine biosynthesis and uptake were stimulated in the kidney to compensate for low carnitine levels. Moreover the activated mitochondrial biogenesis may represent an initial response to energy deficiency correlating to the upregulation of oxidative muscle fibers as a tissue specific effect in skeletal muscle. The observed tissue specific adaptation mechanisms might help to understand part of the great clinical heterogeneity of PCD patients.

Phenotypic variability and clinical, biochemical, histological and molecular genetic characteristics of 17 patients with multiple acyl-CoA dehydrogenase deficiency Tokatli A 1, Yildiz Y 1, Talim B 2, Bilginer Gurbuz B 1, Pektas E 1, Dursun A 1, Sivri H S 1, Coskun T 1 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 2Ped Path Unit, Hacettepe Univ Child Hosp, Ankara, Turkey

Background: Defects in electron transport flavoprotein (ETF) and ETF dehydrogenase cause multiple acyl-CoA dehydrogenase deficiency (MADD). Early-onset MADD usually present in early infancy with acidosis, hypoglycemia, respiratory failure, and sometimes with congenital anomalies. Patients with late-onset may develop muscle pain, weakness, and severe rhabdomyolysis. Here, we present 17 patients with MADD, some exhibiting diagnostic and therapeutic challenges. Methods: Patients diagnosed with MADD in our clinic were retrospectively analysed from hospital records. Results: 17 patients were diagnosed with MADD. Age at onset (antenatal– 23 years) was variable. Five had neonatal onset, presenting with respiratory distress, vomiting or feeding difficulties, and none of whom responded to riboflavin. One patient with congenital anomalies died on the eighth day postpartum. The remaining 12 patients presented later with muscle pain, fatigue and weakness; all were riboflavin-responsive. One was admitted to ICU due to rhabdomyolysis. Genetic analyses were performed in nine patients with lateonset disease, all revealing homozygous mutations in ETFDH. In four patients, initial acylcarnitine analysis was unremarkable except for carnitine deficiency, necessitating carnitine supplementation to elicit the diagnostic profile. Six

S154 muscle and three liver biopsies were performed, showing lipid storage myopathy and diffuse macrovesicular hepatosteatosis. Discussion: Late-onset MADD is treatable, and therefore important to diagnose. Acylcarnitine and organic acid analyses are non-invasive and can obviate the need for biopsy. Short-term carnitine supplementation should be considered if results are non-diagnostic and MADD is suspected. Riboflavin dramatically ameliorates but usually cannot completely reverse symptoms in this subgroup. Early-onset form is a severe disease whose survivors may experience recurrent decompensations.

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Mitochondrial bioenergetics disturbance and increased superoxide production in very-long-chain acyl-CoA dehydrogenase deficient fibroblasts Seminotti B 1, Mohsen A W 1, Karunanidhi A 1, Roginskaya V Y 2, Van Houten B 2, Wipf P 3, Vockley J 1 1

Div Med Genet, Dept Pediat, Univ Pitt, Pittsburgh, United States, 2Dept Pharmacol Chem Biol, Univ Pitt, Pittsburgh, United States, 3Dept Chem, Univ Pitt, Pittsburgh, United States Background: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is considered the most common defect of the mitochondrial fatty acid βoxidation (FAO) of long-chain fatty acids. Patients present with heterogeneous clinical phenotypes, and the common findings include hepatomegaly, cardiomyopathy and hypoketotic hypoglycemia, frequently induced by prolonged fasting and infectious illnesses. Skeletal myopathy associated with rhabdomyolysis may also be precipitated. However, cellular pathophysiology leading to these problems remains unclear. Methods: VLCAD deficient fibroblasts were cultured in medium without glucose for 48–72 h to shift cellular metabolism to fatty acids as their energy source. We evaluated oxygen consumption rate, ATP production, mitochondrial mass, superoxide production, cellular viability and the immunocontent of proteins involved in FAO and mitochondrial function. Results: VLCAD deficient cells showed decreased basal respiration, reserve capacity and ATP production. Superoxide production was increased in VLCAD deficient fibroblasts, which was prevented by JP4039, a novel mitochondrially targeted free radical scavenger, but not by bezafibrate or N-acetylcysteine. We also verified that the mitochondrial mass was significantly increased in the cells cultured in the absence of glucose. Finally, we observed absence of VLCAD antigen, increased VDAC1 and MFN1 antigens, and a decrease of the complex I subunit ND6 antigen, but no changes in CPTII and Hsp60 antigen signal in VLCAD deficient fibroblasts. Discussion: Our findings identify increased superoxide production and decreased respiratory chain function in VLCAD deficient fibroblasts, indicating that the energy metabolism dysfunction in VLCAD deficiency exceeds that of FAO alone. We also verified increased mitochondrial mass and altered protein expression in the fibroblasts. These novel pathophysiological findings should be considered in devising new therapeutic strategies.

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Clinical and genetic aspects of 50 Japanese cases of VLCADD Yamaguchi S 1, Hasegawa Y 1, Furui M 1, Yamada K 1, Bo R 1, Kobayashi H 1, Taketani T 1, Fukuda S 1, Fukao T 2, Nishino I 3 1 Dept Ped, Shimane Univ Sch Med, Izumo, Japan, 2Dept Ped, Gifu Univ Grad Sch, Gifu, Japan, 3Dept NeurMus Res, Nat Inst Neurosci, NCNP, Tokyo, Japan

Background: Very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a disorder of long-chain fatty acid oxidation cycle. Recently, an increasing number of patients have been detected with spread of expanded mass newborn

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 screening (NBS). VLCADD is clinically divided into 3 groups: 1) severe form (early onset) with high mortality; 2) intermediate form (child onset) with hypoglycemia and lower mortality; and 3) myopathic form (adult onset) with muscular symptoms. We investigated clinical and genetic aspects of Japanese cases with VLCADD. Methods: 50 Japanese cases from 48 families with VLCADD, confirmed by gene analysis of ACADVL, were studied. Mutation analysis was done by PCR-amplification of all exons, and direct sequencing. Results: Clinical presentation: 1) Severe form was seen in 6 cases, whose age at onset ranged from 1 d to 4 m after birth; 2) intermediate form was detected in 4 cases, whose ages ranged from 1y,4m to 5 y; 3) myopathic form was seen in 30 cases whose onset ages ranged from 8 m to 29 y. Furthermore, 4) 10 asymptomatic cases were detected by NBS. In this study, the myopathic and asymptomatic cases were combined into “mild” form. Gene mutations were identified in 98 of 100 alleles. The c.790A > G mutation was commonly identified on 16 alleles (16.3 %) exclusively in the mild group. The c.1144A > G was identified in 11 alleles (11.2 %) in the intermediate and mild forms. The c.996_997insT was detected in 10 alleles (10.2 %) in all clinical types. Null mutations were identified in 6 of 12 (50 %), 4 of 8 (50 %), and 11 of 58 (19 %) alleles, in the severe, intermediate, and mild forms, respectively. Discussion: In the previous report of 54 cases by Andresen et al., 25 (46 %) and 21 (39 %) were the severe and intermediate forms, respectively. In contrast, 40 (80 %) of Japanese were the mild form. Only 2 mutations, c.553G > A, and c.1349G > A were identified in both populations. In conclusion, the clinical and genetic aspects are different between Japanese and Caucasian.

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An c.IVS9-9 T > A substitution identified in beta-ketothiolase deficient patients results in exon 10 skipping in most transcripts of ACAT1 gene Fukao T 1 2, Sasai H 1, Otsuka H 1, Aoyama Y 3, Elsayed A 1, Nakama M 2, Hori T 1, Ohnishi H 1, Turner L 5, Sweetman L 4 1 Dept of Pediatr, Gifu Univ, Gifu, Japan, 2Div of Clin Genet, Gifu Univ Hosp, Gifu, Japan, 3Dept of Biomed Sci, Chubu Univ, Kasugai, Japan, 4Inst Metab Dis, Baylor Res Inst, Dallas, United States, 5Memorial Univ of Newfoundland, St John’s, Canada

Background: Mitochondrial acetoacetyl-CoA thiolase (T2; gene symbol ACAT1) deficiency is an autosomal recessive disorder affecting the isoleucine catabolic pathway and ketone body utilization, commonly known as betaketothiolase deficiency. Since 1971, more than 100 patients have been identified. We have identified more than 70 gene mutations, 15 % of which cause aberrant splicing. Case and methods: GK03 is a T2-deficient patient who was previously reported in 1986. Previous analysis showed a decreased amount (less than 10 % control) of T2 mRNA and routine cDNA analysis could not find any mutations at that time. Molecular basis of this patient has not been solved for long time. Genomic PCR-amplified fragment around exon 10 showed a homozygous T to A substitution (c.IVS9-9 T > A) in polypyrimidine tract at splice acceptor site of intron 9. No other mutations were identified. First we were not sure whether this substitution was disease-causing mutation or not. We recently identified another T2-deficient patient with the same mutation. We, hence, hypothesized that c.IVS9-9 T > A mutation caused exon 10 skipping. We performed cDNA analysis using fibroblasts cultured with cycloheximide (CHX). We made wild-type and mutant mini-gene splicing construct and performed a mini-gene splicing experiment. Results: cDNA analysis: When nonsense mediated mRNA decay (NMD) was suppressed by treating fibroblasts with CHX, the smaller cDNA was predominantly amplified in this patient. Sequencing revealed the presence of exon 10 skipping. Mini-gene splicing experiment: Wild-type gave no transcripts with exon 10 skipping but c.IVS9-9 T > A mutant gave transcripts with exon 10 skipping. Discussion: There are few reports that single nucleotide substitutions in polypyrimidine tract of splice acceptor site are proved to cause aberrant splicing. We showed that c.IVS9-9 T > A induces aberrant splicing in ACAT1 gene.

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Galimberti C 1, Gasperini S 1, Brambilla A 1, Pasetti M 1, Tursi S 1, Pretese R 1, Rigoldi M 2, Parini R 1

16 % (22/140) had developmental delay and 10 % (15/149) died. Adverse outcome was significant associated with exposure to intensive treatment (χ2 (1) = 12.78, p < .001), whereas there was no difference in the severity of the episode between those with or without adverse outcome (t(88) = .88, p = .383). Discussion: Intensive treatment is associated with adverse outcome in patients with ketone utilization disorders. Further studies are warranted to delineate cause and consequence.

1 Rare Metab Dis, Ped Dept, San Gerardo Hosp, Monza, Italy, 2Center Rare Dis, Dept Int Med, S Gerardo H, Monza, Italy

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Fatty acid oxidation (VLCADD) defect presenting with ketonuria: two case reports

Background: Very-long-chain acyl-CoA-dehydrogenase deficiency (VLCADD) is the second commonest fatty acid oxidation disorder. It may present early in life or later, precipitated by an infection or prolonged fasting, commonly with encephalopathy, hypo- or a-ketotic hypoglycemia, hypertrophic cardiomyopathy and liver failure. Here we describe two patients affected with VLCADD who presented severe hypoglycemia and massive ketonuria. Case report and results: The first patient is the third child of consanguineous parents; diagnosis of VLCADD was made in neonatal period for severe encephalopathy and hypoglycemia, confirmed by molecular analysis. After a major surgical intervention for bowel occlusion when he was 2 years old, he showed massive ketonuria, hyperglycemia (500 mg/dl) and metabolic acidosis (pH 6.8, HCO3 2, BE −25). The second patient is a 6-years-old child of nonconsanguineous parents. He presented frequent episodes of vomiting and fever. In well-being acylcarnitine profile was normal. During one episode of vomiting he showed metabolic acidosis (pH 7.24, HCO3- 11.9 mEq/L, BE −16) and hypoglycemia (45 mg/dl) with ketonuria (++++), lactic and dicarboxylic aciduria; the acylcarnitine profile was suggestive for VLCADD. Molecular analyses are in progress. Discussion: It is recognised that oxidation of saturated fatty acids is only partially impaired in VLCADD and that fatty acid oxidation disorders could precipitate during infections in severe hypoglycemia with massive ketonuria (Wraige E. et al., Arch Dis Child 2002; 87: 428–429). The biochemical pattern of patients during decompensation is typical while the blood acylcarnitine profile could show normal results when patients are well. Plasma acylcarnitines profile showed accumulation of specific very-long-chain acylcarnitines; urinary organic acid analysis revealed lactic and dicarboxylic aciduria. In conclusion, a diagnosis of a beta-oxidation disorder should not be excluded in the presence of massive ketonuria.

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A systematic review and meta-analysis of case reports in ketoneutilization defects Molema F 1, Hasselt P M 1

Early detection of myocardial diastolic dysfunction in patients with LCHADD—a pilot study Michel M 1, Scholl-Buergi S 1, Pichler K 2, Zlamy M 1, Karall T 1, Karall D 1 1 Pediatrics, Medical University, Innsbruck, Austria, 2Pediatrics, Medical University, Vienna, Austria

Background: In patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) dilative (DCM) or hypertrophic cardiomyopathy (HCM) may occur, transiently or chronically. Myocardial diastolic dysfunction (DD) can be present even if systolic function is still sustained, and has to be considered especially in the case of volume depletion during a metabolic crisis. With the help of serum parameters and new echocardiographic methods we aimed to evaluate which parameter is most accurate for the early detection of DD. Methods: In 6 patients with LCHADD the course of plasma concentrations of N-terminal pro b-type natriuretic peptide (NT-pro-BNP), creatine kinase (CK), troponin T, lactate dehydrogenase (LDH), lactate, liver transaminases (ALT and AST) during a metabolic crisis were examined and correlated with conventional and with 2-dimensional Speckle Tracking (2DST) echocardiography parameters. Systolic function was measured by shortening fraction, ejection fraction and 2DST derived velocity and strain, and diastolic function was determined by atrioventricular inflow Doppler and early and late 2DST derived velocity. Results: HCM was prevalent in 2, DCM in 1 patients. Myocardial DD was present in all 6 patients and was accompanied by systolic dysfunction in 3 of them. DD could be detected accurately by 2DST parameters in all patients, while conventional echocardiography parameters were altered only in 4 patients. An increase in NT-pro-BNP significantly correlated with 2DST parameters indicating DD. Discussion: In patients with LCHADD DD may occur, which is detectable early by 2DST echocardiography and by increase of NT-pro-BNP values. Even when systolic function is sustained, an increase in NT-pro-BNP reliably indicates DD during metabolic crisis. By that the performance of an echocardiographic examination can be accurately timed and volume therapy can be adjusted to guarantee ideal preload in DD especially in prevalent HCM and DCM.

1

Div Metab Dis, WKZ, Utrecht, Netherlands

Background: Ketone-utilization disorders (due to deficiencies of SCOT, T2 and MCT1) are characterized by ketoacidotic bouts precipitated by catabolic stress. Information on the clinical course is limited due to the rarity of these disorders. We aimed to collect all published cases and perform a meta analysis of case reports to better delineate the expected clinical course. Methods: A systematic literature search was performed by including case reports on succinyl-CoA:3-ketocid CoA transferase deficiency (SCOT), beta-ketothiolase (T2) deficiency and monocarboxylate transporter type 1 (MCT1) deficiency, using Medline and Embase. Biochemical characteristics and prognostic parameters of each patient were derived from the included papers. A meta-analysis was performed on clinical, biochemical and adverse outcome (psychomotor developmental delay and mortality) thus retrieved. Results: A total of 149 patients with ketone utilization disorder were included, 111 with T2 deficiency, 27 with SCOT deficiency and 11 with MCT1 deficiency. Ten patients presented within 4 days after birth, the remaining presented in late infancy or early childhood (median 1.17 year). In 84 out of 262 episodes, patients presented with decreased consciousness. In 64 patients treatment was reported, 45 patients received supportive care (i.v. fluid, glucose and/ or bicarbonate) and 19 intensive treatment (mechanical ventilation or dialysis).

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Clinical, biochemical and molecular characterization of very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD) in Saudi Arabia Alsayed M 1, Edrees A 1, AlHassnan Z 1, AlZaidan H 1 1

Dept of Med Gen, KFSHRC, Riyadh, Saudi Arabia

Background: To characterize the clinical, biochemical and molecular data among Saudi patients diagnosed with VLCADD deficiency. Methods: Data on demographics, age of onset of symptoms, type of symptoms, age at diagnosis, medical management and course of illness were collected on 51 patients confirmed to have VLCADD via biochemical testing. Among those 48 were confirmed molecularly as well. Results: Severe early onset phenotype was most common (47). All were homozygous for p.S22X mutation. Age of onset of symptoms in this cohort ranged between the first day of life up to 3 months and were recurrent

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Identification of a novel mutation in Turkish infant with early-onset monocarboxylate transporter 1 (MCT1) deficiency as a cause of recurrent ketoacidosis

Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare, autosomal recessive disease caused by mutations in the TYMP gene encoding thymidine phosphorylase (TP). Clinically it is characterized by progressive external ophthalmoplegia, severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, diffuse leukoencephalopathy and evidence of mitochondrial dysfunction (histologic, biochemical, or genetic abnormalities of the mitochondria). Increased concentrations of thymidine (>3 μmol/L) and deoxyurindine (>5 μmol/L) in plasma or a decrease in buffy coat TP activity to < 8 % relative to controls is considered sufficient to diagnose MNGIE. Case report: We report 3 boys (aged20, 17 and15 years) born to first-cousin parents, who presented with cachexia. Gastrointestinal symptoms in the form of recurrent diarrhoea were present in all of them. The 17-year-old had the most severe symptoms, had sensorineural hearing impairment, severe peripheral axonal neuropathy and symmetric and confluent T2-hyperintensity in the white matter of the semicentumoval on MRI brain. The 20-year-old and 15year-old did not reveal evidence of sensorineural hearing impairment. Results: Plasma thymidine and deoxyuridine were elevated (Patient 1: 13 μmol/L and 16 μmol/L, Patient 2: 14 μmol/L and 25 μmol/L, Patient 3: 15 μmol/L and 27 μmol/L, respectively). A novel homozygous mutation c.798_801dupCGCG (p.Ala268Argfs) in exon 7 of TYMP gene was detected. Discussion: MNGIE is a multisystem disease with a recognizable clinical phenotype, but is often misdiagnosed in the early course of the disease, which may result in the missed opportunity for an allogeneic hematopoietic stem cell transplant. MNGIE disease should be considered in patients presenting with gastrointestinal symptoms and cachexia and should be evaluated for neuropathy, sensorineural hearing impairment and white matter changes on the MRI brain.

Okur I 1, Inci A 1, Keles E 1, Karaoglu A 1, Ceylaner S 2, Biberoglu G 1, Ezgu F S 1, Tumer L 1

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hypoglycemia, lethargy, vomiting, diarrhea, respiratory distress, elevated liver enzymes and creatine kinase and mild hepatomegaly in some patients. 15 have died of various causes. The surviving 32 patients, treated with carnitine and dietary modification using low fat-high carbohydrate diet, medium chain triglycerides (MCT) based formulas and diet, reported recurrent symptoms of feeding intolerance, lethargy, vomiting and diarrhea requiring frequent hospital admission but with full recovery. Positive correlation existed between reduced compliance with dietary modifications and number of metabolic crises. The frequency of symptoms improved dramatically as they grew older. Currently 30 patients out of 32 have normal cardiac function. Retrospective historical data revealed that none of the untreated siblings (54) of this cohort have survived. Four patients had later-onset presentation (3 to 12 yrs) with recurrent rhabdomyolysis triggered by exercise or fasting. They had no cardiac or hepatic manifestations. Discussion: VLCADD is very common among Saudis due to the founder mutation p.S22X. This mutation presents with the severe early onset phenotype. Early management with dietary modification improves survival and preserves cardiac function. Metabolic crisis do continue to occur partially due to decreased compliance and not all patients respond to treatment. Therefore optimization of dietary management is essential.

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1 Dept Ped Metab Nutr, Gazi Univ Hosp, Ankara, Turkey, 2Intergene Gen Diag Cent, Ankara, Turkey

Background: Monocarboxylate transporter 1 (MCT1) deficiency was identified as a novel cause of profound ketoacidosis. MCT-1 deficiency causes episodic ketoacidotic crises and no obvious symptoms between them. We report a Turkish case of early infancy-onset MCT1 deficiency. Case report: The female patient presented a severe ketoacidotic crisis, with blood pH of 6.9 and bicarbonate of 6 mmol/L at the age of 5 months and was successfully treated with intravenous infusion of glucose and sodium bicarbonate. Results: She was diagnosed as MCT1 deficient by enzymatic assay and mutation analysis. At the age of 6 months, she developed a second ketoacidotic crisis, with blood pH of 7.07, bicarbonate of 8.1 mmol/L, and showed 3+ (ketostix). She experienced two milder ketoacidotic crises at the age of 10 months and 11 months. Her urinary ketone bodies usually showed 3+ (ketostix). Hence, this patient does not show permanent ketonuria. She is homozygous for the p.V319. (c.955 _955delG) mutation in the SLC16A1 gene. The p.V319 mutation was not previously reported. The newly found novel mutation was revealed to lead a early stop codon. Discussion: MCT1 (SLC16A1) deficiency, a defect of ketone body utilization, that has only recently been identified (van Hasselt et al., N Engl J Med, 371:1900–1907, 2014) should be considered as a cause for recurrent ketoacidoses.

14. Mitochondrial disorders: nuclear encoded, disorders of pyruvate metabolism and the Krebs cycle

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A novel mutation in three siblings with MNGIE disease from Pakistan Afroze B 1, Chen B C 2, Yusnita Y 3 1 Dept Paed and Child Health Aga Khan Uni, Karachi, Pakistan, 2Unit Bioch Gen, Kuala Lumpur Hosp, Kuala Lumpur, Malaysia, 3Unit Mol Diag and Protein, Inst Med Res, Kuala Lumpur, Malaysia

Clinical, molecular, radiological investigations in patients with SURF1 mutations and muscle biopsy findings Kose M 1, Kagnici M 1, Canda E 1, Eraslan C 3, Diniz G 4, Akinci G 2, Unalp A 2 , Yilmaz U 2, Ceylaner S 7, Kalkan Ucar S 5, Taylor R 6, Coker M 5 1 Div Metab Dis, Behcet Uz Child Hosp, IZMIR, Turkey, 2Div Ped Neurology, Behcet Uz Child Hosp, IZMIR, Turkey, 3Div Neuroradiology, Ege Univ Med Fac, IZMIR, Turkey, 4Div Pathology, Tepecik Hosp, IZMIR, Turkey, 5Div Metab Dis, Ege Univ Med Fac, IZMIR, Turkey, 6Mitochondrial Pathology, Newcastle Univ, Newcastle, United Kingdom, 7Intergen, Genetic Diagnosis Centre, Ankara, Turkey

Background: Leigh syndrome (LS) is a mitochondrial disease that typically presents in infancy with subacute neurodegenerative encephalopathy. It is genetically heterogeneous, but mutations in the complex IV assembly genes, particularly SURF1, are an important cause. In this study, SURF1 gene was sequenced in patients with clinical suspicion of LS, complex IV deficiency, or clinical features of mitochondrial disorders Methods: The study included seven patients with SURF1 mutations identified from a study of 21 children with Leigh syndrome findings in magnetic resonance imaging (MRI) who had undergone muscle biopsy. Diagnosis was based on both the clinical presentation and the characteristic neuropathologic findings of bilateral symmetric necrotizing lesions in the basal ganglia and brain stem as detected using MRI. Results: The patients had various forms of metabolic encephalomyopathy. All of the patients had the typical neuroradiological features of Leigh syndrome, including symmetrical necrotizing lesions scattered within the basal ganglia, thalamus and brain stem. Five patients had severe neuromotor retardation and epileptic encephalopathy. The SURF1 analysis identified previously reported mutations in all the patients. Diffuse decreased activity or total deficit of COX was revealed histochemically in all examined muscles (6 of 7 patients). Discussion: Patients with Leigh syndrome and SURF1 mutation often have skin and hair abnormalities. Bilateral symmetrical hypertrophic olivary degeneration could be a specific clue for SURF1 deficiency. Histological and histochemical features of muscle of genetically homogenous SURF1-deficient LS were reproducible in detection of COX deficit. It should be kept in mind that muscle biopsy changes are not specific if COX staining is not employed.

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Improving the diagnosis of leucoencephalopathy with brain stem and spinal cord involvement and lactate elevation caused by mutations in DARS2 gene Mendes M I 1, Smith D E 1, Licht L 1, Struys E A 1, Wolf N I 2, Van der Knaap M J 2, Salomons G S 1 Metabolic Unit, VU Univ Med Center, Amsterdam, Netherlands, 2Dept of Child Neurol, VU Univ Med Center, Amsterdam, Netherlands 1

Background: Leucoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is caused by mutations in the nuclear encoded mitochondrial aspartyl tRNA synthetase (DARS2) gene. Clinically LBSL shows slowly progressing signs of pyramidal, cerebellar and dorsal column dysfunction. Diagnosis is based on clinical grounds and a specific MRI pattern, but needs genetic confirmation due to clinical heterogeneity. It is the most common aminoacyltRNA synthetase deficiency. About forty mutations have been identified. Here we characterized six novel variants. Methods: Wild-type (WT) DARS2 cDNA was cloned into the pCMV6 expression vector. The variants of interest were introduced by site-directed mutagenesis. The WT was used as positive control. Two previously described missense variants and mock transfected cells were used as negative controls. HEK293 cells were used to express DARS2 variants. Cell homogenates (48 h after transfection) were incubated with E. coli tRNA and [13C415N] aspartic acid (Asp) for 30 min. Aspartyl-tRNA isolation was optimized. A novel cleanup approach based on affinity was used. Labeled Asp was released and quantified by LC-MS/MS. To ensure proper localization of DARS2, mitochondria were isolated from cells transfected with WT DARS2 and subsequently a Western Blot was performed. Results: Four novel variants presented with a severely decreased or undetected activity. Two variants presented with about 50 % of the WT activity. Both variants used as negative control presented with no activity, validating our method. DARS2 was properly located in mitochondria. Discussion: We developed a fast and reliable method to characterize DARS2 variants which is important not only for the confirmation of the diagnosis but also for the interpretation of next generation sequence data.

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A prospective evaluation of whole-exome sequencing as the most effective diagnostic strategy in children with suspected mitochondrial disorders Krajewska-Walasek M 7, Piekutowska-Abramczuk D 7, Ciara E 7, Trubicka J 7 , Rokicki D 1, Karkucinska-Wieckowska A 2, Pajdowska M 3, Jurkiewicz E 4, Halat P 7, Kosinska J 5, Pollak A 6, Rydzanicz M 5, Stawinski P 6, Pronicki M 2 , Ploski R 5, Pronicka E 1 7 1 Dept of Paed Nutr Metab Dis, CMHI, Warsaw, Poland, 2Dept Pathology, CMHI, Warsaw, Poland, 3Dept Bio Exper Med, CMHI, Warsaw, Poland, 4 Dept Radiology, CMHI, Warsaw, Poland, 5Dept Med Genet, Wars Med Univ, Warsaw, Poland, 6Dept Genet, Inst Phys Pathol Hearing, Nadarzyn, Poland, 7Dept Med Genet, CMHI, Warsaw, Poland

Background: Whole-exome sequencing (WES) is revolutionizing diagnostic screening for mitochondrial disorders (MD), particularly those caused by nuclear gene defects. Methods: We performed WES in 113 suspected MD patients from a Polish paediatric reference centre, in whom routine testing failed to identify a molecular defect. Results: Causative mutations were identified in 64 patients; these included variants in mtDNA (6 patients) and nDNA: X-linked (9 patients), autosomal dominant (5 patients), and autosomal recessive (44 patients, 10 homozygotes). Novel variants accounted for 57 % of all

detected changes. In 45 patients, changes in 29 MD-related genes (ACAD9, ADCK3, AIFM1, CLPB, COX10, DLD, EARS2, FBXL4, MTATP6, MTFMT, MTND1, MTND3, MTND5, NDUFS6, NDUFS7, NDUFV1, OPA1, PARS2, PC, PDHA1, POLG, RARS2, RRM2B, SCO2, SERAC1, SLC19A3, SLC25A12, TAZ, VARS2) were found. The genes ACAD9, CLPB, FBXL4, PDHA1 recurred more than twice, suggesting higher general/ethnic prevalence. The percentage of positive WES results rose gradually with increasing probability of MD according to the Mitochondrial Disease Criteria (MDC) scale (from 36 to 90 % for low and high probability, respectively). The percentage of detected MD-related genes compared with non MD-related genes also grew with increasing MD likelihood (from 20 to 97 %). Molecular diagnosis was established in 28/47 neonates and in 18/27 patients with basal ganglia involvement. Mutations in CLPB, SERAC1, TAZ were identified in neonates with 3-MGA aciduria as a discriminative feature. Discussion: Our data show that WES improves diagnostic accuracy for MD in children, including neonates with 3-MGA aciduria, who died without determination of disease cause and with limited availability of laboratory data. There is a strong correlation between the degree of MD diagnosis by WES and MD likelihood expressed by the MDC scale. The study was supported by NCN Projects: Harmonia 4 No. UMO-2013/08/ M/NZ5/00978; 2012/05/B/NZ2/01627.

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Mitochondrial infantile liver disease due to TRMU gene mutations: two cases with different outcome Cimbalistiene L 1, Burnyte B 1, Songailiene J 1, Urbonas V 2, Grabhorn E 3, Hempel M 4, Santer R 3, Haack T 5, Prokisch H 5 1 Dept of Hum Med Gen, Center for Med Gene, Vilnius, Lithuania, 2Clin of Childr Dis, Vilnius University, Vilnius, Lithuania, 3Dept Pediatr, Univ Med Center Eppendorf, Hamburg, Germany, 4Inst Hum Gen, Univ Med Center Eppendorf, Hamburg, Germany, 5Inst Hum Genet, Tech Univ, Munich, Germany

Background: The nuclear TRMU gene encodes a mitochondrial protein, 5methylaminomethyl-2-thiouridylate methyltransferase, which plays a role in the modification of mitochondrial tRNA and thus is important for mitochondrial translation. Case report: We report two unrelated patients with infantile onset acute liver disease and homozygosity for TRMU mutations identified by whole-exome sequencing (WES). Case 1: The female newborn was found through newborn screening with a blood phenylalanine concentration of 460 μmol/l. Repeated Phe and Tyr analyses in blood were normal. At 2 months of age, she presented with faltering growth, vomiting, and poor feeding. Her liver function rapidly deteriorated, with progressive hepatomegaly and cholestasis. At 10 months, she underwent liver transplantation because of progressive liver failure. No serious complications have been observed during 2 years of follow-up and her regular liver tests are normal. Case 2: The previously healthy 3-month-old girl developed rapidly progressing liver disease with mild hypotonia. She recovered spontaneously after 5 weeks and is now 11 months old with normal psychomotor developmental. Regular hepatobiliary ultrasound exams are normal. Common findings in both cases were progressive cholestatic liver failure, hypoglycaemia, lactic acidaemia, elevated plasma alanine and serum ferritin concentrations. Results and discussion: In conclusion, our cases are in line with literature data and confirm that TRMU mutations should be considered in infantile-onset liver disease. Interestingly, the outcome in these patients, both homozygous for the TRMU mutation c.835G > A / p.(Val279Met), previously reported by Zeharia in 2009, was different. The long-term outcome of liver failure in infants with TRMU mutations is yet unclear. Therefore, our cases highlight the need for longterm follow-up of these patients.

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Methods: We used streptozotocin (STZ) to induce diabetes in mice with beta cell-specific deletion of T1D candidate protein tyrosine phosphatase N2 (Ptpn2-βKO). Metabolic studies were combined with proteomic analysis and RNA-sequencing in isolated islets and beta cell lines. Results: Proteomic analysis in beta cells identified PKM2 as a PTPN2interacting factor. PKM2 is a metabolic enzyme that normally resides in the cytosol. In response to STZ, cytosolic PKM2 translocates to the nuclei of diabetic beta cells. Interestingly, PTPN2-deficient beta cells hyperaccumulate PKM2 in the nuclei, suggesting that PTPN2 mediates the nuclear export of PKM2 in stressed beta cells. Moreover, abnormal nuclear accumulation of PKM2 drives the transcriptional activation of key proapototic genes, resulting in the higher susceptibility to STZ-induced diabetes and poorer survival for the animals deficient in beta cell PTPN2. Discussion: The data support the view that loss of PKM2 drives beta cell dysfunction through abnormal metabolic reprogramming that has been shown in other cellular contexts to promote tumorigenesis. The findings reveal a novel mechanism of glycolytic protein PKM2 in a non-proliferating cell-type and suggest that beta cell loss in vivo could be targeted by antitumor therapeutic strategies that promote PKM2 nuclear export.

The fetal and neonatal presentation of CLPB deficiency — a study in 29 patients Pronicka E 1 2, Trubicka J 1, Piekutowska-Abramczuk D 1, Rokicki D 2, Ostergaard E 3 , Saunders C 4 , Van Karnebeek C 5 , Yaplito-Lee J 6 , Distelmaier F 11, Ounap K 7, Rahmans S 8, Mayr J A 10, Iwanicka-Pronicka K 9, Ciara E 1, Wortmann S B 10 1 Dept of Med Gen, CMHI, Warsaw, Poland, 2Dept of Ped, CMHI, Warsaw, Poland, 3Dept of Clin Gen, Univ Hosp, Copenhagen, Denmark, 4Centre for Ped Genomic Med, Child Hosp, Kansas City, United States, 5Dept of Ped, Univ Child Hosp, Vancouver, Canada, 6Dept of Metab Med, Univ Child Hosp, Melbourne, Australia, 7Dept of Genet, Univ Hosp, Tartu, Estonia, 8Inst of Child Health, UCL, London, United Kingdom, 9Dept of Ped Audiol, CMHI, Warsaw, Poland, 10Dept of Ped, SALK and PMU, Salzburg, Austria, 11Dept of Ped, Univ Child Hosp, Dusseldorf, Germany

Background: Recently, CLPB deficiency has been shown to cause a genetic syndrome with cataracts, neutropenia, and 3-methylglutaconic aciduria. Surprisingly, the neurologic presentation ranges from completely unaffected to patients with virtual absence of development. Muscular hypo- and hypertonia, movement disorder and progressive brain atrophy are frequently reported. Methods: We present four new patients and evaluate the fetal, peri- and neonatal features of 29 patients using a newly developed clinical severity scoring system rating clinical, metabolic, imaging and other findings weighted by the age of onset. Our data are illustrated by fetal and neonatal videos. Results: The patients were classified as having a mild (n = 4), moderate (n = 12) or severe (n = 13) disease phenotype. The most striking feature of the severe subtype was the neonatal absence of voluntary movements in combination with ventilator dependency and hyperexcitability. The fetal and neonatal presentation mirrored the course of disease with respect to survival (current median age 17.5 years in the mild group, median age of death 45 days in the severe group), severity and age of onset of all findings evaluated. Discussion: 1) CLPB deficiency should be considered in neonates with absence of voluntary movements, respiratory insufficiency and swallowing problems, especially if associated with 3-methylglutaconic aciduria, neutropenia and cataracts. 2) Being an important differential diagnosis of hyperekplexia, we advise to perform urinary organic acid analysis, blood cell counts and ophthalmological examination in these patients. 3) The neonatal presentation of CLPB deficiency predicts the course of disease in later life, which is extremely important for counselling.

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Pyruvate kinase as a novel metabolic regulator of beta cell loss in diabetes Kim Y 1, Zee T 1, Karsenty G 1, Sussel L 1 1

Columbia University Medical Center, New York, United States

Background: Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by the inflammation of the insulin-producing pancreatic beta cells, eventually leading to beta cell loss and the inability to maintain glucose homeostasis. Pyruvate kinase M2 (PKM2) is an alternatively spliced isoform of pyruvate kinase M that is preferentially expressed in fetal tissues and cancer cells to promote glycolytic metabolism. Unlike most differentiated tissues, pancreatic beta cells also preferentially express PKM2, but the significance of PKM2 function in metabolic reprogramming and diabetogenesis has not been studied.

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NDUFB8 mutations are a novel cause of mitochondrial complex I deficiency in a patient with basal ganglia and white matter brain changes Piekutowska-Abramczuk D 1, Matakovic L 7, Mayr J A 7, Feichtinger R G 7, Prokisch H 3 4, Jurkiewicz E 6, Trubicka J 1, Ciara E 1, Pronicki M 5, Rokicki D 2 , Ploski R 8, Krajewska-Walasek M 1, Pronicka E 1 2 1 Dept Med Genet, CMHI, Warsaw, Poland, 2Dept Ped, Nutr, Met Dis, CMHI, Warsaw, Poland, 3Inst Hum Genet, Helmholtz Zentrum Munch, Munchen, Germany, 4Inst Hum Genet, Techn Univ Munchen, Munchen, Germany, 5 Dept Pathology, CMHI, Warsaw, Poland, 6Dept Diagn Imag, CMHI, Warsaw, Poland, 7Dept Pediatr, Paracelsus Med Univ Salzb, Salzburg, Austria, 8Dept Med Genet, Warsaw Med Univers, Warsaw, Poland

Background: Mitochondrial complex I (C I; NADH:ubiquinone oxidoreductase) is the first and the largest enzyme of oxidative phosphorylation system (OXPHOS). Isolated complex I deficiency appeared the most frequently identified biochemical defect in childhood mitochondrial disease (MD), accounting for ~30 % of OXPHOS disorders. Clinical symptoms range from fatal infantile lactic acidosis to Leigh syndrome and other encephalomyopathies. To date,mutations in 24 C I subunits including all 7 mtDNA-encoded, and 17/ 38 nuclear-encoded, and at least in nine assembly factors have been identified. Methods: Clinical examination, brain MRI, biochemical and molecular analyses, including TaqMan genotyping, Sanger/exome sequencing were performed in our patient. Results: A 2-year old boy presented in his first days with hypotonia, failure to thrive, and delayed psychomotor development. From the 3rd month unconsciousness, seizures, and acute respiratory failure were observed. Progressive changes in the brain basal ganglia and white matter, severe metabolic acidosis and respiratory alkalosis directed molecular studies to MD background searching. Screening for the common POLG, SURF1, SCO2, MT-ATP6, PDHA1 mutations was negative. Spectrophotometric study showed isolated decrease in C I activity in muscle homogenate and fibroblast mitochondria. Whole exome sequencing revealed two parental inherited, likely pathogenic variants in NDUFB8 (c.227C > A and c.432C > G) resulting in missense mutations of conserved amino acids. Western blot analysis and immunohistochemical staining showed decreased complex I protein. Discussion: Recently, NDUFB8 was suggested as a vital structural component in C I formation. We conclude that NDUFB8 mutations can cause an earlyonset Leigh-like disease. Our findings expand the number of nuclear-encoded subunits related to mitochondrial pathology.

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Two sibling cases of aspartate-glutamate carrier deficiency: identifying increased 2-hydrobutyrate and decreased glycolate, glyoxylate and 5oxoproline in CSF Yano S 1, Saitsu H 2, Partikian A 3, Moseley K 1, Bluml S 4, Watanabe Y 5, Matsumoto N 2 1 Genet, Ped, Univ Southern Cal, Los Angeles, United States, 2Hum Genet, Yokohama City Univ, Yokohama, Japan, 3Neuro, Ped, Univ Southern Cal, Los Angeles, United States, 4Radiol, Child Hosp LA, Univ Southern Cal, Los Angeles, United States, 5Med Genet, Ped, Kurume Univ, Kurume, Japan

Background: The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) is a component of the malate-aspartate shuttle which transfers reducing equivalents from NADH from the cytosol to the mitochondria. In humans, AGC1 is expressed in the central nervous system and the other isoform AGC2 is expressed in the liver. Mutations in AGC1 and AGC2 are known to cause hypomyelination and citrullinemia type 2 or neonatal jaundice (NICCD), respectively. AGC1 deficiency is a rare autosomal recessive condition which causes the characteristic brain MRI finding with hypomyelination as well as the MR spectroscopy (MRS) finding with a low N-acetylaspartate signal. Case report: Two Hispanic male siblings (7 y and 11 y) with intellectual disability, seizures, and the characteristic abnormal brain MRI and MRS findings have been followed since they were 9 months and 13 months of age, respectively. The younger brother has no single words and the elder has 8 single words. They can walk with support. They reportedly like high protein food and dislike sweets. Whole exome sequencing study identified a homozygous mutation in SLC25A12 [NM_003705.4:c.1193G > T, p.(Gly398Val)], confirming the diagnosis of AGC1 deficiency. Extensive biochemical genetics studies were obtained. Results: Blood and urine studies showed unremarkable results. Organic acid analysis with cerebrospinal fluid (CSF) in the younger and the elder showed abnormal results consisting of a high 2-hydroxybutyric acid (77 and 80 uM: reference range 0–50) and low glycolate (13, 17 uM: reference range 40–500), glyoxylate (0, 0 uM, reference range 20–86), and 5-oxoproline (14, 11 uM, reference range: 19–150), respectively. Discussion: AGC1 deficiency causes abnormal brain myelination, infantileonset epilepsy syndrome with global developmental delay, hypomyelination and reduced brain N-acetylaspartate. Organic acids analysis in CSF might be useful in diagnosing AGC1 deficiency due to the characteristic findings.

P-345

FARS2 mutations: from early-onset malignant hyperthermia to childhood epilepsy partialis continua and adult intellectual disability Paquay S 1, Renaldo F 2, Germanaud D 2, Perrin L 3, Tabet A C 4, Slama A 5, Auvin S 2, Mignot C 6, Schiff M 1 1 Ref Cent Metab Dis, Robert Debre Hosp, Paris, France, 2Dept Pediatr Neurol, Robert Debre Hosp, Paris, France, 3Dept Genet, Robert Debre Hosp, Paris, France, 4 Dept Cytogenet, Robert Debre Hosp, Paris, France, 5 Dept Biochemistry, Bicetre Hosp, Paris, France, 6Dept Ped Neur and Genet, Trousseau Hosp, Paris, France

Background: FARS2 encodes the mitochondrial phenylalanine-tRNA synthetase, which charges tRNA with phenylalanine in mitochondrial DNA translation. 12 patients were described with FARS2 mutations who exhibited early

Alpers-like encephalopathy or infantile onset epilepsy, global developmental delay with dysarthria and tremor, liver and eye abnormalities or spastic paraplegia. This report further characterizes the FARS2-related phenotype with marked intrafamilial variability. Case report and results: Four siblings were born from non-consanguineous parents. Patient 1 abruptly died at age 15 months in acute conditions including seizures, high fever, disseminated intravascular coagulation, high liver enzymes and rhabdomyolysis. Patient 2 is currently 22 years of age. She exhibited learning difficulties since early childhood and was diagnosed with intellectual disability. She developed epilepsy since the age 12 years and has tremor of upper extremities. Patient 3 is healthy. Patient 4 had learning and behavioural disturbances. At 6 years of age, he presented with an acute episode of hypotonia and pallor. In the following year, recurrent contact losses were noted. At age 8 years, he died from a sudden encephalitic episode with epilepsia partialis continua and severe necrotic lesions on brain MRI. Array CGH revealed a maternally inherited 70 kb deletion in FARS2. FARS2 sequencing found a novel paternally inherited heterozygous mutation, c.467 > T (p.Thr156Met). These combined FARS2 abnormalities were detected in patient 2 and are expected in early expired patient 1. Healthy patient 3 is heterozygous for the paternal mutation. Discussion: Our data further characterize FARS2 phenotype emphasizing high intrafamilial phenotypic variability. This is the third case of FARS2 disease caused by combination of a 6p25.1 deletion with a novel missense mutation, demonstrating that routine array CGH can be the first step prompting the identification of this recessive disorder.

P-346

Pathogenic mutations in FARS2 in a patient with motor regression and epilepsy as first signs, later evolving to spastic paraplegia associated with autonomic disturbances Vantroys E 1, Smet J 1, Vanlander A 1, De Paepe B 1, Vergult S 2, Sante T 2, Menten B 2, Van Coster R 1 1 Div Ped Neur, Univ Hosp Ghent, Ghent, Belgium, 2Cent Med Genet, Ghent Univ, Ghent, Belgium

Background: FARS2 encodes the mitochondrial phenylalanyl-tRNA synthetase. At the moment, 9 patients with pathogenic mutations in FARS2 were reported. The clinical phenotype associated with FARS2 mutations is either early epileptic encephalopathy, or autosomal recessive hereditary spastic paraplegia. Case report: The patient was first seen at the age of 6 months with signs of slight motor retardation. At 9 months, a motor regression was suspected, and myoclonic seizures occurred at 15 months of age. Lactate was increased in CSF and blood. The motor regression became less obvious, and his mental development was relatively well preserved. Spastic diparesis became the more dominating feature. He suffered from severe constipation. A mega-colon with generalized slow colon transit was visualized on RX images. At 3.5y, a neurogenic bladder characterized by instability and high pressure during filling and detrusor sphincter dyssynergia was diagnosed necessitating intermittent catheterization. Results: Spectrophotometric analysis of respiratory chain complex activities (complex I-IV) showed an isolated complex IV deficiency in cultured skin fibroblasts and in skeletal muscle, which was confirmed using BN-PAGE. Sequencing of the mitochondrial DNA did not reveal pathogenic mutations and whole exome sequencing was performed. Filtering of the sequencing data using a panel of known mitochondrial genes revealed two heterozygous missense variants in FARS2 (p.A154V and p.P361L), which have a low prevalence in the population. The second variant was found as likely pathogenic.

S160 Discussion: We report a subject with new mutations in FARS2 with a clinical phenotype reminiscent of previously reported patients. The epileptic encephalopathy dominated in the beginning of the disease while later on the spastic paraplegia was the dominant feature. Unique for the presented subject is that he presented also with signs of autonomic disturbance necessitating intermittent catheterization of the bladder.

P-347

A new mutation of mitochondrial DNAJC19 in a Turkish patient with 3methylglutaconic aciduria, dysmorphic feature, dilated cardiomyopathy, dystonia, anemia, male genital anomalies and deafness Kalkan-Ucar S 1, Wortmann S B 2, Mayr J A 3, Sperl W 3, Canda E 1, Coker M 1 1 Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey, 2Dept Ped Salzburger Lanseskliniken, Salzburg, Austria, 3Paracelsus Medical University Salzburg, Salzburg, Austria

Background: Dilated cardiomyopathy with atxia (DCMA) syndrome belongs to the secondary 3-methylglutaconic aciduria (3-MGA-uria) described in Hutterite patients with childhood onset dilated cardiomyopathy non-progressive cerebellar ataxia(A), testicular dysgenesis, growth failure, and 3-MGA-uria. Here, we report the third patient from non-Canadian Dariusleut Hutterite population group with a new homozygeous mutation in DNAJC19 c.63delC, p.(Tyr21*). Case report: The 29-month-old patient was present with growth failure and moderate mental motor retardation. He was a dysmorphic boy with large, low set protruding ears, thick diffuse eyebrows, long curly eyelashes, short palpebral fissures with upper eyelid retraction, prominent forehead, triangular-fish mouth, thin and parrot-like nose, and hypertrichosis. Profound intentional tremor and dyskinesia, spasticity (particularly at the lower extremities), dystonia and a slightly reduced muscular mass were observed. The other main findings were bilateral cryptorchidism, bilateral sensorineural hearing loss and involvement of basal ganglia on the MRI scan. A microcytic, hypochromic anemia, elevated plasma lactate and lactate–pyruvate ratios (37.01 mg/dl and 97.39) and excretion of urinary 3-methylglutaconic acid (130–120 mmol/ mol creatinine) were laboratory hallmarks for suspicion of mitochondrial dysfunction. Results: Dilated cardiomyopathy stabilized after the second year and the combination of cryptorchidism led to genetic analysis of DNAJC19. Investigation of enzymes of the mitochondrial energy metabolism revealing a decreased activity of cytochrome c oxidase in muscle tissue. Discussion: This is the second report and third patient from non-Canadian Hutterite population with DCMA syndrome reported. The presentation of deafness and dysmorphic feature were two main differences from Hutterite population DCMA syndrome. Furthermore bilateral basal ganglia involvement on brain MRI resembled a characteristic finding for MEGDEL syndrome.

P-348

Clinical, biochemical and genetic spectrum of mitochondrial disorders in Egyptian children: a study of 19 cases. Selim L A 1, Mehaney D 4, Vancoster R 5, Hassan S A 1, Mahmoud I G 1, ElBadawy A I 1, Vanlander A 5, Smet J 5, De Latter E 5, Vandemeulebroeckeb K 5, Abdoh D 4, Nakhla G A 2, Mostafa M 1, Habets D 6, Bakker J 6, Abdel Barry A 3 1 Cairo University, Pediatric Dept, Cairo, Egypt, 2Cairo University, Pathology Dept, Cairo, Egypt, 3Cairo University, Surgical Dept, Cairo, Egypt, 4Cairo University, Clinical Pathology Dept, Cairo, Egypt, 5Gent University, Neurology and Metab Div, Gent, Belgium, 6Amesterdam University, Amesterdam, Netherlands

Background: Mitochondrial encephalomyopathies are a growing group of diseases with a large variety of clinical presentations ranging from well defined

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 clinical syndromes to non specific manifestations such as failure to thrive, hypotonia, seizures, global developmental delay, cardiomyopathy, visual or hearing loss. Methods: This is a retrospective study which included 64 patients with an age ranging from 9 months to 25 years referred to the Neurometabolic Unit at Cairo University Children Hospital to be evaluated for a possible mitochondrial respiratory chain disorder. They were 36 males/28 females. All patients were subjected to thorough neurological examination, basic lab investigations, expanded metabolic screen and urine organic acid profile, muscle biopsy subjected to immunohistopathological stain by COX and SDH, and respiratory chain complexes were assayed spectrophotometrically using specific substrates, and molecular diagnosis for mitochondrial syndrome. Results: 19/64 patients have been confirmed with respiratory chain disorders. Four patients were diagnosed as mitochondrial neurogastrointestinal encephalopathy (MNGIE), 2 patients with mitochondrial encephalopathy lactic acid stroke-like episodes (MELAS), one patient with mitochondrial depletion syndrome, one patient with mitochondrial myopathy, 5 patients with complex I deficiency (1/5 presenting as biotin responsive striatal necrosis), 2 patients with combined complex I and IV deficiency, 2 patients with LHON and one patient with chronic progressive external ophthalmoplegia. Discussion: Mitochondrial disorders have a wide spectrum of clinical presentations accounting for the marked delay in the diagnosis. High degree of suspicion is necessary to start a comprehensive work up combining clinical, biochemical, pathological and molecular data to confirm the diagnosis

P-349

The simultaneous presence of the somatic-specific PDHA1 and the testisspecific PDHA2 proteins in somatic cells of a pyruvate dehydrogenase complex deficient patient Rivera I 1, Pavlu-Pereira H 1, Silva M J 1, Marques B 2, Correia H 2, Tavares de Almeida I 1 1 iMedUL, Fac Phar, Univ Lisboa, Portugal, Lisboa, Portugal, 2Dept Hum Genet, Nat Inst Health, Lisboa,, Lisboa, Portugal

Background: Human pyruvate dehydrogenase complex (PDC) catalyses a key step in the generation of cellular energy and is composed of three catalytic elements (E1, E2, E3), one structural subunit (E3-binding protein), and specific regulatory elements, phosphatases and kinases (PDKs, PDPs). The E1α subunit exists as two isoforms encoded by different genes: PDHA1 located on Xp22.1 and expressed in somatic tissues, and the intronless PDHA2 located on chromosome 4 and only detected in human spermatocytes and spermatids. This study was aimed at the molecular characterization of a female patient carrying PDC deficiency. Methods: Mutation analyses were performed by direct sequencing of PCR products resulting from amplification of individual exons and related intronic boundaries. qRT-PCR and western blot/two dimensional electrophoresis were used for evaluating gene expression at transcriptional and translational levels, respectively. Methylation studies were performed by direct sequencing of bisulfite-treated genomic DNA samples. Results: In this patient, PDC deficiency is caused by two different null mutations in PDHX gene, encoding the E3-binding protein but, unexpectedly, the patient presented the simultaneous presence of fulllength testis-specific PDHA2 and 5’UTR-truncated PDHA1 mRNAs, being both translated into full-length PDHA1 and PDHA2 proteins, resulting in the coexistence of both PDHA isoforms in her somatic cells (lymphocytes and fibroblasts). Moreover, we elucidated that demethylation of a CpG island in its coding region is the molecular mechanism underlying PDHA2 gene activation in somatic tissues. Discussion: This study represents the first case reporting the activation of the testis-specific PDHA2 gene in human somatic cells, and raises some questions related to its somatic activation as a potential therapeutic approach for most forms of PDC deficiency. PEstOE/SAU/UI4013/13FCT; SFRH/BD91729/2012

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Clinical, molecular and genetic characteristics of mitochondrial hepatopathy in Japan Shimura M 1, Murayama K 1, Fushimi T 1, Ichimoto K 1, Matsunaga A 1, Mori M 2, Kishita Y 3, Tokuzawa Y 3, Kohda M 3, Okazaki Y 3, Ohtake A 4 1 Dep Metab, Chiba Child Hosp, Chiba, Japan, 2Dep Pediatr, Matsudo City Hosp, Matsudo, Japan, 3Res Center Genom Medicine, Saitama Med U, Saitama, Japan, 4Dep Pediatr, Saitama Med Univ, Saitama, Japan

Background: Mitochondrial hepatopathies are disorders, presenting as cholestasis, steatohepatitis and acute liver failure in early childhood, caused by mitochondrial dysfunction. Typically, multisystem involvement such as neuromuscular symptoms develops in their course. The aim of this study is to reveal clinical, molecular and genetic features of mitochondrial hepatopathies in Japan. Methods and results: Mitochondrial respiratory chain disorders (MRCDs) were diagnosed by in vitro enzyme assay and BN-PAGE analysis. Subsequently, comprehensive genomic analyses were performed. We’ve diagnosed 455 cases of MRCDs since 2007. 58 cases (13 %) were diagnosed with Mitochondrial hepatopathy that showed liver abnormality as a prominent symptom. Among them, 19 cases were hepatocerebral mitochondrial DNA depletion syndrome (MTDPS). Complex I deficiencies were most frequent, then the second was combined complex deficiency. The most common initial symptoms were failure to thrive, developmental delay and digestive symptoms including poor oral intake and vomiting before liver dysfunction appeared. 14 cases (24 %) were genetically diagnosed. 7 cases had mutations in MPV17, 3 had DGUOK mutations, and 1 had POLG mutations in MTDPS cases. The other 3 cases had GFM1, MRPS23 as a novel causative gene and 6q24.3-q25.1 deletion respectively. Six of our MPV17 cases had c.451dupC mutation. Liver transplantation was performed for 11 cases, but only 5 cases survived. Discussion: MPV17 is the most frequent gene in mitochondrial hepatopathy, and c.451dupC (MPV17) may be hotspot in Japanese population. Our study firstly demonstrates that MRPS23 encoding a component of mitochondrial ribosome small subunit causes mitochondrial hepatopathy.

P-351

High ROS content and biochemical defect of complex I due to a novel mutation in the molecular chaperon NDUFAF4 is partially reversed by the use of antioxidant compounds.

observed in patient’s cells. Treatment with different antioxidants produced variable responses in patient’s cells with n-acetyl cysteine (NAC) being the most efficient compound. Indeed, by lowering the ROS content in patient’s cells, NAC induced the activation of the antioxidant responsive elements, increased the glutathione content, and restored the ATP level and autophagic flux in the patient. Discussion: Overall our results underline the importance to keep a balanced redox environment to preserve the mitochondrial functionality and avoid the failure of other key metabolic pathways.

P-352

Mutations in MRPL57 are associated to OXPHOS defects in a patient with fatal hypertrophic cardiomyopathy Tort F 1, Ferrer-Cortes X 1, Massana M 1, Matalonga L 1, Bujan N 1, LopezGallardo E 2, Lissens W 4, Montoya J 2, Mesa J 3, Fernadez-Burriel M 3, Briones P 1, Ribes A 1 1 Hosp Clinic, IDIBAPS, CIBERER, Barcelona, Spain, 2Univ Zaragoza, CIBERER, Zaragoza, Spain, 3 Hosp Merida, Merida, Spain, 4 Vrije Universiteit, Brussels, Belgium

Background: Mitochondrial translation machinery deficiencies have been associated to multiple defects of the mitochondrial respiratory chain complexes. To this effect, mutations in genes encoding for mitochondrial ribosomal proteins (MRPL3, MRPL12, MRPL44, MRPS16, MRPS22 and MRPS7) have been reported in human disease. Case report: We report a patient from healthy first degree consanguineous parents. Antenatal antecedents were unremarkable. At 2 h of life she was found cyanotic and irritable and died of heart arrest that was unresponsive to the usual measures. Autopsy showed cardiac hypertrophy. Biochemical studies showed a defect in the assembly and activity of complex V and a partial reduction in the activity of complex III in muscle necropsy. Mutations in genes involved in complex V assembly were excluded. Results: We performed whole exome sequencing in the patient and her healthy parents. Using this strategy we identified a homozygous missense mutation in the gene encoding for the mitochondrial ribosomal protein L57 (MRPL57) (c.180C > A, p.Asn34Lys). The function of MRPL57 is not well known. Using cellular fractionation we have demonstrated that MRPL57 is localized into the mitochondria. In addition, MRPL57 depletion with specific siRNA in HeLa cells lead to a defect in the assembly of complex V, mimicking the biochemical defect of our patient. Discussion: We describe for the first time MRPL57 mutations associated to human disease. These findings expand the spectrum of genetic defects targeting proteins involved in mitochondrial ribosomal function.

Torraco A 1, Di Nottia M 1, Verrigni D 1, Petrillo S 1, Rizza T 1, Martinelli D 2, Diodato D 1, Piemonte F 1, Dionisi-Vici C 2, Bertini E 1, Carrozzo R 1 P-353 Unit of Neuromusc Dis, OPBG Child Hosp, Rome, Italy, 2Div Metab Dis, OPBG Child Hosp, Rome, Italy 1

The spectrum of disease and functional implications of TRNT1 mutations Background: Isolated defects of complex I (CI) account for the vast majority of mitochondrial disease. Due to the size of CI, the large number of proteins involved in its construction and the intricate genetic to find a molecular diagnosis is often challenging. In the last years, Next Generation Sequencing (NGS) has proved a powerful tool in the identification of the genetic cause in many undiagnosed mitochondrial defects. Methods: Molecular genetic characterization was performed using next generation sequencing combined with the Sanger method. Biochemical and structural studies were achieved by enzymatic activities and Western blotting, respectively. Treatment with different antioxidant compounds was performed on patient and control fibroblasts. Results: By NGS we have identified a new c.397 T > C [p.W133R] homozygous mutation in NDUFAF4, a gene encoding for an assembly factor of CI in a patient with isolated CI defect. NDUFAF4 loss of function caused a dramatic reduction of CI amount and disassembly of supercomplexes. Moreover high levels of reactive oxygen species (ROS) and an altered autophagic flux were

Wedatilake Y 1, Niazi R 1, Fassone E 1, Powell C A 2, Pearce S 2, Saldanha J W 3 , Kleta R 5, Chong W K 4, Footitt E 4, Mills P B 1, Plagnol V 7, Taanman J W 6 , Minczuk M 2, Clayton P T 1, Rahman S 1 1 UCL Institute of Child Health, London, United Kingdom, 2 MRC Mitochondrial Biology Unit, Cambridge, United Kingdom, 3National Institute for Medical Research, London, United Kingdom, 4Great Ormond Street Hospital, London, United Kingdom, 5Division of Medicine, UCL, London, United Kingdom, 6UCL Institute of Neurology, London, United Kingdom, 7UCL Genetics Institute, London, United Kingdom

Background: TRNT1 (CCA-adding transfer RNA nucleotidyl transferase) is an enzyme responsible for post-transcriptional modification of transfer RNAs (tRNAs). TRNT1 deficiency is a new metabolic disease, where careful clinical collation of symptomatology would aid diagnosis of more cases.

S162 Methods: We used whole exome sequencing and bioinformatics filtering to investigate two siblings with febrile episodes, electrolyte abnormalities, sideroblastic anaemia, low B-lymphocytes, retinitis pigmentosa, hepatosplenomegaly, exocrine pancreatic insufficiency and renal tubulopathy. Functional analysis of CCA-addition to tRNAs was performed in patient fibroblast cells. We describe 2-year follow up of a previously published patient who underwent bone marrow transplantation. A systematic literature review was used to collate all known TRNT1 mutations and to identify the phenotypic spectrum of TRNT1 deficiency. Results: Compound heterozygous TRNT1 mutations (c.668 T > C,p.Ileu223Thr and a novel splice mutation: c.342 + 5G > T) were found in the siblings, segregating with disease in the family. Functional studies demonstrated impaired 3’-CCA addition to mitochondrial tRNAs. The patients were managed using transfusions for anaemia, fluid and electrolyte replacement and immunoglobulin therapy. Bone marrow transplantation in an unrelated patient with a previously reported homozygous TRNT1 mutation resulted in resolution of fever and correction of the abnormal metabolic profile. Other clinical features found by systematic literature review include cerebellar atrophy, sensorineural deafness, brittle hair, partial villous atrophy and nephrocalcinosis. Discussion: This study demonstrates that TRNT1 mutations result in a disease spectrum ranging from adult-onset retinitis pigmentosa to childhood-onset multisystem disease. We found a novel TRNT1 mutation and add metabolic context to the clinical and investigative findings, to enable rapid recognition of the disease and targeted genetic testing.

P-354

Lethal early-onset cardiomyopathy caused by TK2 and AARS2 deficiency. Mazurova S 1, Magner M 1, Kucerova-Vidrova V 1, Vondrackova A 1, Stranecky V 2, Pristoupilova A 2, Zamecnik J 3, Hansikova H 1, Zeman J 1, Tesarova M 1, Honzik T 1 1 Dep Ped, 1st Fac Med,Gen Univ Hosp, Prague, Czech Republic, 2Inst of Inh Metab Disord,Charles Univ, Prague, Czech Republic, 3Dep Pathol Mol Med,Hosp Motol, Prague, Czech Republic

Background: Mitochondrial disorders manifesting in infancy are accompanied by cardiomyopathy (CMP) with an estimated frequency of around 30–40 %. Here we report on two cases of lethal hypertrophic CMP caused by thymidine kinase 2 (TK2) and alanyl-tRNA synthetase (AARS2) deficiency. Case report: A girl with TK2 deficiency manifested with gross motor regression and hypotonia at 13 months of age. Development of hypertrophic CMP contributed to cardiorespiratory failure from which she subsequently died at 22 months of age. A girl with AARS2 deficiency presented with respiratory failure and neurological symptoms in the twelfth hour of life. Progressive hypertrophic CMP and heart rhythm disturbances were noted. Rapid cardiorespiratory failure led to her demise at the age of 9 weeks. Results: In both girls, muscle enzymology showed combined oxidative phosphorylation defects with complex I and IV deficiencies and COX negative fibers at muscle histochemistry. Brain MRI image of leucoencephalopathy and severe neuronal degeneration, together with hepatic steatosis are described for the first time in TK2 deficiency. Muscle histochemistry revealed massive subsarcolemmal accumulation of SDH product, mimicking ragged red fibers, not previously reported in AARS2 deficiency. Next-generation DNA sequencing revealed compound heterozygosity for a novel mutation c.209 T > C and previously published mutation c.416C > T in the TK2 gene and homozygosity for a known mutation c.1774C > T in the AARS2 gene. Discussion: We diagnosed the third known patient with TK2 deficiency and ninth patient with AARS2 deficiency manifesting fatal CMP. Cardiac involvement as a manifestation of mitochondrial disorders has an expanding disease spectrum, suggesting the use of next generation sequencing methods in order to enable rapid diagnostics. Supported by: RVO-VFN 64165, SVV 260256/ 2016, AZV 15-28208A.

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Characterization of the impairment of mitochondrial bioenergetics and dynamics in fibroblasts from patients with complex I deficiency Leipnitz G 1 2, Mohsen A W 2, Karunanidhi A 2, Seminotti B 2, Roginskaya V Y 3, Markantone D M 2, Van Houten B 3, Vockley J 2 1 Dept Bioq, ICBS, UFRGS, Porto Alegre, Brazil, 2Div Med Genet, Dep Pediatr, Univ Pitt, Pittsburgh, United States, 3Dept Pharmacol Chem Biol, Univ Pitt, Pittsburgh, United States

Background: Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation disorders and can result from mutations in either mitochondrial DNA- or nuclear-encoded subunits or assembly factors. Clinical manifestations of CI deficiency include hypotonia, cardiomyopathy, seizures and psychomotor retardation. In the present study, we investigated the effect of CI impairments on the levels of respiratory chain components, mitochondrial bioenergetics and dynamics, and reactive oxygen species production in fibroblasts from patients with NDUFV1, ND6, and ACAD9 deficiencies. Methods: Following 48- to 72-h incubation of patient fibroblast cells in media with or without glucose, protein immunocontent of respiratory chain components, mitofusin 1 (MFN1) and dynamin-related protein 1 (Drp1) were evaluated. Oxygen consumption rate, mitochondrial membrane potential, ATP production, and superoxide generation were also monitored. Results: Our data show a decrease of NDUFV1 and ND6 antigen levels in NDUFV1- and ND6-deficient cells in medium with or without glucose, but no significant decrease was observed in ACAD9 antigen content in the ACAD9deficient fibroblasts. While basal respiration and reserve capacity were decreased in ND6- and ACAD9-deficient cells, in NDUFV1-deficient cells only reserve capacity was decreased in the absence of glucose. Dissipation of mitochondrial membrane potential, alterations in mitochondrial morphology, and increase of MFN1 and Drp1 antigen levels were also observed in all cell lines. Decreased ATP production was confirmed in ND6- and ACAD9-deficient cells. Finally, a significant increase in superoxide production was observed in ACAD9-deficient fibroblasts. Discussion: These findings show that bioenergetics and disruption of mitochondrial dynamics, as well as increased superoxide levels in the ACAD9deficient fibroblasts, contribute to the pathophysiology underlying the symptoms observed in these CI deficiencies.

P-356

A patient with mitochondrial disorder and mutation in MRPS22 gene Kilic M 1, Kilic E 3, Ozgul R K 4, Yucel-Yilmaz D 4, Kavak P 5, Yuceturk B 5, Demirci H 5, Dedeoglu O 2, Yuksel D 2, Sagiroglu M S 5 1 Div Metab Dis, Sami Ulus Child Hosp, Ankara, Turkey, 2Div Pediatr Neurol, Sami Ulus Child Hosp, Ankara, Turkey, 3Div Pediatr Genetic, Hem-Onco Child Hosp, Ankara, Turkey, 4Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 5Bilgem, TUBITAK, Kocaeli, Turkey

Background: Defects in nuclear-encoded components of the translation system comprise a novel group of mitochondrial respiratory chain defects. MRPS22 gene was the second ribosomal protein involved in mitochondrial disease in humans. Here we present an infant, severely affected with mitochondrial disorder caused by a homozygous mutation in MRPS22 gene. Case report: A 4-month-old male infant was admitted to hospital for poor eye contact with his mother and lack of head control. Physical examination showed mild dysmorphism, axial hypotonia, developmental delay, increased deep tendon reflexes. There was second degree cousin marriage and no similar sibling history of family. Laboratory findings showed mild elevated levels of serum lactate and pyruvate, normal ammonia levels. Tandem mass spectrometric analyses were normal, while urinary organic acid analyses showed increased succinic, fumaric, lactic and pyruvic acids. Muscle biopsy was

S163

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 nonspecific but OXPHOS enzymes were not studied yet. Brain MRI revealed bilateral abnormal signal hyperintensity involving the pons central tegmental tractus, mesencephalon tegmentus, dorsomedial nucleus of thalamus in T2 series, decreased diffusion in ADC series. Results: Homozygous c.339 + 5 G > A mutation was found in MRPS22 gene by exome sequencing. Mitochondrial vitamin cocktail theraphy was given. He is 3 years-2 months old now and the treatment outcome is poor with oxygen dependence, tetraspasticity and severe developmental delay. Discussion: The mitochondrial ribosomal protein MRPS22 is a part of the small 28S subunit of the mitochondrial ribosome. So far, only three mutations in three families with altogether five affected patients have been described. The case reported here expands the clinical spectrum of patients with MRPS22 mutations. It suggests that MRPS22 deficiency leads to a severe, early-onset energy metabolism defect with poor prognosis.

P-357

The new autosomal recessive phenotype of mitochondrial disease caused by DNA2 gene mutations Itkis Y S 1, Krylova T D 1, Pechatnikova N L 2, Kakaulina V S 2, Polyakova N A 2, Tsygankova P G 1, Bychkov I O 1, Zakharova E Y 1 1

Research Center for Medical Genetics, Moscow, Russian Federation, Morozov' Moscow City Child Clinical Hosp, Moscow, Russian Federation

2

Background: To date NGS methods have become the main diagnostic tool for searching genetic causes of rare hereditary diseases. As mitochondrial disorders are highly heterogeneous and manifest with a variety of clinical features, NGS could be effective in diagnosis and increase knowledge about phenotypic spectrum in this group of diseases. Case report: Here we present a family of non-consanguineous parents with two affected siblings (13-year-old boy and 7-year-old girl) developing a clinical picture of tetraparesis with generalized truncal and limb hypotonia including plastic rigidity, limb dystonia, increasing after emotional excitation, pseudobulbar syndrome, speech absence, epileptic seizures. The boy had cataract from age of 11.5 years. Brain MRI showed mild cortical atrophy. Methods: We applied targeted sequencing of 62 known mitochondrial nuclear genes using NGS technology on Ion Torrent to define a molecular defect in our patients. MtDNA quantative analysis in blood was conducted with real-time PCR. Fibroblast respirometry was performed on the Oxygraph-2 k (Oroboros). Results: We have identified two substitutions in DNA2 gene c.C1078A (p.Q360K)/ c.C1795T (p.R599C) in compound heterozygous state. Both have high predicted scores of pathogenicity and low (less than 0.5 %) frequency of occurrence in populations. Parents were confirmed to be heterozygous carriers. High score of mtDNA depletion was detected. A measuring of mitochondrial respiration on patient’s intact fibroblasts showed increased ratio of R/E and net R/E in comparison with control cells culture. Mitochondrial inherited disease was proposed. Discussion: We argued that a new autosomal recessive phenotype of mitochondrial disease caused by DNA2 gene mutations was revealed. Previously in the literature only AD inheritance was described, manifesting with completely different clinical features. Our results show that targeted sequencing is an effective approach for routine diagnostics and for revealing new phenotypes as well.

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Unexpected findings from Next Generation Sequencing (NGS) panel suggesting mitochondrial disorder in two patients. Simenson K 1, Pajusalu S 1 2, Kahre T 1 3, Zordania R 1, Rein R 3, Rodenburg R J 4, Ounap K 1 3 1 Dept Genet, United Lab, Tartu Univ Hosp, Tartu, Estonia, 2Dept Biomed, Univ Tartu, Tartu, Estonia, 3Dept Pediat, Inst Clin Med, Univ Tartu, Tartu, Estonia, 4Ctr Mito Dis, Radboudumc, Nijmegen, Netherlands

Background: Since the beginning of routine use of NGS in clinic the order of classical approach and diagnostic methods may be reversed sometimes. We present two cases in whom mitochondrial disorder was not suspected in clinical practice, yet diagnoses were clarified by targeted NGS panel. Case report: Case 1: 14-year-old male who was born by Caesarian section due to fetal overgrowth. He has intellectual disability (ID), verbal dyspraxia, mildly decreased muscle tone, and seizures. MRI and EEG were normal. Case 2: 15-year-old female, who was born with microcephaly (occipitofrontal circumference 31 cm, −3SD). She has dysmorphic features, severe ID, microcephaly, epilepsy and mitral valve prolapse. In infancy lactate and alanine were mildly elevated, but later were in normal range. Muscle immunohistochemical and electronmicroscopical investigations were performed with normal results. CT showed enlargement of lateral ventricle and brain atrophy. RESULTS: Case 1: Compound heterozygous mutations in BCS1L gene (c.232A > G, p.Ser78Gly and c.245C > T, p.Ser82Leu; NM_001257342.1) were found. For phenotype specification, a hearing control was performed and bilateral moderate sensorineural hearing loss was discovered. Enzyme analysis from cultured fibroblasts for complex III deficiency was normal. In muscle biopsy increased number of mitochondria with normal structure were found (enzyme analysis is in process). Case 2: A novel de novo heterozygous frameshift mutation in PDHA1 gene (c.1014_1017dup, p.Arg340Leufs*13, NM_001173454.1) was found. Enzyme analysis from cultured fibroblasts for PDH activity was normal. A Western Blot analysis looking at the expression of the different subunits of PDH complex is in process. Discussion: These cases illustrate that NGS is a powerful tool which enables diagnosis of patients with non-classical phenotypes of mitochondrial disease as well as confirming that normal results of enzyme analysis from cultured fibroblasts do not always exclude diagnosis of a mitochondrial disorder.

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Two novel pathogenic variants in KARS unveil a new phenotype associated with cardiomyopathy and defects of mitochondrial respiratory chain. Di Nottia M 1, Verrigni D 1, Diodato D 1, Torraco A 1, Bellacchio E 2, Rizza T , Bertini E S 1, Carrozzo R 1

1

1 Neuromusc Unit, Child Hosp Bambino Gesu, Roma, Italy, 2Research Lab, Child Hosp Bambino Gesu, Roma, Italy

Background: Aminoacyl-tRNA syntethases (aaRSs) are a group of enzymes responsible for aminoacylation, the first step of protein translation. Depending on their subcellular localization, it is possible to distinguish three groups: cytoplasmatic, mitochondrial or bifunctional. The lysyl-tRNA-synthetase (KARS) is one of the 3-bifunctional aaRSs. KARS codes for the two forms of lysine aminoacyl-tRNA syntethases by alternative splicing. Nowadays KARS mutations have been associated with three different phenotypes: the recessive form of the Charcot-Marie-Tooth peripheral neuropathy; autosomal recessive nonsyndromic hearing loss and congenital visual impairment with progressive microcephaly. Methods: We report a 14 year old girl with mild psychomotor delay, mild myopathy and severe cardiomyopathy; combined defect of CI and CIV was present at muscle biopsy. Mutational analysis was conducted by Illumina TruSight One sequencing panel. The amount of KARS protein and of mitochondrial respiratory chain (MRC) subunits were studied by Western blot analysis. MRC activity was measured by Blue-native in situ enzyme assay in muscle enriched-mitochondria and ATP synthesis was measured in fibroblast mitochondria. Results: We identified two novel pathogenic mutations within the KARS gene: c.1049 T > A [p.350 L > H] and c.1169C > G [p.390P > R], which produce a 50 % reduction of the corresponding protein, and a variable reduction of MRC subunits. Structural studies confirmed the pathological role of these two new variants. Discussion: Here we describe a new clinical phenotype associated with KARS mutations involving a severe form of cardiomyopathy and mitochondrial oxidative phosphorylation defect. These findings expand the phenotypic spectrum associated with mutations in KARS, and confirm the variable clinical phenotype associated with mutations in aaRSs.

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genes associated to mitochondrial disorders. Functional analyses were achieved on patient fibroblasts and the pathogenic role of the TUFM mutation was also investigated by complementation experiment in yeast. A molecular modeling study has been performed to understand the phenotypic effect of the mutation on a structural basis. Results: We identified a novel c.964G > A mutation in the TUFM gene. The OXPHOS activity was reduced, as well as the mitochondrial protein synthesis. In addition, TUFM protein amount was decreased and structural and complementation studies confirmed the pathological role of this new variant. Discussion: To our knowledge this is the second mutation identified in this gene. Our patient displayed overlapping phenotype with the one already reported; in fact, the neurological examination revealed a severe encephalopathy and leucodystrophy with micropolygyria in both patients. Our study strengthens the connection between mutations in TUFM and this peculiar neurological phenotype.

Subcomplexes of complex V in a patient with Perrault syndrome due to pathogenic mutations in C10orf2 Verloo P 1, Smet J 1, Vantroys E 1, Vanlander A 1, Vergult S 2, Sante T 2, Menten B 2, Van Coster R 1 1 Div Ped Neur, Univ Hosp Ghent, Ghent, Belgium, 2Cent Med Genet, Ghent Univ, Ghent, Belgium

Background: Perrault syndrome is a rare disorder characterized by ovarian dysgenesis and sensorineural hearing loss. Five genes were found to be linked to Perrault syndrome (HSD17B4, HARS2, CLPP, LARS2 and C10orf2). Mutations in C10orf2 (encodes twinkle) were shown to be associated with different phenotypes, including progressive external ophthalmoplegia, infantile-onset spinocerebellar ataxia (IOSCA) and Perrault syndrome. Twinkle is essential for correct replication of mitochondrial DNA. Case report: A young girl with ovarian dysgenesis and sensorineural hearing loss was diagnosed with Perrault syndrome. Neurological examination revealed nystagmus and ataxia. Results: Normal activities of oxidative phosphorylation complex I-IV were found in skeletal muscle. Prominent subcomplexes of complex V were detected in BN-PAGE gel, indicating an instability of the holo complex V. Whole exome sequencing was performed and variants in known mitochondrial genes were filtered. Two heterozygous missense variants were found in C10orf2 (p.R400L and p.Y508C). The p.Y508C variant is a known pathogenic mutation previously reported in patients with IOSCA. The second variant, p.R400L, is not seen in a control population and affects an amino acid next to a known mutation in patients with Perrault syndrome (p.N399S). Discussion: Patients with Perrault syndrome caused by pathogenic mutations in C10orf2 have neurological symptoms such as ataxia and nystagmus which are not found in other patients with Perrault syndrome. The presence of these symptoms is suggestive of pathogenic mutations in C10orf2. Also remarkable is the finding of subcomplexes of complex V. This shows the importance of using BN-PAGE in addition to spectrophotometric analysis in diagnosing defects in the oxidative phosphorylation complexes.

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Novel homozygous mutation in TUFM associated to leukodystrophy and defective mitochondrial DNA translation. Rizza T 1, Di Nottia M 1, Verrigni D 1, Montanari A 2, Fernandez-Vizarra E 3, Diodato D 1, Oliva R 4, Torraco A 1, Piemonte F 1, Francisci S 5, Zeviani M 3, Dionisi-Vici C 6, Bertini E S 1, Carrozzo R 1 1 Neuromusc Unit, Child Hosp Bambino Gesu, Roma, Italy, 2Pasteur Inst, Cenci Bolognetti Found, Roma, Italy, 3Mito Biol Unit, MRC, Cambridge, United Kingdom, 4Dept Sci-Tech, Univ Parthenope Naples, Napoli, Italy, 5 Dept Biol-Biotech, Univ Sapienza Rome, Roma, Italy, 6Div Metabolism, Child Hosp Bambino Gesu, Roma, Italy

Background: The oxidative phosphorylation (OXPHOS) system comprises more than 150 proteins of which only 13 subunits are encoded by mitochondrial DNA (mtDNA). In addition, mtDNA encodes for rRNAs and 22 mitochondrial tRNAs, necessary for the translation of the mitochondrial-encoded proteins. The mtDNA genes are translated into proteins within the organelles by a protein-synthesis mechanism which occurs in three steps: initiation, elongation, termination. The mitochondrial Elongation Factor Tu, encoded by the TUFM gene, is a GTPase responsible for the delivery of the aminoacyl-tRNAs to the A site of ribosomes. Impaired mitochondrial translation causes a subgroup of mitochondrial diseases and usually results in severe combined OXPHOS dysfunctions. Methods: We report the case of a young girl, died at 10 months, suffering by lactic acidosis, encephalopathy, leukodystrophy with micropolygyria. The mutational analysis was performed by targeted resequencing of 1381 nuclear

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SPATA5 deficiency in three patients with suspected mitochondrial disease characterized by global developmental delay, hearing loss, visual impairment, and epilepsy Kovacs-Nagy R 1, Kotzaeridou U 2, Makowski C 4, Alhaddad B 1, Braunisch M 1, Wilson C 3, Hoffmann G F 2, Haack T B 1 1 Ins Human Gen, Tech Univ Mun, Munich, Germany, 2 Zentr Kind Jugendmed, Univ Heidelberg, Heidelberg, Germany, 3Nat Met Service, Auck City Hosp, Auckland, New Zealand, 4 Kinderklinik, Klin Mun Schwabing, Munich, Germany

Background: SPATA5 is a member of the AAA-protein family (ATPase associated with diverse activities) with a putative function in spermatogenesis and neurodevelopment. Bi-allelic SPATA5 variants in 4 familial and 6 sporadic cases have been recently established as the molecular correlate underlying “Epilepsy, hearing loss, and mental retardation syndrome”. We report on the identification of pathogenic SPATA5 variants in three sporadic patients referred to clinical exome sequencing with a suspected mitochondrial disorder. Case report: Case #1 developed tonic-myoclonic seizures, sensorineural hearing loss, dyskinesia, spasticity, dystonia, muscular hypotonia and cortical visual impairment. He is not able to speak and cannot sit unsupported. MRI showed hypoplasia of corpus callosum, delayed myelination, and supratentorial white matter atrophy. Case #2 suffered from neonatal myoclonic seizures, hearing loss, spasticity, dystonia and delayed motor milestones. Case #3 presented with similar clinical features plus global leukodystrophy. A combined respiratory chain enzyme defect was diagnosed in a muscle biopsy. Results: Exome sequencing identified bi-allelic variants in all three patients: two novel nonsense variants (Gln132* and Gln234*), two novel missense variants (Gly185Glu and Pro795Arg) and one previously described inframe-deletion (Thr330del). Discussion: Clinical exome sequencing is a suitable first line diagnostic test for cases with suspected mitochondrial disorders. The prediction of pathogenic variants in SPATA5 is restricted in the absence of functional assays. The respiratory chain defect in skeletal muscle of index case #3 lends further support to the hypothesis of a mitochondrial dysfunction being involved in the pathogenesis of the disease.

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A novel mutation in the human MPV17 gene is responsible for the high incidence of mitochondrial neurohepatopathy in Black South Africans. Meldau S 1, De Lacy R 3, Riordan G 3, Pillay K 2, Van der Watt G F 1 1 Div Chem Path, Univ of Cape Town, Cape Town, South Africa, 2Div of Histopath Univ of Cape Town, Cape Town, South Africa, 3ICH, Red Cross Child Hosp, UCT, Cape Town, South Africa

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: MPV17 Mitochondrial neurohepatopathy is considered a rare metabolic disorder caused by dysfunction of the human MPV17 mitochondrial membrane protein. The MPV17 protein functions as an inner membrane pore that controls mitochondrial membrane potential and mitigates mitochondrial oxidative damage. Mutations are associated with mitochondrial-DNA depletion and a hepatocerebral syndrome. We have long suspected that a common mutation may be responsible for the regular similar clinical cases of mitochondrial neurohepatopathy seen only in Black children at our institution. Methods: EXOME sequencing confirmed by Sanger sequencing in two unrelated patients, identified homozygosity for a c.C106T (p.Q36X) mutation in the MPV17 gene. In both cases parents were confirmed as carriers. Anonymised leukocyte DNA, obtained from 750 self-identified black South African controls from the Cape Town area was screened by PCR/RFLP for this mutation to estimate the carrier frequency in this population. Results: Subsequently we have indentified an additional 11 unrelated patients, all homozygous for the same c.C106T mutation. Eleven patients presented between 4 and 12 months with progressive fatal neurohepatopathy and 2 patients as neonates with fulminant hepatic failure. One of these neonates was exposed to azidothymidine perinatally. Heterozygosity for the c.C106T mutation was confirmed in 11/750 of controls, estimating a carrier frequency of 1/68 and a newborn incidence of 1/18496 in this population. Discussion: Here we describe the first identification and phenotype of 13 unrelated Black South African patients, all homozygous for a novel c.C106T mutation in the human MPV17 gene, and demonstrate a strikingly high carrier frequency of this mutation in this population. These data suggest that this previously unrecognized disorder contributes significantly to the metabolic disease burden in Southern Africa. Further research into treatment modalities is urgently required.

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ECHS1 and HIBCH mutations : valine metabolism disorders or multiple mitochondrial dysfunction syndromes? Lebigot E 2, Mehler-Jacob C 1, Noughes M C 3, Gaignard P 2, Drira L 2, Therond P 2, Sevin C 1, Slama A 2, Boutron A 2 1 Div Neurol Dis, Univ Bicetre Hosp, Le Kremlin Bicetre, France, 2Div Bioch and Metab, Univ Bicetre Hosp, Le Kremlin Bicetre, France, 3Div Neurol Dis, Trousseau Hosp, Paris, France

Background: Evidence of elevated pyruvate and a lactate/pyruvate ratio of < 20 can indicate PDHc deficiency (PDHD) in children presenting with early neurodegenerative disorders. PDHD is predominantly caused by mutations in PDHA1, encoding the E1 alpha subunit, but also by mutations in genes related to the PDHc cofactors. Another pathway recently associated with Leigh syndrome is the valine degradation pathway in which L-valine is converted to succinylCoA. Mutations have been described in 2014 in two genes encoding enzymes involved in this pathway, named 3- hydroxyisobutyryl-CoA hydrolase (HIBCH) and short-chain enoyl-CoA hydratase (ECHS1). Methods: We used targeted NGS to perform a retrospective genetic study of our cohort of PDHc-deficient individuals with no mutations in genes encoding PDHc subunits or genes related to cofactors. The genes on the panel were selected on the basis of metabolic encephalopathy disease–causing genes. Results: In our cohort we identified 4 unrelated individuals carrying mutations in HIBCH and 5 others with mutations in ECHS1. Evidence for the pathogenicity of the respective mutation was provided by clinical and biochemical features, functional investigations in patient-derived fibroblast cell line including immunoblotting, and biochemical investigation of PDHc and respiratory chain (RC) complexes activity. RC studies and/or PDHc activity were strikingly abnormal in our patients. Discussion: An accumulation of toxic reactive metabolites has been suggested to be the mechanism by which ECHS1 and HIBCH mutations impair RC and PDHc activity. Although it may not be the primary consequence of mutation, an indirect effect on mitochondrial energy generation can therefore be sufficient to cause the development of encephalopathy with lactic acidosis. Our results highlight the need to suspect those diseases in patients presenting comparable biochemical and clinical features.

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Could Arginine Aspartate be a promising treatment for Fumaric Aciduria? Pinto P L 7, Janeiro P 1, Moreno T 2, Mexia S 3, Jardim I 3, Grazina M 6, Vilarinho L 5, Almeida I T 4, Gaspar A 1 1 Div MetabChild Dis,Santa Maria Hosp,CHLN, Lisbon, Portugal, 2Div Neuroped, Santa Maria Hosp, CHLN, Lisbon, Portugal, 3Div Nutr Diet, Santa Maria Hosp, CHLN, Lisbon, Portugal, 4Met Ge, Fac Pharm, Lisb Univ, Lisbon, Portugal, 5Met Gen Unit,Hum Gen Depart,Nat Inst H, Porto, Portugal, 6Neurosci cell Biol centre,Coimbra Univ, Coimbra, Portugal, 7Ped Dep, Santa Maria Hosp, CHLN, Lisbon, Portugal

Background: Fumaric aciduria is a rare autosomal recessive metabolic disease, characterized by developmental delay, hypotonia, seizures, dysmorphic facial features. No specific treatments are available and many children don’t survive childhood. The authors report a 3-year-old-boy with fumarase deficiency on arginine aspartate supplementation and a low glycaemic index diet. Case report: First child of non-consanguineous couple, unremarkable family background, uneventful gestation with adequate somatometry. During postnatal period he developed tachypnoea and asphyxia. By 2 months, he lost weight and at 6 was noted to have global developmental delay, microcephaly, hypotonia, hyperlaxity and distal dystonic postures. MRI showed delayed myelination, subcortical bifrontal/bioccipital white matter hyperintensity and thinning of corpus callosum. Metabolic studies showed persistent elevated urinary fumaric and succinic acids. Molecular FH gene test revealed compound heterozygosity c.431G > A / c.1431_1433dupAAA confirming the diagnosis. Fumarase enzymatic activity showed reduced level (11.7 %). At 22 months he was hospitalized with a severe metabolic acidosis and started a low glycaemic index diet supplemented by MCT-oil, DHA, L-carnitine and arginine aspartate 5–10 g/day. Results: Six months later, glutamine, lactate levels in blood and urinary fumaric acid decreased and he had a slight improvement in motor development showing cervical control, ability to sit and communicate. Discussion: No specific treatment showed good efficacy. Arginine aspartate suplementation might be a solution. The malate-aspartate shuttle converts aspartate and oxoglutarate into oxaloacetate and glutamate acting as a bypass. Glutamate is degraded to enter the cycle as oxoglutarate, increasing ATP production. A low glucose diet may minimize lactic acidosis once ATP production is maintained by anaplerotic metabolites added to the diet. Further studies are necessary to assess the clinical impact of this therapy.

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Imitating the pretender: inborn errors of metabolism mimicking mitochondrial disorders Flynn N 1, Hogg S L 1, Parker A P J 2, Chitre M 2, Calvin J 1 1 Biochem Genet, The Pathology Partnership, Cambridge, United Kingdom, 2 Paed Neurology, Addenbrooke’s Hospital, Cambridge, United Kingdom Background: The literature stresses that mitochondrial cytopathies may mimic other diseases but the reverse is rarely mentioned. Three cases are presented where a primary mitochondrial disorder was strongly suspected clinically, yet the final diagnosis proved to be an alternative inborn error of metabolism (IEM). Case report: Child 1 (female, 14 years) presented with an 8 month history of dysarthria and increasing cognitive impairment. MRI brain showed bilateral basal ganglia changes and a mitochondrial work-up was initiated. Child 2 (female, 2 years 6 months) was admitted with decreased consciousness and metabolic acidosis, following a 3 month history of milestone regression and increasing hypotonia. Raised CSF lactate (7.4 mmol/L) and extensive symmetrical, abnormal intensities, involving multiple vascular territories on MRI head were suggestive of a mitochondrial cytopathy. Child 3 (male, 10 months) was admitted with generalised seizures, neuroregression, hypotonia and

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Watanabe Y 1 2, Fukui K 2, Harada N 2, Tashiro K 1, Inokuchi T 1, Yano S 3, Yamashita Y 2

Background: Deficiency of coenzyme Q10 biosynthesis associated with COQ4 mutations represents a very rare cause of respiratory chain disorders. Infantile epileptic encephalopathy is a possible clinical manifestation. Case report: After uneventful pregnancy and delivery, seizures started at the age of 2 months. Drug resistant epileptic encephalopathy with multifocal seizures, severe developmental delay and secondary microcephaly were diagnosed in the course. EEG demonstrated abnormalities similar to Malignant Migrating Partial Seizures of Infancy. Brain MRI revealed cortical atrophy and cerebellar cysts. Muscle biopsy showed COX negative fibers as well as slightly decreased activities for respiratory chain complex I and IV. Results: Whole exome analysis identified compound heterozygous mutations in COQ4: c.[469C > A];[718C > T], p.[Gln157Lys];[Arg240Cys]. Subsequent coenzyme Q10 measurement showed decreased concentration in muscle. In addition, depletion of mtDNAwas detected. We have found association of Q10 and mtDNA depletion in several myopathic patients. Supplementation with high dosed coenzyme Q10 provided no positive effect. Discussion: We report a 1.5 y old girl with severe infantile epileptic encephalopathy and cerebellar cysts associated with pathogenic sequence variants in COQ4. To our knowledge, cerebellar cysts in mitochondrial diseases were not reported before.

1 Research Inst GCMS, Kurume Univ, Kurume, Japan, 2Dept Pediatrics, Kurume Univ, Kurume, Japan, 3Div Med Genetics, Dept Pediatrics, USC, Los Angeles, United States

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encephalopathy. MRI showed bilateral basal ganglia and external capsule changes, suggestive of a mitochondrial disorder. Results: In child 1, ophthalmic examination revealed Kayser Fleischer rings and the diagnosis of Wilson disease was confirmed by biochemical and genetic testing. Urine organic acids and blood spot acylcarnitines in child 2 were consistent with partial biotinidase deficiency (confirmed by enzyme assay and genetic testing). Child 3 was diagnosed with succinic semialdehyde dehydrogenase deficiency (4 hydroxybutyric aciduria) on urine organic acids. Discussion: Biochemical screening for other IEMs should be undertaken in suspected mitochondrial disease as this may avoid invasive investigations and identify treatable conditions.

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Two sibling cases of aspartate-glutamate carrier 2 (Citrin) deficiency: Does diet affect prognosis?

Background: Citrullinemia Type 2 (CTLN2) is due to the defective mitochondrial aspartate-glutamate carrier isoform 2 (AGC2 or citrin). Citrin is a component of the malate-aspartate shuttle which transfers reducing equivalents from NADH from the cytosol to the mitochondria and is expressed in the liver. Patients with CTLN2 develop sudden onset hyperammonemia with neuropsychiatric symptoms in adulthood. Mutations in citrin are also known to cause neonatal intrahepatic cholestasis (NICCD). Patients with citrin deficiency often have characteristic food preference for high protein and high fat diet, and aversion to carbohydrate rich diet. Case report: Two Japanese full siblings (62y male and 59y female) with citrin deficiency showed significantly different clinical courses, possibly due to their diet. The elder brother is an owner chef of a “Tempura” restaurant for more than 30 years. He had been well until age 19y when he developed recurrent pancreatitis. Liver biopsy at age 56y due to chronic liver disease, showed the findings suggestive of Hemochromatosis. At age 62y, he developed hepatic coma with hyperammonemia (312uM). Plasma amino acid analysis showed a high citrulline, suggestive of CTLN2. He has been on protein rich diet; however, he cannot tolerate high fat diet due to chronic pancreatitis. His sister has been well without any specific citrin deficiency related symptoms. She likes high protein food and “Tenkasu”, crunchy surface of Tempura, and avoids high carbohydrate diet. Results: Compound heterozygous mutations in SLC25A13[g.IVS13 + 1G > A/g.IVS16ins3kb] were identified, confirming the diagnosis of citrin deficiency in the two siblings. Implementation of MCT oil in the brother improved the liver function including total bile acid metabolism. Discussion: Sodium pyruvate has been used to treat Citrin deficiency. Supplementing fatty acids including MCT oil may also have beneficial effects.

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Severe infantile epileptic encephalopathy with cerebellar cysts associated with COQ4 mutations and primary coenzyme Q10 deficiency Ahting U 1, Leiz S 5, Petrova S 5, Baethmann M 5, Alhaddad B 1, Rolinski B 3, Haack T 1, Freisinger P 4, Meitinger T 1 2 1

Human Genetics Technische Univ Munich, Munich, Germany, 2Human Genetics Helmholtz-Zentrum, Munich, Germany, 3Laboratory Pathology Elblandliniken, Meissen, Germany, 4Pediatrics Kreisklinikum Reutlingen, Reutlingen, Germany, 5Pediatric Neurol Klinikum Dritter Orden, Munich, Germany

Severe motor neuronopathy: a clinical hallmark in two young brothers with SUCLG1 mutations Kuster A 1, Caillaux G 2, Benbrik N 3, Ceballos I 4, Benoist J F 7, Pereon Y 5, Lebre A S 6 1

Div Metab Dis, Univ Child Hosp, Nantes, France, 2Neonatology, Univ Child Hosp, Nantes, France, 3Pediatric cardiology, Univ Child Hosp, Nantes, France, 4 Biochemistry, Hop Necker Enfants Malades, Paris, France, 5Neuromusc Ref Center, Univ Hosp, Nantes, France, 6Genetics, Univ Hosp, Reims, France, 7 Biochemistry, Robert Debre, Paris, France Background: Succinate-CoA ligase (SUCL) deficiency is responsible for mitochondrial DNA depletion syndromes, a most prevalent cause of mitochondrial dysfunction in early childhood. SUCL catalyses conversion of succinyl-CoA and ADP/GDP to succinate and ATP/ GTP.Mutations in SUCLG1, encoding α-subunit of the enzyme, are rarer than SUCLA2 and SUCLG2 mutations encoding the β-subunit, and clinical phenotypes are more severe due to combined impaired ATP and GTP synthesis. Case report: The first son of French parents had neonatal surgery for anomalous pulmonary venous return. Follow-up revealed a spinal muscular atrophy phenotype with neurosensory deafness, attributed to perioperative complications. The second boy of the family presented with a similar neurological phenotype and work-up was indicated. Results: Repetitive electromyography and nerve conduction studies, performed because of marked hypotonia, reduced voluntary movements and absent tendon reflexes, showed pure motor axonal neuronopathy with normal velocities, but reduced amplitudes. Mild methylmalonic aciduria with Krebs cycle intermediates led to sequencing of SUCLA2 and subsequently SUCLG1, finding a heterozygous mutation (exon7,c.787G > A). Complementary cDNA tests revealed mRNA decay due to second intronic mutation. Discussion: Clinical phenotype and nerve conduction studies were indicative of a primary pathologic process in neuron cell body of the anterior horn. Motor neurons depend substantially on mitochondrial oxidative phosphorylation for normal function. Spinal muscular atrophy-like phenotypes have previously been reported in other mitochondrial depletion syndromes (TK2) and in mutations in COX assembly gene (SCO2). Encephalomyopathy, peripheral neuropathy and deafness are common features of mitochondrial disease. Here, we report motor neuronopathy as a hallmark of SUCL deficiency. Genes responsible for mitochondrial depletion syndromes should be integrated in gene panels offered for investigation of motor neuronopathy.

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UQCRC2 mutation in a patient with mitochondrial complex III deficiency with recurrent hepatic failure, lactic acidosis and hypoglycemia Gaignard P 1, Eyer D 2, Lebigot E 1, Oliveira C 1, Therond P 1, Boutron A 1, Slama A 1 1 Div Bioch and Metab, APHP-Bicetre Hosp, Le Kremlin Bicetre, France, France, 2Div Ped Haguenau Hosp, Strasbourg, France

Background: Complex III deficiencies are a relatively rare cause of primary mitochondrial defects. Mutations in the mitochondrial gene encoding the cytochrome b and in nuclear genes encoding assembly proteins BCS1L and TTC19 are the most often reported, but the molecular origins of C III deficiencies remain frequently not elucidated. We present here the clinical, biochemical and molecular investigations from a patient with a mitochondrial respiratory chain complex III deficiency. Case report: The patient was born at term after an uneventful pregnancy. At few days of age, he presented poor feeding and laboratory investigations showed a lactic acidosis associated with hypoglycemia and hepatocellular deficiency. A favorable outcome occurred in 10 days within glucose serum perfusion. During the first year of life, he exhibited 5 episodes of hypoglycemia with ketonuria, hyperlactatemia and high lactate/pyruvate ratio. A mitochondrial defect was suspected and investigated by respiratory chain function analysis (oxygen consumption, enzymatic activities and BNPAGE), mitochondrial DNA sequencing and targeted resequencing of the coding exons and flanking intronic stretches of 150 nuclear genes involved in mitochondrial defects. Results: Patient’s fibroblasts analysis showed a strong decrease of C III activity and amount with low oxygen consumption in presence of CIII substrate (decylubiquinol). Molecular analysis revealed a homozygous mutation c.547C > T, p.Arg183Trp in UQCRC2 gene. The parents are heterozygous carriers. Discussion: We present here the second reported case of mutation in UQCRC2 gene, encoding the core protein 2. The mutation is the same as that found in the only other described family with UQCRC2 deficiency. The clinical presentation was nearly similar and marked by neonatal lactic acidosis with favorable longterm neurological outcome. Our study expands the very few descriptions of patients with primary C III deficiencies by nuclear mutations in structural genes.

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COX5 related Complex IV deficiency: primary pulmonary hypertension, failure to thrive, and lactic acidosis. Al-Murshedi F 1, Al-Sineidi K 1, Joshi N 2, Al-Thihli K 1, Bruwer Z 1, Al Kharusi K 1, Al-Mawali A 1, Rodenburg R 3 1 Dept of Genetics, Sultan Qaboos Unv Hosp, Muscat, Oman, 2Dept of Child Health, Sultan Qaboos Unv, Muscat, Oman, 3Dept of Lab Med, Radboud Univ Nijmegen M, Nijmegen, Netherlands

Background: Cytochrome c oxidase (COX) catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. The human cytochrome c oxidase is composed of 13 subunits, ten of which are nuclear-encoded. Case report: We hereby describe the clinical presentation of two female sisters due to homozygous mutations in COX5 nuclear subunit identified through whole-exome sequencing. The first child was born at full term with normal growth parameters at birth but progressed to severe failure to thrive and severe global developmental delay. Upon her presentation at the age of 7 months, her weight was 3.5 kg and she had attained only social smile. She presented with progressive lethargy, vomiting and progressive respiratory distress. Investigations revealed severe supra-systemic pulmonary hypertension with right side heart failure and persistent elevation of lactate (2.3–4.2 mmol/L). The child passed away a few days later as she developed pulmonary hemorrhage during intubation. Her second sister had normal birth growth parameters as well. Persistent elevation of lactate (4.1–10.5 mmol/L) was observed since

birth. The child progressed to severe failure to thrive with current weight at age of 15 months of 3.8 kg and did not achieve head support as yet. Her echocardiography at birth showed mild pulmonary hypertension. Results: Exome sequencing on the first child revealed homozygous c.319C > T, p.Arg107Cys variant of unknown significance in COX5 gene. Oxidative phosphorylation assay on skin fibroblasts of her sister revealed isolated complex IV deficiency of 457 mU/U CII (600–2627). Discussion: This is the first description of human disease due to the subunit COX5 deficiency of the cytochrome oxidase. Our patients share some features to other autosomal recessive COX deficiencies with severe early onset phenotypes including Leigh’s disease and fatal infantile lactic acidosis. However the early onset of pulmonary hypertension is a marked clinical feature in these siblings.

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Two cases with ELAC2 mutations presenting with isolated infancy-onset cardiomyopathy Mastantuono E 1, Seidel H 2, Eichinger A 3, Alhaddad B 1, Prokisch H 1, Haack T 1 1 Inst Hum Genet, Helmholtz Zentrum, Munich, Germany, 2Div Hum Genet, Klinikum Rechts Der Isar, Munich, Germany, 3Div Ped Card, LudwigMaximilians Univ, Munich, Germany

Background: The mitochondrial (mt) genome encodes 13 subunits of the respiratory chain as well as RNA components of the translational machinery. Transcription of the mitochondrial genome produces large polycistronic transcripts and maturation of the mt-tRNAs requires the RNase P-complex and the RNase Z activity of ELAC2 for cleavage at 5’ and 3’ ends, respectively. Defects in nuclear genes encoding factors involved in the maturation and post-transcriptional modification of mt-tRNAs (ELAC2, MTO1, GTPBP3, TRMT5) have been associated with mitochondrial disorders affecting multiple organ systems. Methods: Exome sequencing was performed in two unrelated patients with severe early-onset cardiomyopathy as the key clinical features. The first case, a 4-year-old female, presented with a rapidly progressive cardiomyopathy and muscular dystrophy. Similarly, in a second 6-month-old infant, the predominant feature observed was a severe form of hypertrophic cardiomyopathy leading to death a few months later. Results: Clinical exome sequencing identified bi-allelic ELAC2 variants in both patients. The first case carried two rare heterozygous missense variants (Glu310Ala and Arg781His) affecting highly conserved amminoacid residues. In the second index patient we detected two rare heterozygous variants, one frameshift (Cys670Serfs*14), and one missense (Met343Arg). Discussion: Our findings underline the efficacy of next-generation sequencing approaches in the diagnostic process of individuals with atypical disease presentation. In both patients, mitochondrial disease was initially not regarded a likely differential diagnosis and clinical features such as failure to thrive and occasionally elevated lactate levels have been considered consequences of heart failure. Our findings underline the importance of including mitochondrial cardiomyopathy, and ELAC2 deficiency in particular, in the differential diagnosis of infantile forms of heart failure.

P-373 Functional characterization of a novel mitochondrial translation defect Gardeitchik T 1, Mohamed M 1, Dalloyaux D 1, Brandt U 1, Guerro Castillo S , Karall D 2, Morava E 3, Wevers R A 1

1

1 Radboud University Medical Center, Nijmegen, Netherlands, 2Medical University Inssbruck, Innsbruck, Austria, 3Tulane University, New Orleans, United States

Background: Mitochondria are essential organelles responsible for the generation of energy, mainly through the oxidative phosphorylation

S168 (OXPHOS) system. With their own genetic material and translation machinery mitochondria take a unique place in cellular metabolism. Translation is tightly regulated by an intriguing interplay between mitochondrial and nuclear encoded proteins with a central role for the mitochondrial ribosome (MRP). All reported defects in subunits lead to combined OXPHOS complex deficiencies and many of the patients die in the first months. Recently, we identified a novel genetic defect in one of the mitochondrial ribosomal protein subunits (MRPS2) in a patient presenting with wrinkled skin, hypotonia, sensorineurinal deafness, chronic lactic acidosis and multiple OXPHOS complex deficiencies. At the age of 11 she does not show progressive disease. Methods: To evaluate cellular consequences of this defect we studied the assembly and abundance of MRP and OXPHOS complexes, mitochondrial translation and OXPHOS enzyme activities in patient fibroblasts using complexome profiling, pulse labeling of mitochondrial protein synthesis and OXPHOS enzyme measurements. Results: Decreased levels of the fully assembled 28S ribosomal subunit and the subassembly of this complex were seen in the patient cells. Profiles of the OXPHOS complexes showed a decrease in the amount of fully assembled complex I and IV as well as an accumulation of subassemblies of complex I and complex V. In vitro pulse labeling of mitochondrial protein synthesis showed a generalized translation defect resulting in decreased OXPHOS enzyme activities. Discussion: We identified a new metabolic cutis laxa defect and using a combination of different state-of-the-art techniques we show that this defect in MRPS2 defect leads to a decreased level of functional mitochondrial ribosome, leading to impaired mitochondrial translation with reduced enzyme activity of several OXPHOS complexes.

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Personalized medicine approach confirms a new case of ABAT deficiency Bonnen P E 2, Besse A 2, Appadurai V 2, Lalani S 2, Koenig M K 1 1 Dept Mito Med, Univ Texas Health Science, Houston, United States, 2Dept Mol Hum Gen, Baylor College Med, Houston, United States

Background: ABAT deficiency (OMIM 613163) is a rare inborn error of metabolism caused by recessive mutations in the gene 4aminobutyric acid transaminase (ABAT). To date, only a few patients have been reported worldwide. Their clinical presentation has been remarkably consistent with primary features of severe psychomotor retardation, hypersomnolence, hypotonia, and infantile-onset refractory epilepsy. We report a new case of ABAT deficiency that marks an important departure from previous clinical findings. Case report: The patient presented at age 6 months with global developmental delay, hypotonia, hypersomnolence and mild choreiform movements. At age 18 months, the subject’s clinical presentation was still milder than all previously reported patients and, most notably, did not include seizures. Whole exome sequencing revealed two heterozygous ABAT variants that are rare and predicted damaging, but never before reported in a patient. Leveraging a personalized medicine approach all ABAT variants in previously reported patients plus the variants in this new case were expressed in cells and both functions of ABAT were assessed via GABA transaminase enzyme activity and mtDNA copy number assays. Results: This work confirmed the novel variants compromised ABAT function to similar levels as variants in previously characterized cases with more severe clinical presentation. Additionally, functional studies on patient fibroblasts showed similar levels of compromise in mitochondrial bioenergetics and mtDNA depletion as patients with the classical, severe clinical presentation of ABAT deficiency. Discussion: These results illustrate how cell-based functional studies can aid in the diagnosis of a neurological disorder. Importantly, this patient marks an expansion in the clinical phenotype for ABAT deficiency to a milder presentation that is more commonly seen in pediatric genetics and neurology clinics.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 15. Mitochondrial disorders: mtDNA P-375

Sarcopenic obesity frequently occurs in adult patients with mitochondrial disease Zweers H E E 1, Van de Vorst L A 1 3, Huisman S 1, Leij S 1 2, Wanten G J A 1, Janssen M C H 1 Radboudumc, Nijmegen, Netherlands, 2HAN, Nijmegen, Netherlands, WUR, Wageningen, Netherlands

1 3

Background: Adult patients with mitochondrial disease (MD) are known to be at risk of malnutrition, but data on body composition and physical functioning are lacking. The aim of this study is to describe the nutritional status and food intake, body composition and physical functioning in MD. Methods: Body composition of adult MD patients was measured by DXA, anthropometric characteristics and food intake were also assessed. Nutritional status was evaluated by the Patient Generated Global Assessment (PG SGA). Sarcopenia was defined as low fat free mass index (FFMI) combined with a low score in at least one of the following tests of physical functioning: handgrip strength (HG)-, 30 sec sit to stand-test (30SCT) and 6 min walk tests (6MWT). Central obesity was defined as high waist circumference, World Health Organization (WHO) reference values were used to assess body composition. Results: 35 MD patients (age 44 ± 10 yr; 14 males) had an average BMI of 23 kg/m2 (range 18–28). FFMI in women was 14.3 ± 1.7 kg/m2 and 16.4 ± 2.4 kg/m2 in men, 60 % had a FFMI below reference values. The majority of patients had low physical functioning according to HG test (78 %) 30 SCT (81 %) and 6 MWT (88 %). Sarcopenia was present in 54 % and malnutrition in 38 %. Central obesity was observed in 57 % of patients and 97 % had a high fat%. Food intake covered 79 ± 23 % and 95 ± 33 % of energy and protein requirements, respectively. Discussion: In spite of BMI not qualifying MD patients as being obese, sarcopenic obesity was frequently observed in adult MD patients. Furthermore, malnutrition and low physical functioning seem to be common in this study population. Therefore, in order to provide optimal support to MD patients, it appears mandatory to do a complete nutritional assessment in these patients including measurement of food intake, body composition and physical functioning.

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Acyl-carnitine profile mimicking multiple acyl-CoA dehydrogenase deficiency in a patient with mitochondrial myopathy and a mutation in the MT-CO2 gene Kollberg G 1, Asin-Cayuela J 1, Hedberg-Oldfors C 1, Oldfors A 1, Tulinius M 1 1

Sahlgrenska Academy Univ of Gothenburg, Gothenburg, Sweden

Background: Mitochondrial fatty acid β-oxidation (FAO) and the respiratory chain (RC) are connected via the ubiquinone pool and the citric acid cycle. Thus, defects in either pathway may affect the other secondarily. Whilst FAO disorders are inherited in an autosomal recessive manner, RC disorders can present with autosomal dominant, recessive, X-linked or maternal patterns of inheritance depending on whether nuclear DNA or mitochondrial DNA (mtDNA) is mutated. The two groups of disorders share some common clinical and biochemical features e.g. myopathy, exercise intolerance, encephalopathy and lactic acidosis, but differ regarding treatment and genetic counseling. Case report: Our patient is a 16-year-old girl with a life-long history of exercise intolerance which has accentuated during the past 3 years. Laboratory investigations showed increased urinary excretion of lactate and 2hydroxyglutarate. Her acyl-carnitine profile in serum was consistent with multiple acyl-CoA dehydrogenase deficiency (MADD). Her clinical phenotype differed slightly from MADD and she was referred for mitochondrial investigation. A biopsy from the quadriceps femoris muscle was taken for histochemical and biochemical analyses.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Results: Biochemical analysis showed an isolated and extremely low activity of cytochrome c oxidase (COX). Histochemical examination revealed that only a few fibers exhibited normal COX activity and abnormal mitochondrial proliferation. Sequencing of mtDNA revealed a one-base pair deletion (m.8087delT) leading to a premature stop codon in the MT-CO2 gene encoding subunit II in complex IV of the RC. The level of heteroplasmy was >95 % in the examined muscle tissue. Discussion: The presented case illustrates the clinical and biochemical overlap between FAO and RC disorders, and particularly the need to expand the differential diagnosis of MADD-like acylcarnitine profiles to RC disorders, especially those affecting components of the RC downstream of ubiquinone.

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Unusual intragenic MT-ND1 inversion detected in the eye lens sample from patient with Leigh-like presentation Ciara E 1, Pelc M 1, Kowalski P 1, Jurkiewicz E 2, Piekutowska-Abramczuk D , Trubicka J 1 , Prost M 3 , Halat P 1 , Rokicki D 4 , Jurkiewicz D 1 , Siestrzykowska D 1, Pajdowska M 5, Iwanicka-Pronicka K 6, Stawinski P 7 8 , Ploski R 7, Krajewska-Walasek M 1, Pronicka E 1 4 1

1 Dept Med Genet, CMHI, Warsaw, Poland, 2Dept Radiol, CMHI, Warsaw, Poland, 3Dept Ophthal, MIAM, Warsaw, Poland, 4Dept Paed Nutr Metab Dis, CMHI, Warsaw, Poland, 5Dept Biochem, CMHI, Warsaw, Poland, 6Dept Aud Phon Lar, CMHI, Warsaw, Poland, 7Dept Med Genet, WMU, Warsaw, Poland, 8 Dept Genet, IPPH, Warsaw, Poland

Background: Mutations in mtDNA are an important cause of mitochondrial disorders (MD) in pediatric patients. Numerous heteroplasmic pathogenic variants including point mutations and large-scale rearrangements responsible for various MD phenotypes have been identified. They are usually maternally inherited except for sporadic mtDNA large deletions. An intragenic inversion in MTND1 have been reported only twice; in an adult man presenting with exercise intolerance and in a female infant with biventricular cardiomyopathy, aortic coarctation and severe lactic acidosis. Complex I deficiency was confirmed in both. Case report: A 2-year-old boy, presented in infancy with hypotonia, severe failure to thrive, cardiomyopathy and cataracts requiring surgery. There were lactic acidemia and tendency to neutropenia, hyponatremia and hypocapnic alkalosis. In second year of life Leigh-like changes developed. Muscle biopsy examinations showed subsarcolemmal densities, type I fibers’ predominance and lipid accumulation. OXPHOS assessment could not be done due to a technician’s fault. After negative results of genetic testing for common mtDNA mutations (m.3243A > G, m.8344A > G, m.8993 T > G/C, m.8470_13447del4977), the whole-exome and mtDNA genome sequencing were performed. Results: A rare m.3902_3908invACCTTGC alteration in the MT-ND1 gene was found in the patient’s muscle, urine, eye lens (heteroplasmy level >90 %) and blood samples (50 %). The mutation occurred de novo, as it was excluded in his mother, brother and grandmother. Discussion: We present the third case of a de novo mtDNA intragenic inversion in a patient with Leigh-like syndrome expanding the phenotypic range of this unusual mtDNA alteration. Its sporadic occurrence in the family resemble the inheritance pattern of large mtDNA deletions. This is the first report on detection of mutant mtDNA heteroplasmy in the eye lens sample. The study was supported by: UMO-2013/08/M/NZ5/00978, 216/12, S136/13, S134/13, S140/14.

P-378

Mitochondrial Network properties in Cultured Myoblasts from Patients with Mitochondrial Disorders Hansikova H 1, Krizova J 1, Sladkova J 1, Capek M 2, Tesarova M 1, Honzik T 1 , Martinek J 3, Zamecnik J 4, Kostkova O 1, Langer J 1, Zeman J 1 1 Dep Ped, 1st Fac Med, Charles Univ, Prague, Czech Republic, 2Dep Biomathematics, Inst Physiol, CAS, Prague, Czech Republic, 3Inst Histol

Embryol, 1st Fac Med, CUNI, Prague, Czech Republic, 4Dep Pathol Mol Med, Hosp Motol, Prague, Czech Republic Background: Mitochondrial disorders represent a heterogeneous group of more than 250 diseases. Although an increasing number of patients may be diagnosed directly by molecular analyses, muscle biopsies still serve as the gold standard for diagnostics. Cultured muscle progenitor cells, myoblasts, may be used in the structural and biochemical analyses, and thus can meet the need to limit the volume of invasively obtained bioptic samples, especially in small children. Methods: Cultivated myoblasts were isolated from muscle biopsy of patients with mitochondrial disorders, including CPEO, MELAS and TMEM70 deficiency. Mitochondrial networks and ultrastructure were visualized by fluorescence microscopy and transmission electron microscopy, respectively. Quantitative analysis of the mitochondrial networks morphology in cultivated cells was performed with the software package Fiji (fiji.sc; UW-Madison,WI). Mitochondrial phenotypes were correlated with the biochemical findings in muscle and the results of molecular genetic analysis in each patient. Results: A heterogeneous picture of abnormally sized and shaped mitochondria with fragmentation, shortening, and aberrant cristae, lower density of mitochondria and an increased number of “megamitochondria” were found in patient myoblasts. Morphometric Fiji analyses revealed significantly decreased mitochondrial network parameters such as the mitochondria/cell ratio, the aspect ratio, the form and the branching factor in myoblasts from patients in comparison to controls (P < 0,05). Discussion: Although image analysis based on exact algorithms it is not sufficient to distinguish between specific mitochondrial disorders, the results of our study suggest that cultivated myoblasts and semiautomatic morphometric analyses are useful supplemental tools for the estimation and clarification of mitochondrial disorders. Supported by PRVOUK P49, P24/LF1/3 and RVOVFN64165

P-379

3-methylglutaconic aciduria in a patient with fatal infantile cardiomyopathy due to a m.3303C > T MT-TL1 mutation. Joost K 1, Poder H 2, Kall K 3, Taylor R W 4, Blakely E L 4, Jalas T 2, Zordania R 1 Tartu Univ Hosp, Gen Dept, Tallinn, Estonia, 2Tallinn Child Hosp, Tallinn, Estonia, 3Central Lab of Health Board, Tallinn, Estonia, 4Wellc Trust Cent Mit Res, Newcastle Univ, Newcastle, United Kingdom 1

Background: 3-methylglutaconic aciduria (3-MGA-uria) is relatively common finding in patients with a possible metabolic disorder often related to primary or secondary mitochondrial dysfunction. The association of 3-MGA-uria with some nuclear genes (AUH, TAZ, TMEM70 etc.) is known, but also the association with some mitochondrial genes is possible. Hypertrophic cardiomyopathy (hCMP) is the most frequent cardiac manifestation of mitochondrial disease with the m.3303C > T MT-TL1 gene mutation causing a rare, but severe form of the disease—fatal infantile hCMP. Objective: to present a patient with fatal infantile hCMP with moderate 3-MGA-uria due to the m.3303C > T MTTL1 gene mutation. Case report: The patient presented at the age of 6 months with extrasystolia observed during routine check-up. Subsequent investigations revealed hCMP. Metabolic investigations revealed high lactate in blood and ketosis with increased excretion of 3-MGA and Krebs cycle intermediates in urine. Results: MT-TL1 gene mutation m.3303C > T was identified at very high levels of heteroplasmy, possibly homoplasmic, in the patient’s blood and fibroblast derived DNA samples (99 % in both tissues). Subsequent quantitation of mutation load in blood and urinary epithelia from maternal relatives identified high levels of the m.3303C > T MT-TL1 mutation in the patient’s mother (85 % and 86 % respectively), whilst the mutation was present at low levels in the patient’s maternal grandmother (5 % blood and 9 % urinary epithelia).

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A review of anaesthetic outcomes in a cohort of patients with genetically confirmed Mitochondrial Disorders

Case report: Here, we present the case of a young woman with childhoodonset seizures, developmental delay, left hemiparesis, tremor, migraines, optic atrophy, hypertrophic cardiomyopathy, episodic ventricular tachycardia, and premature death at 23 years of age due to multiple stroke-like episodes. Postmortem brain examination showed multifocal stroke-like lesions of grey and white matter, and areas of marked spongiosus with neovascularization. Results: Muscle examination showed negative COX staining without raggedred fibres. Sequencing of mtDNA in muscle showed 97 % heteroplasmy for a m.5537_5538insT insertion mutation in the tRNA(Trp) gene MT-TW, previously reported in the context of Leigh disease. Discussion: Based on the few available previous reports of this mutation, complex IV deficiency in muscle appears to be a consistent feature. As far as we are aware, this is the first reported occurrence of stroke-like episodes in this condition, which should be considered in the differential diagnosis of MELAS.

Smith A 1, Dunne E 2, Mannion M 1, O’Connor C 3, Knerr I 1, Monavari A 1, Hughes J 1, Eustace N 2, Crushell E 1

P-382

Despite a relatively high mutation load, echocardiographic investigations revealed no hCMP in the patient’s mother. Discussion: Fatal infantile hCMP due to the m.3303C > T mutation can be accompanied by moderate excretion of 3-MGA. The exclusion of mitochondrial DNA mutations in infantile cardiomyopathy is important in familial recurrence risk evaluation. Relatively high mutation load of m.3303C > T in MTTL1 may have an asymptomatic presentation.

P-380

1 Nat Cent for Inher Metab Dis, Temple St, Dublin, Ireland, 2Dept Anaesthesia, Temple St, Dublin, Ireland, 3Dept Met Medicine, OLCHC, Dublin, Ireland

Background: General anaesthesia (GA) poses a potential risk of metabolic decompensation in children with mitochondrial disorders and there is little guidance for anaesthetists and clinicians regarding optimal anaesthetic agents and perioperative management to provide to patients with mitochondrial disease. The literature to date consists of case reports/series which included patients with various mitochondrial diseases with and without genetic confirmation. The aim of this study was to document adverse events and complications from GA in paediatric patients with genetically confirmed mitochondrial disorders. Methods: A retrospective chart review of paediatric patients with genetically confirmed mitochondrial disorders who had undergone GA was undertaken. The indication for GA, anaesthetic agents utilised, length of admission and post anaesthetic complications were documented and analysed. Results: 26 patients disease underwent 65 GAs. 57 (87 %) were under GA for less than 1 h. 34 (52 %), received propofol as their induction agent. 33 (51 %) patients received sevoflurane for the maintenance of anaesthesia, 8 (12 %) received isoflurane and 24 (37 %) received propofol for maintenance. Complications occurred in 5 cases (7 %) while under GA, including ST segment depression in one, hypotension and mild metabolic acidosis. All were stabilised successfully and none required ICU admission. Discussion: Adverse events directly attributable to GA were uncommon in this cohort. No relationship between choice of anaesthetic agent and complications was observed. It is likely that individual care optimisation on a case-by-case basis is more important overall than choice of any one particular anaesthetic technique.

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MELAS-like presentation of mitochondrially-encoded tRNA(Trp) deficiency Lines M A 1 2, Rupar C A 3 4, Geraghty M T 1 2 1 Dept Peds, Fac Med, Univ Ottawa, Ottawa, Canada, 2Div Metab, Child Hosp East Ontario, Ottawa, Canada, 3Dept Peds, LHSC and Univ West Ontario, London, Canada, 4Dept Path and Lab Med, Univ West Ontario, London, Canada

Background: Transfer RNAs and their cognate aminoacyl-tRNA synthetases govern both the substrate availability and the specificity of protein translation. The mitochondrial genome, which uses a genetic code distinct from that of the nucleus, includes 22 mtDNA-encoded tRNA genes (mt-tRNAs). The best recognized clinical mt-tRNA disorder is mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes (MELAS), ~80 % of which is caused by mutations in the tRNA(Leu(UUR)) gene MT-TL1.

Evidence of multiple carboxylase deficiencies in seven unrelated patients with mutations in MT-ATP6 Weisfeld-Adams J D 1 2, Heese B A 3 4, Hauser N 5, Balasubramaniam S 6, Christodoulou J 9, Glamuzina E 7, Van Karnebeek C 10, Mattman A 8 10, Kluijtmans L A 11, Williamson A L 13, Diaz G A 13, Rutledge S L 12, Van Hove J K L 1 2, Larson A 1 2 1 University of Colorado, Denver, United States, 2Children’s Hospital Colorado, Aurora, United States, 3University of Missouri-Kansas City, Kansas City, United States, 4Children’s Mercy Hospital, Kansas City, United States, 5Valley Children’s Hospital, Madera, United States, 6Metab Service, Child Hosp at Westmead, Sydney, Australia, 7Metabolic Service, Starship Child Hosp, Auckland, New Zealand, 8Adult IMD Clinic, UBC, Vancouver, Canada, 9Murdoch Child Research Institute, Melbourne, Australia, 10Center for Mol Med and Therapeutics, UBC, Vancouver, Canada, 11Radboud University Medical Centre, Nijmegen, Netherlands, 12University of Alabama at Birmingham, Birmingham, United States, 13Icahn School of Medicine at Mount Sinai, New York, United States

Background. Co-existent elevations ofpropionylcarnitine (C3) and 3hydroxyisovalerylcarnitine (C5OH) on acylcarnitine profile (ACP) are evidence of deficient activity of multiple biotin-dependent carboxylase enzymes. Typically, multiple carboxylase deficiency (MCD) results from biallelic mutations in either BTD (biotinidase) or HCLS (holocarboxylase synthetase). Methods. Follow-up urine organic acid (UOA) analysis and molecular genetic testing including mtDNA analysis in unrelated individuals with co-existent C3 and C5OH elevations on ACP. Results. Seven individuals with abnormal ACP suggestive of MCD were found to have mutations in MT-ATP6, namely m.8993 T > G (all >87 % heteroplasmy in blood) in six patients, and m.9176 T > G in one patient (homoplasmic in blood/urine). Most were initially ascertained with abnormal ACP on newborn screening through tandem mass spectrometry. All had normal enzymatic and/or molecular genetic testing to exclude biotinidase and HCLS deficiencies. All patients were treated with supplemental biotin; 6/7 showed no biochemical response. 5/7 patients had evidence of MCD on UOA analysis. Outcomes ranged from death in early childhood from neurodegenerative disease or cardiomyopathy to clinically asymptomatic with normal development over 5 years of follow up. Discussion: These cases expand the biochemical phenotype associated with MT-ATP6 mutations, especially m.8993 T > G. Mechanisms of inhibition of biotin-dependent carboxylases in the setting of MTATP6 mutations remain unclear, although 3-methylcrotonyl-CoA and propionyl-CoA carboxylase are located in proximity to Complex V in the mitochondrial matrix. There is no historical precedent of reports associating MT-ATP6 mutations with biochemical evidence of MCD, but clinicians should be aware of this association and consider mtDNA sequencing for patients with combined elevations of C3 and C5OH acylcarnitines or evidence of MCD on UOA, once biotinidase/ HCLS deficiencies are excluded.

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Mitochondrial optic neuropathies—optimization of diagnosis and management strategy Kolarova H 1, Liskova P 2, Forgac M 3, Dvorakova V 1, Havrankova P 3, Tesarova M 1, Honzik T 1, Zeman J 1 1 Dpt of Pediatrics and Adolescent med, Prague, Czech Republic, 2Dpt of Ophthalmology, Prague, Czech Republic, 3Dpt of Neurology, Prague, Czech Republic

Background: Leber hereditary optic neuropathy (LHON) and dominant optic atrophy (DOA), caused by mutations in OPA1, represent the two most common mitochondrial optic neuropathies (MON) responsible for progressive visual failure. Although plus symptoms are reported in up to 30 % of patients with DOA, in patients with LHON, these are rather rare findings. The aim of our work was to establish a specialized medical centre in the Czech Republic, dedicated to patient care and research of MON. Methods: A detailed neuroophthalmic examination of 42 patients with LHON, 14 patients with DOA, 33 patients with MELAS and 6 patients with MON of unknown origin was performed within a period of 2 years. Results: All patients with LHON had one of the 3 prevalent mutations m.11778A > G (35), m.3460G > A (4) and m.144884 T > C (3) in one of the complex I subunits. A total of 20 out of 42 patients (from 12 families) were symptomatic. Interestingly, plus symptoms were present in 10 patients (hearing loss, peripheral neuropathy, myopathy, tremor). In the group of 14 patients (from 7 families) with DOA, all had pathogenic mutations in OPA1. Plus symptoms were observed in 3 patients (hearing loss, peripheral neuropathy, myopathy and multiple sclerosis-like phenotype). Thanks to the early diagnosis in our centre, we were able to initiate therapy with coenzyme Q analogue (idebenone) in 5 symptomatic patients in a period of 4 weeks– 9 months after the disease onset. This treatment was further commenced in 5 patients with subclinical signs of the disease. Optic atrophy, in association with retinitis pigmentosa, was observed only in 2/33 patients with MELAS. In one patient with unknown cause of MON a rare m.13046 T > C mutation was revealed, manifesting with unique phenotype of LHON/MELAS overlap syndrome. Discussion: We report the high incidence of plus symptoms in the Czech LHON patients and benefits of centered care. Supported by GAUK 38515, AZV 16-32341A, VFN-RVO 64165 and SVV 260256.

P-384 Withdrawn

16. Disorders of purines, pyrimidines, nucleic acids and porphyrias

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Hypoxanthine intrastriatal administration alters redox status and provokes DNA damage in striatum of infant and young adult rats Biasibetti H 1, Pierozan P 1, Rodrigues A F 1, Prezzi C A 1, Manfredini V 2, Wyse A T S 1 UFRGS, Porto Alegre, Brazil, 2Universidade Federal do Pampa, Bage, Brazil

1

Background: Deficiency of the enzyme hypoxanthine-guanine phosphoribosyltranferase (HPRT) characterizes the Lesch-Nyhan Dis e as e ( LND) , w h ic h is an inb or n e r ro r o f m et ab ol ism . Hypoxanthine is the major oxypurine metabolite involved in the purine salvage pathway in the brain and accumulates in this condition.

The aim of this research was to study the effects of hypoxanthine intrastriatal administration on infant and young adult rats submitted to stereotactic surgery. We investigated the effect of hypoxanthine on some oxidative parameters, including reactive oxygen species production, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, as well as DNA damage. We also analyzed astrocyte and microglial markers for inflammation, including ionized calcium-binding adapter molecule 1 (IBA1) and glial fibrillary acidic protein (GFAP). Methods: Wistar rats of 21 and 60 days of life underwent stereotactic surgery and were divided into two groups: control (infusion with saline 0.9 %) and hypoxanthine (10 μM). Animals were decapitated 30 min after the injection and the striatum was utilized for further analysis. Results: Hypoxanthine increases reactive species production in both ages of rats, and decreases SOD, CAT and GPx activities in 21-dayold rats. We also analyzed DNA damage and detected a massive increase in the DNA damage index in the striatum of 21 and 60day-old rats. Microglial and astrocyte activation was seen by the increase in IBA1 and GFAP immunocontent, respectively, in striatum of infant rats. Discussion: According to our results, hypoxanthine intrastriatal administration alters redox status suggesting a strong neurotoxic role in oxidative stress. Hypoxanthine also increases neuroinflammatory parameters, perhaps through oxidative misbalance, suggesting that this process may be involved, at least in part, to neurological disorders found in patients with LND. Supported by CNPq.

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Development of a LC-MS/MS method for the quantitation of purine and pyrimidine metabolites in human urine Cremonesi A 1, Perkins N 1, Haeberle J 2, Hersberger M 1 1 Div Clin Chem Biochem, Univ Child Hosp, Zurich, Switzerland, 2Div Metabolism, Univ Child Hosp, Zurich, Switzerland

Background: Purines (Pu) and pyrimidines (Py) are biologically ubiquitous compounds involved in a multitude of biochemical processes. Their physiological importance is underlined by the fact that they can be synthetized by nearly all cells and that errors in their metabolism often lead to very debilitating diseases. To date, more than 30 enzyme defects in the metabolism of Pu/Py have been characterized. It is therefore crucial to accurately quantify these metabolites for diagnostic and therapeutic purposes. Methods: Urine samples are warmed up at 37 °C for 30’, filtered, diluted with 0.1 % formic acid to 0.5 mM Creatinine and spiked with an internal standard mix. For each sample, 2 μL are separated on an Acquity UPLC HSS T3 column (Waters) coupled to a Sciex TripleQuad 5500 (AB Sciex). Results: The final LC-MS/MS method includes 32 clinically relevant Pu/Py metabolites. This method is linear (r 2 > 0.99), sensitive (LLOQ = 0.040–1.00 μM), precise (intra-assay CV = 1.0 %–15 %; inter-assay CV = 5.0–17.0 %) and has a broad dynamic range. The accuracy was assessed by analyzing 8 ERNDIM EQA samples with established consensus concentrations and was within ±3σ and ±2σ in 99.7 % and in 94.0 % of the measurements, respectively. Ion suppression was notable for certain analytes but its impact was minimized using isotopically-labelled internal standards. Discussion: The validated LC-MS/MS method was used for selective screening of clinical samples and allowed the identification of patients with Adenylosuccinate lyase deficiency, Lesch-Nyhan syndrome or Adenine phosphoribosyltransferase (APRT) deficiency, who have been further confirmed by mutation analysis. Treatment with Allopurinol was started in the patients suffering from Lesch-Nyhan syndrome and APRT deficiency and led, in the latter case, to an improvement

S172 of the clinical picture, which was confirmed by a marked reduction in the excretion of 2,8-dihydroxadenine.

P-387

Altered pre-mRNA splicing due to a novel intronic mutation c.1443 + 5G > A in the dihydropyrimidinase (DPYS) gene Nakajima Y 1, Meijer J 2, Zhang C 3, Wang X 4, Ito T 1, Van Kuilenburg A B P 2 Dept of Pediatr, Fujita Health Univ, Toyoake, Japan, 2Lab Genet Metab Dis, Academic Med Cente, Amsterdam, Netherlands, 3 Dept of Res and Development, MILS Int, Kanazawa, Japan, 4Beijing Children’s Hospital, Beijing, China 1

Background: Dihydropyrimidinase (DHP) deficiencyis an autosomal recessive disease causedby mutations in the DPYS gene and patients present with strongly elevated levels of dihydrouracil and dihydrothymine in urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Case Report: The first patient was a girl who experienced an episode of loss of consciousness at 3 months old. After the incident, she showed normal development. When she was 10 years old, she presented with dizziness, unconsciousness and seizures. The second patient was a 22-month-old girl who presented with seizures and mental and motor developmental delay. The level of pyrimidine metabolites in urine were determined using HPLC-MS/MS. Exons and their flanking sequences of DPYS were amplified. To investigate the function of mutant dihydropyrimidinase, the mutations were introduced into pcDNA3.1-DHP and expressed in HEK293. Results: Urine analysis showed the characteristic pattern for DHP deficiency. Molecular analysis showed that both patients were compound heterozygous for a novel intronic mutation c.1443 + 5G > A in intron 8 and a previously described missense mutation c.1001A > G (p.Q334R) in exon 6. Wild type and the mutated minigene constructs, containing exon 7, exon 8 and exon 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443 + 5G > A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Discussion: Thus, the minigene analysis suggests that the c.1443 + 5G > A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

P-388

Preparation of individual intermediates of de novo purine synthesis as standards for LC-MS/MS Krijt M 1, Skopova V 1, Baresova V 1, Souckova O 1, Zikanova M 1 1

IIMD 1 Fac Med Charles Uni Prg, Prague, Czech Republic

Background: Purines are basic compounds of all living organisms. To date there are known two genetic disorders in de novo purine synthesis (DNPS) caused by mutations in individual genes coding enzymes of this pathway. Known disorders cause neurological symptoms and accumulation of substrates for affected enzyme. Defects in other four DNPS enzymes have not yet been found, but their existence is expected. These defects may remain hidden owing to the absence of diagnostics methods. The major constraint is

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 the commercial unavailability of DNPS intermediates applicable as standards for a technique of LC-MS/MS, one of the suitable methods for analysis of accumulated compounds in body fluids of patients. Methods: First we prepared constructs with individual sequences of DNPS enzymes, introduced them into E. coli and expressed recombinant enzymes. Then we generated intermediates of DNPS pathway by enzymatic reactions and dephosphorylated them by calf intestine phosphatase. We purified all products by LC and used them as standards for LC-MS/MS analysis. Results: We prepared all six enzymes of DNPS pathway and utilized them to produce seven intermediates of DNPS. Then we prepared their dephosphorylated products. For more accurate development and validation of LC-MS/MS detection methods we synthetized also six multiple isotopically labelled forms of DNPS intermediates. Discussion: Specialized laboratory screening of patients with neurological symptoms for metabolic disorders may not show satisfactory results due to the limits of diagnostic methods. We validated diagnostic methods for all plausible genetically determined DNPS disorders based on detection of all possible accumulated substrates of DNPS enzymes by LC-MS/MS. This work was supported by grant AZV 15-28979A from the Ministry of Health, CR, grant GAUK 818416 and programs: UNCE 204011, PRVOUK-P24/LF1/3, SVVUK 260256/2016 from the Charles University in Prague, CR.

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Genetic and transcriptomic approaches of Lesch-Nyhan Disease Ceballos-Picot I 1, Mockel L 1, Ledroit M 1, Dauphinot L 2, Petitgas C 1, Potier M C 2, Auge F 3, Olivier-Bandini A 3 1 Metab Lab Necker Hosp, Paris, France, 2ICM Pitie Salp Hospital, Paris, France, 3Sanofi, Chilly Mazarin, France

Background: Lesch-Nyhan disease (LND) is an X-linked metabolic disease caused by various mutations in the HPRT1 gene encoding an enzyme of purine metabolism, hypoxanthine guanine phosphoribosyltransferase (HPRT). In its most severe form, LND patients suffer from overproduction of uric acid along with neurological or behavioural difficulties including self-injurious behaviours. Methods: In France a cohort of 139 LND patients from 112 families were diagnosed using HPRT enzymatic assay in red blood cells and HPRT1 gene analysis (from 1987 to 2015). To gain more insight into pathogenesis, we compared the transcriptome from human LND fibroblasts and induced Pluripotent Stem cells (iPS) to human fibroblasts and iPS cells without HPRT deficiency. Results: Ninety-eight patients displayed LND full phenotype (86 families) and 41 (26 families) had attenuated clinical phenotypes named LN Variants (LNV). Genotype/phenotype correlations show that LND full phenotype was correlated to genetic alterations resulting in null enzyme function, while variant phenotypes are often associated with missense mutations allowing some residual HPRT activity. A microarray with 60,000 probes corresponding to the entire human genome was used and comparison using stringent criteria identified transcripts, confirmed by quantitative RT-PCR, to be dysregulated in LND cells. Moreover, bioinformatic analysis of microarray data using gene ontology (GO) highlighted clusters of genes displaying biological processes most significantly affected in LND cells. DISCUSSION: The affected genes belonged to specific processes such as cell cycle and cell-division processes, metabolic and nucleic acid processes, demonstrating the specific nature of the changes and providing new insights into LND pathogenesis.

17. Peroxisomal, sterol, bile acid, lipid and lipoprotein

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Blood lysophosphatidylcholine: a diagnostic marker for X-linked adrenoleukodystrophy Mashima R 1, Tanaka M 1, Sakai E 1, Kumagai T 1, Kosuga M 1, Okuyama T 1 1

National Center Child Health Development, Tokyo, Japan

Background: X-linked adrenoleukodystrophy (X-ALD) is associated with an extracellular accumulation of very-long chain fatty acids due to an impaired peroxisomal membrane transporter ABCD1. Accumulating evidence ha suggest that 1-hexacosanoyl-2-hydroxy-snglycero-3-phosphocholine (Lyso-PC 26:0) may be a selective biomarker for X-ALD. Although approximately a 10-fold increase in the concentration of Lyso-PC 26:0 in the dried blood spots (DBSs) from XALD-affected individuals were reported, it remained unclear whether the population of carriers might be correctly identified in the clinical samples . To answer this question, we examined the concentrations of Lyso-PC 26:0 in DBSs from carriers using LC-MS/MS. Methods: A 3-mm DBS from ALD-affected individuals, carriers and healthy controls was treated with methanol containing deuterated internal standard. The concentration of Lyso-PC 26:0 were measured using an LCMS-8040 equipped with a Nexara UPLC (Shimadzu). Recovery study was performed by measuring the recovered concentration of Lyso-PC 26:0 in the QC DBS which had been prepared by the whole blood supplemented with known amounts of Lyso-PC 26:0. Results: Under our experimental conditions, the amounts of Lyso-PC 26:0 in the DBSs from the healthy controls and ALD-affected individuals were 0.090 ± 0.004 (n = 11) and 1.078 ± 0.217 pmol/DBS (n = 4), respectively. The concentration of Lyso-PC 26:0 in the DBSs from the carriers were 0.548 ± 0.095 pmol/DBS (n = 3), indicating that the population of the carriers were also identified. The recovery of Lyso-PC 26:0 from the QC DBSs was 73.6 ± 0.3 % when 2 μM of Lyso-PC 26:0 was spiked into the blood. Discussion: These results suggest that blood Lyso-PC 26:0 can be used as a diagnostic biomarker for X-ALD.

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Successful living donor liver transplantation (LDLT) for a Korean infant with oxysterol 7α-hydroxylase deficiency Yoo H W 1, Lee B H 1, OH S H 1, Namgung J M 2, Kim D Y 2, Hwang S 2, Kim K M 1 1 Dept Ped, Asan Med Ctr, Univ Ulsan, Seoul, Republic of Korea, 2Dept Surgery, Asan Med Ctr, Univ Ulsan, Seoul, Republic of Korea

Background: Inborn errors of bile acid synthesis constitute an expanding category of rare genetic disorders accounting for up to 1–2 % of cases of neonatal cholestasis. Nine inborn errors of bile acid synthesis have been reported, including oxysterol 7α-hydroxylase deficiency. This rare inborn error of bile acid synthesis often progresses to fatal liver failure during infancy. So far, only a few patients with an oxysterol 7α-hydroxylase deficiency treated with LDLT were reported . Case Report: A 3 month old Korean male infant was brought to our Institute because of protracted neonatal cholestasis with progressively deteriorating liver function. Jaundice was noted 1 week after birth and persisted. Dark yellow jaundice was apparent on entire body with mild hepatosplenomegaly. Laboratory tests showed highly elevated AST/ALT, normal GGT, conjugated hyperbilirubinemia and profound coagulopathy, indicating hepatic failure. Giant cell hepatitis with moderate cirrhosis was observed in liver biopsy.

Results: Urine bile acids analysis by LC/MS/MS using dried urine spots (kindly performed by Drs. Kimura & Nittono, Japan), revealed profound excretion of 3-β-monohydroxy-Δ5-bile acid derivatives, strongly suggesting an oxysterol 7α-hydroxylase deficiency. We sequenced the CYP7B1 gene and identified compound heterozygosity for p.Arg388Ter and p.Tyr469Ilefs*5 mutations., He was initially put on urdeoxycholic acid but his liver function continued to worsen. He eventually received living donor liver transplantation (LDLT) at the age of 4 months. At present (8 months of age), his l i v e r f u n c t i o n i s p e r f e c t l y n o r ma l a s a r e h i s g r o w t h a n d development. Discussion: The oxysterol 7α-hydroxylase deficiency is a rare inborn error of bile acid biosynthesis, causing fatal cholestatic liver failure during infancy. The chenodeoxycholic acid therapy might be beneficial before irreversible liver damage occurs. Our patient has severe mutations and hepatic failure, necessitating LDLT.

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Long term outcome of early liver transplantation for a peroxisome biogenesis defect: 15 years follow up. Van Maldergem L 1, Stephenne X 2, Fagnart D 7, Kestens C 7, Deltenre P 3, Bosschi A 4, Ferdinandusse S 6, Deggouj N 5, Debray F G 8, Nassogne M C 9, Moser A 10, Wanders R J 6, Sokal E 2 1 Ctr genet hum, Univ Franche-Comte, Besancon, France, 2Dept Medicosurg Gastroenterol, U Louvain, Brussels, Belgium, 3Dept Neurol Rehabilit, Univ Hosp, ULB, Brussels, Belgium, 4Dept Ophtalmol, U Louvain, Brussels, Belgium, 5Dept Otorhinolaryngol, U Louvain, Brussels, Belgium, 6Lab Genet Metabol Dis, Acad Med Ctr, Amsterdam, Netherlands, 7 Med Dept,Instit Royal Sourds et Aveugles, Brussels, Belgium, 8Metabol Unit, Univ Hosp, Liege, Belgium, 9Dept Ped Neurol, U Louvain, Brussels, Belgium, 10Peroxisomal Lab, Kennedy-Krieger Instit, Baltimore, United States

Background: We described, 10 years ago, a successful orthotopic liver transplantation (OLT) from a related donor in a 6-m old female infant suffering a PEX1-related generalized peroxisomal disorder. Although a significant biochemical improvement, including C26/C22 ratio, clearance of abnormal plasma bile acids and phytanic acid plasma concentration decrease, was obtained, no long term information on her clinical course was provided. Case Report: She walked without assistance in due course and showed normal developement of her communication skills and social interaction. Sphincter control was achieved normally. At 16 y, she is able to sustain a conversation. Following a transient electrophysiological improvement, hearing loss leading to moderate deafnes was observed : 50 dB at 4000Hz after cochlear implant performed at 12y. No overt retinitis pigmentosa developed but ERG amplitudes remained decreased and her visual acuity is currently < 1/10 on both eyes with concentric narrowing of her visual fields. Results: This is in sharp contrast with the course of her affected sister who did not receive any treatment until the age of 4.5 y, when she received regular infusions of hepatocytes,which allowed her biochemical parameters to correct to a similar level to that of her younger sister. However, the improvement in her social interaction appeared quite limited compared to the level achieved by her sister, even though a cochlear implantation at 14y was successful in terms of hearing recovery. Her visual acuity is under 1/20. At 18 y, she functions in the severe to profound intellectual deficiency range (IQ < 35) with an ASD . Her extraneurological complications seem nonetheless significantly milder than those observed in untreated PBD patients Discussion: We speculate that postnatal toxic effects of metabolites (VLCFA, THCA, DHCA) induce irreversible CNS damage that can be at least partially prevented by early OLT.

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P-393

P-395

Lipidomic analysis of fibroblasts from Zellweger Spectrum disorder patients identifies disease-specific phospholipid ratios

Clinical diversity of cerebrotendinous xanthomatosis between pediatric and adult patients: report of 7 cases

Herzog K 1, Pras-Raves M L 1 2, Vervaart M AT 1, Luyf A C M 2, Van Kampen A H C 2, Wanders R J A 1, Waterham H R 1, Vaz F M 1

Zubarioglu T 1, Kiykim E 1, Gunduz A 3, Yesil G 4, Cansever M S 2, Saip S 3, Uygunoglu U 3, Yalcinkaya C 3, Aktuglu-Zeybek A C 1

1 Lab GMD, AMC, Amsterdam, Netherlands, 2Bioinformatics lab, Dept Clin Epid, AMC, Amsterdam, Netherlands

1 Div Nutr Metab Dis, Ist Univ Cer Med Fac, Istanbul, Turkey, 2Cent Lab, Ist Univ Cer Med Fac, Istanbul, Turkey, 3Div Neur, Ist Univ Cer Med Fac, Istanbul, Turkey, 4Div Med Genet, Bezm Vak Univ Med Fac, Istanbul, Turkey

Background: Peroxisomes are subcellular organelles involved in various metabolic processes, including fatty acid and phospholipid homeostasis. The Zellweger Spectrum disorders (ZSDs) represent a group of diseases caused by a defect in the biogenesis of peroxisomes. Accordingly, cells from ZSD patients are expected to have an altered composition of fatty acids and phospholipids. Methods: Using an LC/MS-based lipidomics approach, we show that the phospholipid composition is characteristically altered in cultured primary skin fibroblasts from ZSD patients when compared to healthy controls. Results: We observed a marked overall increase of phospholipid species containing very long chain fatty acids, and a decrease of phospholipid species with shorter fatty acid species in ZSD patient fibroblasts. In addition, we detected a distinct phosphatidylcholine profile in ZSD patients with a severe and mild phenotype when compared to control cells. Discussion: Based on our data, we present a set of specific phospholipid ratios for fibroblasts that clearly discriminate between mild and severe ZSD patients, and those from healthy controls. Our findings will aid in the diagnosis and prognosis of ZSD patients, including an increasing number of mild patients in whom hardly any abnormalities are observed in biochemical parameters commonly used for diagnosis.

P-394

The role of C26:0-lysophosphatidylcholine in the diagnosis of Zellweger spectrum disorders Klouwer F C C 1 2, Ferdinandusse S 1, Van Lenthe H 1, Poll-The B T 2, Wanders R J A 1, Waterham H R 1, Vaz F M 1

Background: Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid storage disorder that is caused by mutations in the CYP27A1 gene. Common clinical manifestations as progressive neurologic dysfunction, tendon xanthomas and cataract are usually recognized in adulthood period. Since early diagnosis and chenodeoxycholic acid treatment can prevent clinical deterioration, knowledge about early findings of disease has a great importance. In this study, clinical diversity of CTX between pediatric and adult patients is described among 7 patients. Methods: Seven patients in whom the diagnosis of CTX was made by molecular analysis of the CYP27A1 gene enrolled in this study. Data of patients including demographic features, history of chronic diarrhea and neoanatal jaundice, physical examination findings and abnormal EMG and MRI were collected retrospectively. Results: 4 of 7 patients were diagnosed before 18 years of age whereas diagnosis of CTX was made in adulthood in the other 3 patients. Pediatric patients were between 7 and 18 years old, and all these patients had abnormal cerebellar examination, intellectual disability and abnormal EMG findings. Pyramidal signs and tendon xanthomas were not detected in this group. 2 of the patients were operated for cataract and one patient had been followed for disc drusen. Chronic diarrhea, osteoporosis and history of recurrent bone fractures were additional findings in this group. 3 adult patients who were all diagnosed after the 3rd decade of life had severe pyramidal signs in addition to cerebellar signs. All of these patients were operated because of tendon xanthomas and cataract. Brain MRI’s were abnormal. In detailed anamnesis, chronic diarrhea in infancy was noted in 2 patients. Discussion: CTX is a treatable disorder of bile acid metabolism. Recognition of early and diverse systemic signs should allow the diagnosis at an initial stage of the disease and prevent progressive neurologic dysfunction.

1

Lab Genet Metab Dis, AMC, Amsterdam, Netherlands, 2Dept Ped Neur, Emma Child Hosp, AMC, Amsterdam, Netherlands Background: Zellweger spectrum disorders (ZSD) are caused by recessive mutations in different PEX genes, resulting in a defect of peroxisome formation. This leads to multiple metabolic abnormalities, including elevated very long chain fatty acid (VLCFA) levels. It is known from adrenoleukodystrophy newborn screening programs, measuring C26:0 lysophosphatidylcholine (C26:0 LysoPC) in dried blood spots, that elevated levels of C26:0 LysoPC can also be found in ZSD patients. However, little is known about the sensitivity of this analysis in ZSD patients. Methods: We measured C26:0 LysoPC in 89 dried blood spots of 35 known ZSD patients with combined liquid chromatography-tandem mass spectrometric (LC-MS/MS). VLCFA levels in plasma measured at the same time points were available and specific PEX mutations were known for all patients. Results: Elevated C26:0 LysoPC levels (>72 nmol/l) were found in 86 ZSD blood spots (n = 33) with normal levels ( G (p.Leu177Arg) (NM_153818.1)) in PEX10 gene predicted to be pathogenic. Discussion: We have identified a novel mutation in PEX10 gene in a patient with a late-onset slowly progressive peroxisomal disorder and a mild biochemical phenotype.

Moreau C 1, Jeoual A 1 2, Briand G 2, Deprez B 1 3, Dobbelaere D 4, Beghyn T 1 P-399 Apteeus, Lille, France, 2RaDeMe - Universite de Lille, Lille, France, 3Inserm U1177 - Institut Pasteur de Lille, Lille, France, 4CR MHM - CHRU, Lille, France 1

Apheresis therapy in patients with homozygous Familial Hypercholesterolemia Background: ID2STOP Orphan, this is the name we gave to the program cofinanced by Apteeus and the French Government through the Concours Mondial de l’Innovation. This program is dedicated to evaluate if Apteeus technology could be of interest for patients suffering from orphan diseases and to measure its rendered service. Methods: This technology allows the systematic testing of all marketed drug (>1500) directly on a culture of primary patient cells bearing the causative defect of the disease. Drugs that are able to correct the cellular defective function are rapidly identified. If their safety and pharmacokinetic profiles are compatible, a treatment can be proposed to the patient and evaluated in clinic. Results: The technology is currently well adapted for inherited metabolic diseases. Ten patients have already been included in in vitro studies in 6 different diseases. One patient suffering from pseudo-adrenoleukodystrophy should benefit from a treatment soon. Incubation of his skin fibroblasts with TEE886, a marketed drug with good tolerance, is able to restore the profile of very long chain fatty acids that usually accumulate in all cells of the organism. Discussion: This program is running for 12 months. We are continously developing new assays. We hope to be able to include more than 50 patients for in vitro experiments in more than 20 diseases and hopefully manage to discover some efficacious treatments for most of them during that period. Conflict of Interest declared.

P-398

Identification of a novel mutation in PEX10 in a patient with attenuated Zellweger spectrum disorder

Kekec I 1, Seker Yilmaz B 1, Kor D 1, Bulut F D 1, Tekinturhan F 2, Eren H 1, Onenli Mungan N 1 1 Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Apheresis Unit, Univ Cukurova, Adana, Turkey

Background: Homozygous familial hypercholesterolemia is an inherited metabolic disorder characterized with an 2–3 times increment in serum total cholesterol and LDL cholesterol levels. Xanthoma, xanthelasma, and corneal arcus are the important physical findings. The most striking feature of this disorder is the association with the multiplied risk of cardiovascular disease. Methods: From 50 genetically confirmed homozygous familial hypercholesterolemia pediatric patients, 10 patients who needed lipid apheresis therapy, despite dietary modifications and antihyperlipidemic medication were included into this report. Results: Among 10 patients, 7 were homozygous for LDLR and 3 for LDLRAP1 mutations. One year treatment with dietary modification and antihyperlipidemic agents lowered total and LDL cholesterol levels of 20.3 % and 20.1 %, respectively. Therefore, these patients were included into a lipid apheresis programme. The median age of onset of apheresis treatment was 118 (85–204) months. The reduction in total cholesterol levels after the first procedure and at the end of first year were 64.2 % and 61.5 %, respectively. After the first apheresis the decrement in LDL cholesterol level was 67.5 % and at the end of the year it was 65.6 %. There were no adverse events and complications. Discussion: The goal of apheresis treatment is to achieve 60 % reduction in total and LDL cholesterol levels in the first few procedures and to maintain 56 % reduction at the end of first year to reduce the multiplied cardiovascular risks. We provided this decrement in our patient group. Further studies in larger groups are needed to predict long-term effects of lipid apheresis.

Blomqvist M K 3, Ahlberg K 2, Lindgren J 3, Ferdinandusse S 1, Asin-Cayuela J 3 1 Lab Gen Metab Dis, Dep Clin Chem, AMC, Amsterdam, Netherlands, 2Dep Paediatrics, Central Hosp Karlstad, Karlstad, Sweden, 3 Clin Chem, Sahlgrenska Univ Hosp, Gothenburg, Sweden

Background: The Peroxisomal biogenesis disorders include the Zellweger spectrum disorders which are caused by mutations in any of 13 different PEX genes. Severe defects in one of these PEX genes result in the absence of functional peroxisomes which is seen in the classical Zellweger syndrome. Other, milder clinical and biochemical phenotypes are often associated with partial loss of peroxisomal function. Case Report: A now 15 year old girl presented with sensorineural hearing impairment at the age of 5. At the age of 8 her motor skills were below her age and brain imaging showed a lesion in the mesencephalon from the nucleus ruber dorsocaudally down to the cerebellar peduncles. At the age of 12 she

P-400

Glycerol-3-phosphate dehydrogenase 1 deficiency presenting with hepatosplenomegaly and pseudohypertriglyceridemia Verloo P 1, De Bruyne R 2, Vanhouteghem K 3, Delanghe J 3 Div Ped Neur, Univ Hosp, Ghent, Belgium, 2Div Ped Gastr, Univ Hosp, Ghent, Belgium, 3Div Clin Chem, Univ Hosp, Ghent, Belgium 1

Background: Deficiency of glycerol-3-phosphate (G3P) dehydrogenase 1 (GPD1) is a disorder in which the NADH/NAD+ dependent cycling between dihydroxyacetone phosphate (DHAP) and G3P, a key metabolite in

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triglyceride synthesis, is dysregulated. Deficiency of GPD1 has only been described in 11 infants having hypertriglyceridemia and a fatty liver with tendency to improve with age. Case Report: A 17-year-old woman presented in the joined hepatologymetabolic clinic. At the age of 4 years she was referred to an other centre with growth retardation and hepatosplenomegaly. Liver function tests were normal at all occasions. Liver histology revealed liver cirrhosis. Despite an extensive work-up no underlying cause was found. Of note, no steatosis was seen on biopsy. Follow-up showed a gradual improvement of the hepatosplenomegaly. Results: During a routine control a triglyceride level of 687 mg/dL was noted (reference interval 37–131 mg/dL). The non-lipemic nature of the serum prompted us to check for hyperglycerolemia as previously described in glycerol kinase (GK) deficiency. However, since GK deficiency is a X-linked disorder, we checked glycerol dehydrogenase activity in serum, which was only about 10 % of normal. Discussion: This case shows that GPD1 deficiency can present as hepatosplenomegaly without increased aminotransferase or gamma glutamyltransferase levels. The hyperglycerolemia noted in this patient could mean that the other described patients had a pseudohypertriglyceridemia and that hyperglycerolemia or -uria might be an important clue to the diagnosis. Since this is the oldest patient described up to date, this case seems to confirm that the liver disease in GPD1 deficiency improves with age and that liver steatosis might only be a transient finding early on in life.

P-401

Homozygous missense mutations in GPD1 presenting with ichthyosis, hepatosteatosis, and hyperlipidemia 1

1

1

Zubarioglu T , Aktuglu Zeybek A C , Kiykim E , Yesil G

Case Report: We desrcribe 4 patients - 2 unrelated female girls and 2 monozygotic twin sisters with peroxisomal D-bifunctional protein (DBP) deficiency. Results: Patient 1 presented neonatally with seizures, and later on developed global developmental delay and regression, sensorineural hearing loss, nystagmus and cortical blindness. The brain MRI demonstrated mild bilateral cortical irregularities. Whole exome sequencing revealed 2 mutations in HSD17B4 gene (c.752G > A;c.868 + 1delG). Patient 2 presented hypotonia, motor delay, and sensorineural hearing loss in infancy, developmental regression during her fourth year, nystagmus, and peripheral neuropathy. Brain MRI demonstrated cerebellar atrophy and abnormal basal ganglia and white matter signal. Whole exome sequencing revealed 2 mutations in HSD17B4 gene (c.14 T > G;c.752G > A). Patients 3 and 4, two female monozygotic twins, presented hypotonia, developmental delay, and macrocephaly from birth, and later on also sensorineural hearing loss, seizures, and adrenal insufficiency. Brain MRI demonstrated delayed myelination, and an assay of peroxisomal beta oxidation suggested DBP deficiency. Sequencing of HSD17B4 revealed 2 mutations (c.752G > A;c.868 + 1delG). Discussion: We describe 4 patients with variable clinical picture of DBP deficiency. The clinical pictures were not completely consistent with previous reports and expand further the clinical spectrum of this disorder, which should be considered in the differential diagnosis of every patient with seizures, developmental delay, sensorineural hearing loss, nystagmus, and adrenal insufficiency.

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2

1

Div Metab Dis Nut, Cerr Med Fac, Ist Uni, Istanbul, Turkey, 2Dep Med Gen, Bezmialem Univ, Istanbul, Turkey Background: Next generation sequencing (NGS) is a cost-effective and time saving approach for rapid and accurate detection of genetic mutations in patients with suspected genetic disorders, especially when no mutations are detected by target gene analyses. Case Report: A 10 years old female patient was referred to our clinic because of incidentally detected hyperlipidemia during her follow-up in dermatology clinic because of ichthyosis. Her physical examination was unremarkable except for the ichthyosis. Laboratory findings revealed hypercholesterolemia, increased LDL-cholesterol, hepatosteatosis and Jordan anomaly in white blood cells. Due to clinical and laboratory findings, Chanarin-Dorfman syndrome was considered but no pathogenic variants were detected by ABHD5 gene analysis. Whole exome sequencing revealed homozygous missense mutation in GPD1 gene. Result and Discusion: GPD1 mutations have been first described in 2012 in 10 patients from 4 different families. Although hepatomegaly, steatosis and marked hypertriglyceridemia in infancy has been reported before, to our knowledge, our patient is the first case presenting with icthyosis and hypercholesterolemia.

P-402

D-bifunctional protein deficiency: expanding the phenotypic spectrum Landau Y E 1, Heimer G 1, Shalva N 1, Marek-Yagel D 1, Veber A 1, Ben-Zeev B 1, Anikster Y 1 1

Sheba Medical Center, Tel Aviv Universit, Tel Aviv, Israel

Background: Peroxisomal D-bifunctional protein (DBP) deficiency is an autosomal recessive disorder historically described as a Zellweger-like syndrome comprising neonatal seizures, retinopathy, hearing loss, dysmorphic features, and other complications. HSD17B4 gene encodes DBP which is essential for oxidation of peroxisomal substrates.

Clinical and Neuroradiological Findings of X-ALD Patients Yildiz M 1, Karaca M 1, Balci C 2, Cakar E 1, Demirkol M 1, Gokcay G 1 1 Dep Ped Nutr and Metab, Istanbul Univ, Istanbul, Turkey, 2Tokat Goverment Hospital, Tokat, Turkey

BACKGROUND: Adrenoleukodystrophy (X-ALD; OMIM #300100) is one of the X-linked genetic disorders presenting with myelin sheath breakdown in the brain and progressive adrenal insufficiency. The aim of the study is to evaluate clinical features, neuroradiological findings and prognosis of XALD cases. METHODS: X-ALD patients followed between 1990 and 2016 at the Istanbul Medical Faculty Pediatric Nutrition and Metabolism Department were enrolled. The diagnoses were confirmed by clinical, neuroradiological, biochemical, and genetic studies. We evaluated age, family history, plasma very long chain fatty acid levels and neuroradiological findings, retrospectively. RESULTS: 39 male patients from 31 families were diagnosed with XALD. The rate of consanguinity was 12 % and 50 % had positive family history. Mean age of diagnosis was 87.1 ± 46.5 months. 23 % of patients who had family history were asymptomatic at the time of diagnosis. Behavioral changes and poor school performance (38 %), visual complaints (23 %), and neurological problems (20 %), adrenal insufficiency symptoms (15 %) were reported. Phenotypes were cerebral ALD (66 %), addison only (12 %), adrenomyeloneuropathy (AMN) (2 %) and asymptomatic (20 %). The most frequent initial radiologic finding was parietooccipital involvement (70 %) followed by frontal (22 %) and frontoposterior (8 %) involvement. Lorenzo’s oil was used in 66 % of patients and bone marrow transplantation (BMT) was performed in 20 % of patients. 46 % of the patients died. 58 % of patients who had BMT died. Mean Loes scores of the surviving patients after BMT and lost patients were 8.5 and 12.7, respectively. DISCUSSION: Cerebral X-ALD is the most common phenotype. Patients with behavioral changes and poor school performance should be evaluated for X-ALD. At initial diagnosis most patients had parietooccipital involvement but the percentage of frontal involvement was higher than reported in the literature implicating poor prognosis.

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Cerebrotendinous Xanthomatosis: Response to Treatment in Late Diagnosed Cases Karaca M 1, Balci C 2, Cakar E 1, Demirkol M 1, Gokcay G 1 1 Dep Ped Nutr and Metab, Istanbul Univ, Istanbul, Turkey, 2Tokat Goverment Hospital, Tokat, Turkey

Background: Cerebrotendinous xanthomatosis (CTX) (OMIM#213700) is a very rare autosomal recessive lipid storage disorder affecting bile acid biosynthesis caused by mutations in CYP27A1 gene, coding for sterol 27-hydroxylase. Clinical manifestations are related to the presence of cholesterol and cholestanol deposits and include tendon xanthomatosis, premature cataracts, chronic diarrhea, progressive neurologic impairment, psychiatric disorders and less frequently coronary heart disease and early onset osteoporosis. Early diagnosis and long term treatment with chenodeoxycholic acid (CDCA) is important for the reversal of symptoms and disease progression. Response to treatment has been evaluated in three CTX cases to determine the value of early diagnosis. Case Report: Three cases, one female two males, presented with premature cataracts, chronic diarrhea, learning disabilities during childhood. All had elevated cholestanol levels and psychiatric symptoms such as anxiety, obsessive compulsive disorder and depression prominent after 10 years of age. Ankle xantomas were detected at around the age of 20. All had varying degrees of speech and gait disturbances. Age of diagnosis was 26, 19 and 30 years, respectively Results: The youngest case at diagnosis received the longest CDCA treatment for 14 years. He has no motor deficit and obsessive compulsive symptoms greatly diminished and speech disorder disappeared with treatment. The case diagnosed at 30 years followed for 4 years has still serious motor, speech and psychiatric problems. The case diagnosed at 26 years, is on follow-up for 6 months and still symptomatic. Discussion: CTX should be considered in patients with chronic diarrhea, juvenile cataract, and learning disabilities. Early diagnosis may prevent disease progression with reversal of psychiatric and dysabiliating neurologic problems.

18. Lysosomal disorders: mucopolysaccharidoses, oligosaccharidoses

P-405

Clinical, biochemical, and genetic findings in 8 infants affected with Sandhoff disease Zaman T Z 1 2, Moarefian S H 2, Fazeli A S 3, Sanati M H 3, Hushmand M 4, Aryan R 3 4 1

Metab Div Dis,Tehran Univ of Medical Sc, Tehran, Iran, Islamic Republic of, Iranian National Research Unit, Tehran, Iran, Islamic Republic of, 3National Inst for Gene engin and Biotec, Tehran, Iran, Islamic Republic of, 4Special Medical Centre, Tehran, Iran, Islamic Republic of

Methods: 8 SD infants diagnosed by clinical data, enzyme assay and gene sequencing on combination therapy. Results: Eight cases, male 6/8; consanguinity 7/8; age at onset 2–9 months; developmental delay/regression 8/8; convulsions 3/8; vertical nystagmus 7/8; horizontal nystagmus & abnormal Mongolian spots 1/8; cherry red spots 8/8; hepatosplenomegaly 1/8; mean hexosaminidases activities A 8.6 %, B < 1 %, A B, 1.5 %; sequencing of HEXB, compound heterozyte c.1341_1342insCCTT and c.530_533 delAGTT(p.Gln177GlnfsX29) in exon 11&4, c:delT,c.1552 delG(3 cases) and GC:518 in exon13 and insertionA in exon 14,most of them were novel and their pathogenicity were confirmed by investigation in their parents and one hundred controls; response to combination therapy, 3/4; death by 5 years, 6/8. Discussion: Sandhoff disease does not have an effective treatment . Mutation detection faciliates prenatal diagnosis in at-risk families.

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Clinical, radiographic, and genetic features of Korean patients with Morquio A syndrome Cho S Y 1, Kim J S 1, Cho E K 1, Park H D 1, Jin D K 1 1

Samsung Medical Center, Sunkyunkwan Univ, Seoul, Republic of Korea

Background: Mucopolysaccharidosis type IVA (MPS IVA; Morquio A syndrome) is a rare autosomal recessive lysosomal storage disorder characterized by the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S) resulting from the deficient activity of N-acetyl-galactosamine-6-sulfatase (GALNS). The most common characteristics of patients with MPS IVA are progressive skeletal dysplasia, short stature and motor dysfunction with normal intelligence. We studied clinical, radiographic and genetic features of Korean MPS IVA patients for determining factors that may allow early diagnosis. Methods: MPS IVA was confirmed via assay for enzymatic activity of leukocytes in 13 patients. GALNS gene was analyzed. Patients’ medical records were retrospectively reviewed for obtaining clinical features and evaluated for radiological skeletal surveys, echocardiography, pulmonary function test, and ophthalmologic test results. Results: Eleven patients had severe clinical phenotype, and one had an intermediate phenotype, and one had an attenuated form on the basis of clinical phenotype criteria. Radiologic findings indicated skeletal abnormalities in all patients, specifically in the hips and extremities. Eight patients had an odontoid hypoplasia, and two showed mild atlantoaxial subluxation and cord myelopathy. Genetic analysis indicated 14 different GALNS mutations. Two mutations, c.451C > A and c.1000C > T, account for 33 % and 19 % of all mutations in this samples, respectively. Discussion: An understanding of the clinical and radiological features involved in MPS IVA may allow early diagnosis of MPS IVA. Adequate evaluations and therapy in the early stages may improve the quality of life of patients suffering from skeletal abnormalities and may reduce lifethreatening effects of atlantoaxial subluxation.

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Background: Sandhoff disease, a GM2 gangliosidosis is a lysosomal storage disorder due to mutations in HEXB gene encoding subunit- beta of hexosaminidases A and B (O variant), resulting in pathological accumulation of GM2 gangliosides in lysosomes of CNS, manifests as classic infantile, juvenile and adult late onset. Infants with the classical form appear normal until the age of 2–9 months when development slows, muscles weaken, motor skills show regression, seizures, hearing and vision loss develop (charactristic cherry red spot), and often results in death by the age of 5 years. The juvenile form start at age 3–10 years, presenting autism, ataxia, motor skill regression, spasticity, learning disorders and death by 15. The adult onset occurs in older individuals, with motor dysfunction and not yet known life span. There is not standard treatment for SD but some studies suggest that ketogenic diet and miglustat may increase survival, improve motor function in mouse model, and control seizures and improve cardiac function in one SD patient.

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Design and Rationale of the Clinical Study Programs for BMN 250, a Novel Investigational Enzyme Replacement Therapy for Sanfilippo B Syndrome Shaywitz A 1, Oh M 1, Kent S 1 1

BioMarin Pharmaceutical Inc, Novato, United States

BACKGROUND: Sanfilippo B syndrome (mucopolysaccharidosis [MPS] IIIB) is a lysosomal storage disease characterized by rapid and progressive neurological decline due to deficiency of the human alpha-Nacetylglucosaminidase (NAGLU) enzyme. Children with the severe form of disease are initially symptom-free but begin to manifest developmental delay

S178 between ages 1 and 4 years, progressing to severe behavioral problems, sleepwake disturbance, and intellectual decline. Severe decline of all motor functions ensues and death usually occurs in the second or third decade of life. BMN 250 is a novel enzyme replacement therapy (ERT) designed to restore functional NAGLU activity to the brain. Understanding cognitive decline in untreated MPS IIIB patients would help assess clinical relevance of treatment effects. Development quotient (DQ) has been validated as a cognitive measure in children with MPS III. While studies have demonstrated marked changes in DQ in younger MPS III patients, information regarding DQ trajectory in young MPS IIIB patients is scarce. Methods: BMN250-901 (NCT02493998) is an observational study designed to quantify the progression of MPS IIIB over time in affected children primarily aged 1–5 years at baseline and to correlate changes in clinical features of the disease, in particular changes in DQ, with both MRI characteristics and biochemical markers of disease burden. A concurrent treatment study (BMN250201) is also planned: Part 1 is a dose-escalation period to establish safety; Part 2 is a dose-expansion period. Eligible patients from Study 901 may be able to roll over into Part 2 of Study 201. Results: Efficacy will be assessed by comparing changes in disease progression in the observational study vs changes observed in Part 2 of Study 201. Discussion: Data from the BMN250 Study Program will provide valuable information on both the natural history of untreated MPS IIIB patients and the efficacy and safety of BMN250. Conflict of Interest declared.

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Clinical features, molecular analysis and outcome of ERT in Korean patients with Mucopolysaccharidosis type VI Cho E K 1, Wichajarn K 2, Kim J S 1, Yang A R 1, Sohn Y B 3, Kim S J 4, Park S W 5, Cho S Y 1, Jin D K 1 1 Dep Ped, Samsung Medical Center, Seoul, Republic of Korea, 2Div Gen, Dep Ped, Khon Kaen Univ, Khon Kaen, Thailand, 3Dep Med Gen, Ajou Univ Hosp, Suwon, Republic of Korea, 4Dep Ped, Myungji Hosp, Seonam Univ, Goyang, Republic of Korea, 5Dep Ped, Jeil Hosp, Dankook Univ, Seoul, Republic of Korea

Background: Mucopolysaccharidosis type VI (MPS VI) is a rare disease caused by the mutation of ARSB with prevalence range from 1/5,000 in northeast Brazil to 1/2,057,529 births in Czech Republic. In Asia, there is only onepublished data in Taiwan about 1/833,000 births. The exact prevalence in Korean population is unknown but we estimated the incidence of MPS VI is about 0.03/100,000 live births. Enzyme replacement therapy (ERT) with recombinant human Arylsulfatase B (rhASB) is a modality for treatment of MPS VI that reduce excretion of urine glycosaminoglycan (GAG) and improve joint motion, pulmonary function and endurance Case Report: We presented clinical features, molecular analysis and outcome of ERT in 3 Korean MPS VI patients. All patients had the typical characteristic clinical features of MPS IV. Short stature, dysostosis multiplex, corneal opacity and valvular heart diseases was found at first presentation, while restrictive lung disease and carpal tunnel syndrome developed later in all patients. Results: Molecular analysis demonstrated novel missense and nonsense mutation in Korean including, p.Ile67Ser,p.Gly328Arg, p.Arg191*, p.Asp352Asn and p.Gly17Asp. After ERT, urine GAG was decrease in all patients. Skeletal involvement, corneal opacity, heart valve abnormalities and pulmonary function were not improved with ERT but it had better outcome on joint motion and endurance. One patient had allogenic bone marrow transplantation (BMT) prior ERT but clinical response was not quite improved after BMT. Discussion: This study would demonstrate clinical phenotypes and molecular analysis of severe form MPS VI Korean patients. ERT in Korean patients has better outcome in urine GAG excretion, joint movement and endurance. But it cannot improve skeletal involvement, valvular heart disease and respiratory function in this study.

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Mucopolysaccharidoses and oligosaccharidoses: biochemical diagnosis in clinically suspected Egyptian children. Ibrahim M M 1, Fateen E M 1, Radwan A M 1 1

Div Hum Gent Geno Res, Nation Res Cent, Cairo, Egypt

Background: Lysosomal storage disorders (LSDs) are a group of inherited disorders caused by deficiency of lysosomal enzymes. Mucopolysaccharidoses (MPS) and oligosaccharidoses are among these disorders and share many clinical features. This study was designed to establish a protocol for the differential diagnosis of these disorders and their subtypes in order to prepare patients for enzyme replacement therapy and to provide proper genetic counseling for families of affected cases. Methods: Two hundred and eighty urine samples from patients with coarse facial features, mental retardation and/ or hepatospleenomegaly were investigated by a procedure that included a quantitative measurement of glycosaminoglycans (GAGs), a qualitative GAGs analysis by two dimensional electrophoresis (2-DEP), thin layer chromatography (TLC) separation of oligosaccharides and assessment of plasma or leukocyte enzyme activity according to the abnormal pattern of the 2-DEP or TLC to confirm the diagnosis of different types of MPS or oligosaccharidoses. Fifty healthy subjects were included with matching age and sex. Results: Eighty four patients had abnormal patterns of GAGs separation in 2DEP and the types were confirmed by measuring the specific deficient enzyme activity. Sixteen patients had abnormal pattern of oligosaccharides separation in TLC, 2 of them proved to have α-mannosidosis and 2 with GM1 gangliosidosis diagnosed on the basis of deficient enzyme activity. Discussion: Results showed that MPS should be excluded before suspecting oligosaccharidoses as they share similar clinical features. 2-DEP and/or TLC alone cannot rule out the diagnosis of either MPS or oligosaccharidoses and confirmation must be done by enzymatic assay.

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The reduction of heparan sulfate level in the cerebrospinal fluid and of heparan sulfate content in the brain tissue of IDS knockout mice by an intracerebroventricular injection of idursulfase-beta are correlated Sohn Y B 1, Ko A 2, Cho S Y 3, Sakaguchi M 4, Nakazawa T 4, Kosuga M 5, Seo J H 5, Okuyama T 5, Jin D 3 1 Ajou University Hospital, Suwon, Republic of Korea, 2Samsung Biomedical Research Center, Seoul, Republic of Korea, 3Samsung Medical Center, Seoul, Republic of Korea, 4AnGes MG, Osaka, Japan, 5National Center for Children Health and, Tokyo, Japan

Background: Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessively inherited lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase. In patients with severe forms of MPS II, current intravenous enzyme replacement therapy (ERT) is not effective in improving CNS pathology. This study aims to examine the dose–response relationship and pharmacological effects of a single intracerebroventricular(ICV) administration of idursulfase-beta (IDS-β) and to investigate the correlation of heparan sulfate (HS) content in the cerebrospinal fluid (CSF) and the HS and glycosaminoglycans (GAGs) levels in the brain tissue of IDS knockout (KO) mice. Methods: Three doses of ICV IDS-β injections (3, 10, and 30 μg) were performed, and the tissue GAGs (brain, heart, lung, liver, spleen, and kidney) were measured at 7, 14, and 28 days after injection. HS was measured by using LC/MS-MS in the CSF and brains of mice. Results: The total GAGs in the brain and other somatic tissues of all the IDS-β-treated groups were significantly reduced. The significant reduction was maintained for 28 days in the 30-μg injection group. We also demonstrated that HS content was reduced in both the CSF and brain tissue of all IDS-β-

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 treated groups. Furthermore, we demonstrated that HS concentration in the CSF was significantly correlated with brain HS and brain tissue GAGs. Discussion: A single ICV injection of IDS-β was generally well tolerated, and it produced a significant reduction of HS and GAGs in the brain and other somatic tissues. A significant positive correlation of HS content in the CSF between brain HS and brain GAGs suggests that CSF HS concentration could be a useful biomarker for representing brain pathology in MPS patients with CNS involvement.

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were evaluated in 4 MPS II untreated patients (mean age: 12.5 ± 4.41 years) and were correlated with their concentrations of urinary dermatan sulphate measured by LC-MS/MS. Results: Our data showed that DS concentrations were significant elevated in the urine of MPS II patients in relation to the controls (514.11 ± 374.99 μg/mg creatinine, mean ± SD) and were positively correlated with the levels of urinary di-tyrosine (r = 0.99, p < 0.05) and urinary nitrate/nitrite (r = 0.96, p < 0.05), which were also elevated in these patients in relation to normal subjects. Discussion: These preliminary results allow us to infer that oxidative protein damage and nitrosative stress occur in MPS II patients, probably contributing to the pathophisiology of this disease, and may be related with the DS accumulation in tissues and biological fluids of these patients.

Molecular analysis of 24 patients with mucopolysaccharidosis IIIA from Ukraine P-413 Trofimova N S 1, Olkhovych N V 1 2, Mytsyk N I 1, Pichkur N A 2, Gorovenko NG1 1

Div Genet Diagn, Inst Genet Regener Med, Kiev, Ukraine, 2Cent Orph Dis, Nat Child Hosp OKHMATDET, Kiev, Ukraine Background: Mucopolysaccharidosis type IIIA (MPS IIIA,Sanfilippo A syndrome) occurs due to the deficiency of lysosomal enzyme heparan-N-sulfatase (sulfamidase), which is caused by mutations in the SGSH gene. Patients who carry two mutated alleles of the SGSH gene have a decreased ability to catabolise heparan sulfate, which results in its accumulation and leads to disorder in the functioning of different organs, the nervous system, most notably. We aimed to analyze the spectrum of mutations in the SGSH gene responsible for the disorder in Ukraine. Methods: Twenty four patients with MPS IIIA from different regions of Ukraine, in whom diagnosis was conferment biochemically and enzymatically, were studied. Results: In total, eight different disease-causing mutations were identified. No mutations were identified on 9/48 alleles. The analysis of the results of molecular investigation of the patients’ samples showed that the incidence of mutation p.R74C was 29/48 (60.4 %). Three mutations: p.E369K, p.N389K and c.1135delG were showed in 2/48 (4.17 %) each. The incidence of two mutation: p.245H and p.T271M were 1/48 (2.08 %) each. It was find one novel mutation c.216delC. All patients, except one (homozygous for p.R74C) presented with a severe phenotype. Discussion: The p.R74C mutation in the SGSH gene is the most common in Ukraine (60.4 %). It is appropriate to have screening of this mutation in the diagnostic algorithm along with the definition of biochemical sulfamidase activity to prevent false-negative or false-positive diagnosis of Sanfilippo syndrome A.

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Nitrosative stress and protein damage in Mucopolysaccharidosis type II patients is correlated with dermatan sulphate. Ribas G S 1 2, Jacques C 1 2, Donida B 1 2, Marchetti D 1 2, Deon M 1 2, Mescka C 1 2, Giugliani R 1 2 3, Vargas C R 1 2

Multiplex MS/MS method to measure MPS II, MPS IIIB, MPS IVA, MPS VI and MPS-VII enzyme activities in dried blood spots Rehnberg J R 1, Potier A 2, Cournoyer J 2, Trometer J 2, Schermer M 2, Vranish A 4, DiPerna J 4, Yi F 3, Chennamaneni N 3, Spacil Z 3, Kumar A B 3, Liao H C 3 , Gelb M H 3, Scott C R 3, Turecek F 3 1

PerkinElmer, Turku, Finland, 2PerkinElmer, Waltham, United States, University of Washington, Seattle, United States, 4PerkinElmer, Bridgeville, United States 3

Background: The mucopolysaccharisodes (MPS) family of lysosomal storage disorders (LSDs) is caused by defects in the metabolic breakdown of glycosaminoglycans (GAGs). Our previous work demonstrated the ability to distinguish samples with low enzyme activities for I2S (MPS II), GALNS (MPS IVA) and ARSB (MPS VI) using a single 3.2 mm dried blood spot (DBS) punch and one incubation cocktail. The current work will focus on also measuring enzyme activities of NAGLU (MPS IIIB) and GUSB (MPS VII). This five-plex assay is incubated overnight at 37 °C followed by a post-incubation workup that is less than 30 min per plate. Sample-to-sample time using MS/ MS analysis is only 3 min. Methods: To assess optimized assay performance, a study consisting of DBS from de-identified presumed healthy subjects, confirmed low I2S, NAGLU, GALNS, ARSB or GUSB activity and PerkinElmer control DBS was completed. Precision was determined using 13 replicates of CDC basepool/low/ mid/high control DBS. Linearity was determined using 3 replicates each of a 30 level DBS series prepared by controlled white-cell/red-cell/plasma dilutions. Results: The assay showed good linearity and precision across all five enzymes. Furthermore, the comparison between de-identified healthy subjects, confirmed low activity DBS and control DBS showed excellent resolution and a clear distinction between the different enzyme activity levels. Discussion: A new tandem mass spectrometric multiplex assay for measuring I2S, NAGLU, GALNS, ARSB and GUSB enzyme activities in DBS was demonstrated. This method was able to clearly differentiate between the activities in samples collected from presumed healthy subjects and in samples confirmed to have low activity. Good precision and linearity within the expected range of each method was demonstrated. Conflict of Interest declared.

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Clinicas Hosp of Porto Alegre, Porto Alegre, Brazil, 2Federal Univ of Rio Grande do Sul, Porto Alegre, Brazil, 3Department of Genetics and Molec Biol, Porto Alegre, Brazil Background: Mucopolysaccharidosis type II (MPS II) is an inherited lysosomal storage disorder caused by deficiency of iduronate-2-sulfatase. As consequence of the metabolic block, the glycosaminoglicans heparan sulphate (HS) and dermatan sulphate (DS) accumulate within cellular lysosomes, leading to the progressive multi-systemic build-up, followed by cell, tissue and organ damage and, in particular, neurodegeneration. The deficient degradation of these macromolecules may disrupt a variety of physiological and biochemical processes. Methods: In this work, we investigated the effects of DS on protein damage and nitrosative stress in MPS II. Urinary nitrate/nitrite and urinary di-tyrosine

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Dramatic mobility improvement on galsulfase ERT: a case report Zakharchuk J 1 1

Cit Neur Cent f Child to Neur a Vert Pat, Chelyabinsk, Russian Federation

Background: Mucopolysaccharidosis type VI (MPS VI) is a rare, autosomal recessive lysosomal storage disease. Symptoms include elevated urine glycosaminoglycan (uGAG), skeletal abnormalities, spinal cord compression,

S180 compromised cardiac and respiratory function, reduced endurance and mobility, and reduced joint range of motion Case Report: The case of a female MPS VI patient, born 23.01.1997, is reported. In September 2010, the patient had a closed traumatic brain injury. Afterwards, the patient started to complain of weakness in her left extremities. In April 2011, the patient was not able to walk long distances. In June 2011, she was diagnosed with MPS VI by genetic testing. By August 2011, the patient was unable to walk independently. On 24.10.2011 the patient underwent surgical treatment of the vertebral column, zone C1-C2-C3. After the surgery, she could walk with support. On 01.04.13 she started enzyme replacement therapy (ERT) with galsulfase. Results: After 2 months and 13 days of ERT, assessment results were as follows: 12MWT (12-min walk test)—310 meters; 3MSC (3-min stair climb)—20 steps; uGAG—178 CPC U/gCr.; FVC and FEV1—unable to perform. After 1 year of ERT, results improved dramatically: 12 MWT—670 meters; 3MSC—80 steps; uGAG—116 CPC U/gCr. The patient also started to climb stairs without support and was able to restart school after missing a year due to mobility limitations. After 2 years and 5 months of ERT, the patient’s results improved further: 12MWT—875 meters; 3MSC—120 stairs, uGAG—124 CPC U/gCr. The patient was now able to perform pulmonary assessments; FVC was 31 % predicted and FEV1 was 33 % predicted. An increase in finger extension range was also noted. Discussion: The combination of surgical treatment and ERT allowed for substantial recovery of mobility in this patient. Conflict of Interest declared.

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Genistein: an effective Oral Substrate Reduction Therapy for MPS III Jalan A B 1, Kudalkar K V 1, Jalan R A 1, Shinde D H 1, Borugale M A 1, Joshi M M 1, Shirke S M 1, Mahamunkar A P 1, Tawde R J 1, Eichler S 2, Schmidt K 3 , Zielke S 3, Giese A 3, Rolf A 2 3 1 NIRMAN, Div of Biochemical Genetics, Navi Mumbai, India, 2Albrecht Kossel Institute,Univ Rostock, Rostock, Germany, 3 Centogene AG, Rostock, Germany

Background: MPS are inherited metabolic disorders caused by mutations leading to dysfunction of enzymes involved in degradation of glycosaminoglycans(GAG). Substrate reduction therapy(SRT) is one of the possible treatments that could be effective in management of CNS related symptoms of MPS. The goal of SRT is to inhibit production of the stored substrates. The compound Genistein has been demonstrated to be an inhibitor of GAGs synthesis in fibroblasts of patients suffering from various types of MPS. Objective: To evaluate efficacy and safety of oral natural Genistein(>98%purity) in patients with MPS III. Methods: This is a prospective observational study. Patients with MPS III were provided with the information regarding possibility of oral Genistein. Informed consent was obtained. Parents themselves ordered Genistein. Initial dose was 5 mgm/kg/day and later it was escalated to 10–15 mgm/kg/day. All the cases of MPS III were confirmed by DNA studies and specific types were identified. 5 patients out of 7 opted for Genistein therapy and 2 refused. Pre treatment 24 hr urine GAG and EPP for HS % and quantitation were performed. Later all the children were clinically evaluated, GAG and HS were repeated and CBC, LFT and RFT were performed for evaluation of safety of the drug. Results: 7 patients, 5 are males and 2 females. MPS IIIA (2/7), MPS IIIB (2/7), MPS IIIC (1/7) and MPS IIID (2/7). Family of first pair of sibs (A)# 1 and 2 refused treatment. Genistein was started for patients # 3–7(B,C,D). All of them showed reduction in HS and great improvement in texture of skin & hair, reduced hyperactivity, improved cognition and reduction in upper respiratory infections. Discussion: None of the 5 patients showed any side effects clinically or biochemically Conclusion: Genistein therapy is economical, easily available and safe. In India only natural Genistein is available. Though there is improvement in cognition, learning does not improve.

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Mucopolysaccharidosis type 2: Information on 19 Hunter syndrome patients in Iran. Hadipour F 1 2, Rolf A 3, Shafeghati Y 1 2, Verheijen F W 4, Hadipour Z 1 2 1 Sarem Cell research Center, Hospital, Tehran, Iran, Islamic Republic of, 2Med Gen, Sarem Fertility infertilty Cent, Tehran, Iran, Islamic Republic of, 3 Albrecht-Kossel-Institute for Neuroregen, Rostock, Germany, 4Dept Clin, Fun Gen Lab ErasmusMC, Rotterdam, Netherlands

Background: Mucopolysaccharidosis type 2 is a lysosomal storage disease leading to accumulation of glycosaminoglycans and a variety of symptoms including distinctive coarse facial features, short stature, cardio-respiratory involvement and skeletal abnormalities; macrocephaly; respiratory tract infections; umbilical and inguinal hernia; intractable diarrhea; hepatosplenomegaly; and skin lesions resembling an orange peel; may become evident between 2 and 4 years of age. MPS2 results from iduronate-2-sulfatase (I2S) deficiency, which leads lysosomal accumulation of dermatan sulfate (DS) and heparan sulfate (HS). The causative gene, IDS, is located on Xq28, approximately 320 mutations have been reported. Prevalence is 1 in 100,000 to 1 in 170,000 males. Progression varies from a severe form with early psychomotor regression to an attenuated form. Case Report: Our Data including genotypes, clinical, biochemical and phenotypes, is being collected on of 19 Hunter patients from IRAN. All the patients’ diagnosis has been confirmed by enzyme assay and 4 patients have molecular analysis, with 2 novel mutation. Results: Our findings to date reinforce the validity of the characteristic symptoms of Hunter syndrome. However, they also reveal that there are patients who present with only one or two of these symptoms, and that the spectrum of disease includes remarkably attenuated forms with relatively little associated disability. Discussion: These insights provide an indication for diligent screening of certain pediatric and adult patients for Lysosmal storage disorders such as Hunter syndrome. Enzyme replacement therapy (ERT) which has been shown to alleviate somatic symptoms is not available in Iran yet.

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34 Maroteaux-Lamy (Mucopolysaccharidosis type 6) Patients Diagnosed in Iran. Hadipour F 1 2, Rolf A 3, Shafeghati Y 1 2, Verheijen F W 4, Hadipour Z 1 2 1 Sarem Cell research Center, Hospital, Tehran, Iran, Islamic Republic of, 2Med Gen, Sarem Fertility infertilty Cent, Tehran, Iran, Islamic Republic of, 3 Albrecht-Kossel-Institute for Neuroregen, Rostock, Germany, 4Dept Clin, Fun Gen Lab ErasmusMC, Rotterdam, Netherlands

Background: Mucopolysaccharidosis type 6 is a lysosomal storage disease leading to accumulation of glycosaminoglycans and a variety of symptoms including skeletal dysplasia includes short stature, dysostosis multiplex and degenerative joint disease, Coarse facial features, cardiac valve disease and reduced pulmonary function, macrocephaly; umbilical and inguinal hernia; hepatosplenomegaly, otitis media, hearing loss, sleep apnea, corneal clouding, and carpal tunnel disease. Intellectual deficit is absent in MPS 6, central nervous system findings such as cervical cord compression, communicating hydrocephalus, optic nerve atrophy and blindness. MPS6 results from autosomal recessive manner and is caused by mutations in the ARSB gene, located in 5q13-5q14. Over 130 ARSB mutations have been reported. Case Report: Our Data including genotypes, clinical, biochemical and phenotypes, is being collected on of 34 MPS type 6 patients from IRAN. All the patients’ diagnosis has been confirmed by enzyme assay and 13 patients have molecular analysis, with 3 novel mutation.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Results: Our findings to date reinforce the validity of the characteristic symptoms of Maroteaux-Lamy disease. However, they also reveal that there are patients who present with only one or two of these symptoms, and that the spectrum of disease includes remarkably attenuated forms with relatively little associated disability. Discussion: These insights provide an indication for diligent screening of certain pediatric and adult patients for Lysosmal storage disorders such as Maroteaux-Lamy disease. Enzyme replacement therapy (ERT) with galsulfase (Naglazyme®), clinical management,supportive care and hematopoietic stem cell transplantation are suggestive manegments for Maroteaux-Lamy disease.

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Mucopolysaccharidosis: orofacial findings in a series of 35 cases Kalkan-Ucar S 1, Cogulu D 2, Canda E 1, Atila E 2, Alpoz A R 2, Coker M 1 1 Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey, 2Div Ped Dent, Ege Univ Fac Dent, Izmir, Turkey

Background: Mucopolysaccharidoses (MPS) are a group of inherited metabolic disorders characterized by intralysosomal storage of glycosaminoglycans. The aim of this study was to investigate the orofacial features of 35 (18 M, 17 F) patients with MPS. Methods: Following consent, oral and radiographic examinations were performed. The decayed, missing and filled surfaces (dmfs/DMFS) index, and the Silness & Löe Index were assessed. The clinical data and radiographic findings were interpreted and the incidence of each findings was recorded. Results: The sample group was composed of 3 MPS-I (8 %), 6 MPS-II (17 %), 8 MPS-III (23 %), 10 MPS-IV (29 %), 7 MPS-VI (20 %) and 1 MPS-VII (3 %) with mean age of 12.63 ± 6.48 (3–30) year. The mean DMFT/DMFS and dmft/dmfs were 1.34 ± 2.03/2.29 ± 3.83 and 2.91 ± 3.77/6.74 ± 10.49 respectively. The most common oral features were detected as; orthodontic requirement (91.4 %, 32/35), tongue thrusting (80 %, 28/35), limited mouth opening (71 %, 25/35), macroglossia (71 %, 25/35) and anterior open-bite (54 %,19/35). Radiographic examination was not performed in 9 patients because of severe mental retardation. Thin mandibular cortex (38 %, 10/26), impacted teeth (27 %, 7/26) and short mandibular ramus (23 %, 6/26) were detected as common radiographic findings. Talon cusp was detected in one patient with MPS-IV as an unusual dental finding. Discussion: Orofacial findings of patients with MPS were encountered in almost all MPS patients, which lead to conclusion that each MPS patient needs to be referred to the dentist. Early detection of the dental disease and appropriate management through a multidisciplinary approach is recommended to improve the quality of their life.

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Mucopolysaccharidosis type I from the perspective of phenotypegenotype-therapeutical response correlation Coker M 1, Canda E 1, Ozkaya B 1, Ben E 1, Onay H 2, Ozkinay F 2, Sozmen E 3 , Kalkan-Ucar S 1 1 Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey, 2Div Med Gen, Ege Univ Child Hosp, Izmir, Turkey, 3Div Med Bioch, Ege Univ Child Hosp, Izmir, Turkey

Background: This study aimed to investigate the relation between phenotypical diversity-IDUA gene mutations and therapeutic response in Turkish patients diagnosed with MPS type I disease. Methods: Phenotypical findings were obtained from the hospital records of eight patients enzymatically diagnosed as MPS I. Genetic

analysis was carried out in 7 patients using direct DNA sequencing. Therapeutic reply was evaluated by GAG excretion, 6 min walking and 3 min climbing tests, and visceral, cardiac and ophthalmological evaluation. Results: Phenotype: The female/male ratio was 7/1 and median age of the patients was 24mo (3 mo-5 yrs). The course face was the main clinical presentation encountered in all MPS I patients followed by skeletal and visceral findings; short neck (75 %), hypertrichosis (25 %), claw hand (37 %), flattened nose (87 %), gibbous (62 %), macroglossia (62 %), inguinal hernia (37 %), hepatomegaly (87 %). Hearing loss was found in 87 %; corneal clouding was presented in all patients, whereas retraction defects were detected in 25 %. Developmental delay and/or cognitive impairment were found in half of the patients. Cardiac involvement was determined as cardiomyopathy (87 %) or valve diseases: PS(25 %), ASD (12 %), AS(12 %), MVP(62 %), MS(12 %). Genotype: In five patients already reported homozygous mutations (nonsense:Y64X, missense:P533R;L490P;N350S;deletion: 619delG) were identified. One patient had a heterozygous nonsense mutation (W68X) and another a deletion(c.826_828del3).Therapeutics: All patients receiving ERT (4.6 yrs and 6mo-9 yrs)) demonstrated decrease in GAG levels, improvement at visceral measurements and functional tests. However, ophtalmological and cardiac problems continued to get worse despite ERT. Discussion: The intermediate group had a better treatment response with intact cognition. Progression in ophthalmological findings despite ERT was the main problem in the intermediate group.

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Sialidosis type I with nonspecific biochemical and enzymatic patterns caused by two novel, compound heterozygous variants in NEU1 gene Muetze U 1, Buerger F 1, Hoffmann J 2, Tegetmeyer H 4, Nickel P 3, Lemke J R , Syrbe S 1, Beblo S 3

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Div Metab Dis, Univ Child Hosp, Heidelberg, Germany, 2CeGaT, Tuebingen, Germany, 3Div Metab Dis, Univ Child Hosp, Leipzig, Germany, 4Dep Ophtalmol, Univ Hosp, Leipzig, Germany, 5Inst Hum Genet, Univ Hosp, Leipzig, Germany Background: Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. Case Report: We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. Results: Cherry red macular spots were first noted at 6 years of age after routine screening revealed myopia. Physical examination, psychometric testing, laboratory investigations as well as cerebral MRI were unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialid osis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was minimally elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to less than 1 %; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene, leading to amino acid changes predicted to impair protein function. Discussion: Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

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Methods: In this study 36 MPS patients as patient group and 30 healthy children as control group were included. Plasma chitotriosidase and hs-CRP levels were studied in these patient and control groups. Results: Although plazma chitotriosidase levels were found significantly increased levels in MPS patients than control groups (p ≤ 0.05 was significant) there were no significantly difference between hs-CRP levels in MPS patients and control groups. Discussion: As chitotriosidase is one of the parameter showing inflammatory p r oc e s s , i t ma y b e a he l p fu l d i a g n os t i c m a r ke r t o e v a l u a t e mucopolysaccaridosis.

Iminosugar based pharmacological chaperones: selecting new leads to target Gaucher, Morquio A and Hunter disease. Matassini C 1, D’Adamio G 1, Parmeggiani C Morrone A 3, Cardona F 1

1 2

, Catarzi S 4, Goti A 1,

1 Dip Chemistry U Schiff, Univ Florence, Florence, Italy, 2CNR INO and LENS, Florence, Italy, 3Dip Neurofarba, UNIFI, AOU Meyer, Florence, Italy, 4Paed. Neur. Unit and Lab, AOU Meyer, Florence, Italy

P-423 Background: Pharmacological chaperone therapy (PCT) is emerging as a treatment for many lysosomal storage disorders. It offers several advantages, such as oral administration and reduced costs. PCs are able to recover endogenous enzyme activity and are thus the only treatment that targets the functional origin of LSDs at present. Unfortunately, a great hurdle is represented by the challenging identification of compounds which are suitable for use as chaperones, especially because the mechanisms behind these processes are still far from clear. The most promising PCs for LSDs reported to date are iminosugars, sugar mimetics with a nitrogen atom replacing the endocyclic oxygen of carbohydrates. Methods: The inhibitory activity of a library of natural iminosugar analogues was initially assayed on a panel of 14 human lysosomal glycosidases and sulfatases, with the aim of identifying target enzyme(s) and the most promising molecules. On the basis of these findings, new properly functionalized compounds were designed and synthesized resulting in more potent inhibitors, suitable for testing as PCs in vivo. Results: Among the screened glycosidases, glucocerebrosidase (GCase, the deficient enzyme in Gaucher disease) has proved to be the most affected. In particular, two piperidines functionalized in different positions with a lipophilic pendant were found to rescue the residual GCase activity in N370S/RecNcil mutated human fibroblasts up to 1.5 fold. Moreover, sulfatase enzymes such as N-acetylgalactosamine-6sulfatase (GALNS) and iduronate-2-sulfatase (IDS) were inhibited in the micromolar range by multivalent compounds (obtained by grafting 9 bioactive units on the same scaffold). Discussion: The close collaboration between synthetic chemists and molecular biologists allowed the identification of two new GCase PCs and two potent reversible inhibitors of GALNS and IDS, excellent candidates as PCs or enzyme stabilizers for Morquio A and Hunter syndrome. Grant RF-2011-02347694

Type 1 hypersensitivity reaction and desensitization with Elosulphase alpha Inci A 1, Kan A 2, Topuz B 3, Okur I 1, Ezgu F S 1, Bakirtas A 2, Tumer L 1 1

Div Ped Metab Dis,Gazi Univ Hosp, Ankara, Turkey, 2Div Ped Allergy Dis, Gazi Univ Hosp, Ankara, Turkey, 3Dep Ped, Gazi Univ Hosp, Ankara, Turkey Background: Elosulphase alpha is an enzyme used in Mucopolysaccaridosis type 4-A (MPS 4-A). In phase III studies hypersensitivity reactions of elosulphase were discussed. Case Report: An 8 year old patient diagnosed with MPS 4-A had thirteen doses of elosulphase enzyme with 2 mg/kg per dose weekly. He had no history of hypersensitivity reaction until thirteenth dose of enzyme. In 13th dose, he had papulomacular, urticerial reaction over the foot and also he had fever. In 14 th dose, he had itchy, urticerial lesions and 15 th dose he had angioedema. Pediatric allegy department performed skin reaction test with 1/100 dilution as 6x6 mm induration was measured and accepted as positive reaction. Total enzyme amount were calculated and desensitisation with dilution of 1/100, 1/10 and whole concentration were performed within 6 h. Results: No complication or reaction were seen after desensitization. He has been treated with 26 doses of enzymes with desensitisation protocol. Discussion: Elosulphase alpha is the only drug that needs to be used in MPS 4A patients that have been no other avaible treatment. In this report we used a succesful desensitisation protocol in the MPS 4-A patient, having type I hypersensitivity reaction.

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Evaluation of chitotriosidase and high sensitivity c—reactive protein levels in mucopolysaccaridosis 1

1

2

1

1

1

Inci A , Genc B , Demirtas C Y , Udgu B , Karaoglu A , Okur I , Ezgu F S 1 , Biberoglu G 1, Tumer L 1

Bone marrow transplantation from heterozygous donor improve IDS deficiency in mucopolysaccharidosis type II mice Akiyama K 1 2, Shimada Y 2, Higuchi T 2, Yokoi T 2 3, Yokoi K 2 3, Fukuda T 4 , Iizuka S 2, Kobayashi H 2 3, Ishii M 1, Ida H 2 3, Ohashi T 2 3

1

Div Ped Metab Dis,Gazi Univ Hosp, Ankara, Turkey, 2 Div Med Biochemistry, Gazi Univ Hosp, Ankara, Turkey

1 Dep Ped, kitasato Univ, Kanagawa, Japan, 2Div Gene Ther, The Jikei School of Med, Tokyo, Japan, 3Dep Ped, The Jikei School of Med, Tokyo, Japan, 4Div Neuropatho, The Jikei School of Med, Tokyo, Japan

Background: Mucopolysaccaridosis (MPS) are a group of lysosomal storage diseases caused by deficiency of lysosomal enzymes. In pathogenesis of mucopolysaccaridosis, inflammatory pathway have been showed playing major role due to intralysosomal storage of glycosaminogylycans and then inflammation is being increased as oxidative stress and other inflammatory mediators are increased. Chitotriosidase is secreted mainly by active macrophages and is synthesized both in normal healthy conditions and also in inflammatory situations. Increased levels of chitotriosidase are found in lysosomal storage diseases especially in Gaucher disease and Niemann-Pick diseases. High sensitivity c reactive protein (hs-CRP) is an inflammatory biomarker found in high levels in cardiovascular disorders and inflammation. In this study, aim was to evaluate the chitotriosidase and hs-CRP levels in MPS patients as these parameters could show proinflammatory process.

Background: Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder,caused by deficient activity of iduronate-2-sulfatase (IDS), resulting in accumulation of glycosaminoglycans (GAGs) in various tissues. Although bone marrow transplantation (BMT) remains controversial in regard to indication for patients with MPS II, we previously demonstrated that BMT has a therapeutic effect on several tissues in murine model of MPS II. Given the clinical implementation of BMT to MPS II, HLA-matched sibling, who is heterozygous carrier, seems to be promising donor. However, utility of bone marrow cells derived from heterozygous donor as a therapeutic tool is still unclear. In this study, we analyzed whether BMT from heterozygous donor rescues IDS deficiency in MPS II model mice. Methods and Results: Irradiated MPS II mice were transplanted with bone marrow cells obtained from wild type or heterozygous mice. No significant differences were detected in engraftment between BMT from wild type

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 (WBMT) and heterozygous donor (HBMT). Both increased serum IDS activity and decreased levels of glycosaminoglycans (GAGs) in several tissues were observed in MPS II mice treated by HBMT, but less than that in mice treated by WBMT. This observation was consistent with the result of pathological analysis. On the other hand, the levels of LC3 lipidation, which is marker of autophagosome, were normalized in liver from MPS II mice treated by HBMT as well as WBMT. Discussion: These results indicate that HBMT is less effective than WBMT in elimination of accumulated GAGs in tissues from MPS II mice. However, our observations also suggest that HBMT may be able to rescue the secondary alterations such as autophagic dysfunction caused by IDS deficiency in MPS II mice. HBMT may have potential application to therapy for MPS II.

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Sulfated disaccharides improve iduronate-2-sulfatase function in fibroblasts from patients with mucopolysaccharidosis type II Hoshina H

12

2

2

, Shimada Y , Higuchi T , Kobayashi H

12

, Ida H

12

, Ohashi T

Results: The main clinical signs included dysostosis multiplex, genu valgus, short stature and mild coarse faeces. In addition, mild cardiac involvement, hepatosplenomegaly and corneal opacity were also detected in some cases. Biochemical assays revealed urinary KS excretion, detected by LC-MS/MS spectrometry, and a partial beta- galactosidase enzyme deficiency in all patients. GLB1 molecular analysis detected the common p.Trp273Leu mutation at a heterozygous level in three patients. Also, three novel mutations were detected: a splicing defect and two missense mutations. Standard sequencing procedures, that include analysis of the GLB1 coding regions and intron- exon boundaries, failed to identify the second disease causing mutation in one patient’s DNA. The investigation of deletions/ insertions and GLB1 mRNA analysis are ongoing in this patient’s samples. Discussion: We report a significant number of patients affected by this rare disease. The clinical features of Morquio B are less severe than for Morquio A so diagnosis is more challenging. We provide new insights for the clinical, biochemical and molecular evaluation of these patients.

P-427

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1

Depart Pedi, Jikei Univ Sch Medi, tokyo, Japan, 2Divi Gene Thera Res Cent Medi Scie, tokyo, Japan

Could propionylcarnitine and free carnitine be used as antioxidative markers in Mucopolysaccaridosis? Inci A 1, Biberoglu G 1, Genc B 1, Karaoglu A 1, Okur I 1, Ezgu F S 1, Tumer L 1

Background: In recent years, pharmacological chaperone (PC) therapy has attracted considerable concern as potential treatment for several lysosomal storage disorders such as Fabry disease and Gaucher disease. However, PC therapy for mucopolysaccharidosis type II (MPS II), which is an inherited metabolic lysosomal storage disorder that caused by a deficiency of iduronate-2-sulfatase (IDS), has not been established yet. We previously showed that sulfated disaccharide derived from heparin is a candidate chemical of PC for IDS. In this study, we investigated whether sulfated disaccharide improves the function of IDS in patient fibroblasts and transiently expressed cells. Methods and Results: Addition of sulfated disaccharide alleviated the thermal degeneration of IDS in cell extracts obtained from HEK293T cells overexpressing wild type human IDS. To analyze whether sulfated disaccharide stabilize intracellular mutated enzyme, skin fibroblasts derived from MPS II patient showing mild phenotypes were treated with the disaccharide for 4 days. Sulfated disaccharide significantly increased the IDS activity in patient fibroblasts. Furthermore, decreased levels of glycosaminoglycan accumulation were observed in patient fibroblast after treatment with sulfated disaccharide. Discussion: These results suggest that sulfated disaccharide may have a potential application for the PC therapy of MPS II.

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From clinical to biochemical- genetic characterisation of Morquio B disease Caciotti A 1, Tonin R 1 3, Catarzi S 1, Vasarri M 1, La Marca G 1, Forni G 1, Paoli A 1 , Bechini S 1, Procopio E 3, Donati M A 3, Rigoldi M 4, Di Rocco M 5, Andaloro A 5, Antuzzi D 6, Rampazzo A 7, Scarpa M 7, Renzo G 1 2, Morrone A 1 2 1 Paed Neurol Unit, Meyer Child Hosp, Florence, Italy, 2 Dept of NEUROFARBA, Univ of Florence, Florence, Italy, 3Metab and Musc Unit, Meyer Child Hosp, Florence, Italy, 4Center Rare Dis,Dep Int Med,S Gerardo H, Monza, Italy, 5Unit Rare Dis, Dept Ped, IRCCS Gaslini, Genova, Italy, 6Ped Clinic, Catholic Univ, Gemelli Hosp, Rome, Italy, 7Dept of Ped, Univ Child Hosp, Padova, Italy

Background: Morquio B is the milder form of the lysosomal storage disorder caused by beta galactosidase (GLB1) deficiency. Patients mainly exhibit connective- skeletal features due to selective keratan sulphate (KS) storage. We aim to provide a clinical, biochemical and molecular overview of this extremely rare, and probably underestimated disorder. Methods: We analysed 9 patients affected by the disease at a clinical, biochemical and molecular level. The patients have not been reported elsewhere.

1

Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey

Background: Oxidative stress may play an important role in the pathophysiology of mucopolysaccaridosis (MPS), since accumulation of glycosaminoglycans may lead to excessive production of free radicals. Human and animal studies have showed that free carnitine and propionylcarnitine had some antioxidant properties as free radical scavengers, protecting cells from radical oxygen species, preventing cells from oxidative damage. Malondialdehyde could be used as an indicator of lipid peroxidation and oxidative stress processes. In this study, aim was to show antioxidative properties of propionylcarnitine and free carnitine in acylcarnitine profile with respect to oxidative markers in MPS. Methods: We included 27 MPS as patient group and 24 healthy child as control group. We studied free carnitine and propionylcarnitine as antioxidation markers by tandem mass spectroscopy and malondialdehyde as oxidative marker measured by spectrophotometric method. Results: When we compared free carnitine, propionylcarnitine and malondialdehyde between MPS and control groups. We found free carnitine and propionylcarnitine levels were significantly lower in MPS than control groups (p = 0.02 and p = 0,00 respectively. Malondialdehyde levels were found significantly increased in MPS(p = 0.000).Wen we compared these parameters between enzyme treated MPS and no enzyme treated MPS, there were no significantly differences (p = 0,54, p = 0, 21, p = 0.94).Also there were significantly difference in propionylarnitine and malondialdehyde between MPS type III and control groups (p = 0.001,p = 0.000). Discussion: Mucopolysaccaridosis is an inflammatory process that oxidative stress has major effect. In MPS patients, oxidative parameters like malondialdhyde and antioxidant parameters like propionylcarnitine and free carnitine could not be in balance and it could be through the oxidative side. Propionylcarnitine and free carnitine could be used as an antioxidative marker in mucopolysaccaridosis.

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High-throughput determination of urinary hexosamines in newborns of 2– 3 days of age: application for the early diagnosis of mucopolysaccharidoses Volpi N 2, Maccari F 2, Galeotti F 2, Concolino D 3, Marchesiello R L 1, Galeazzi T 1 1 Dep Odont Spec Cl Sc, UNIVPM, Ancona, Italy, 2Dep Life Sc, UNIMORE, Modena, Italy, 3Div Ped, Univ Magna Graecia, Catanzaro, Italy

S184 Background: In this study, we propose a high-throughput procedure for the simultaneous determination of glucosamine and galactosamine generated from urinary glycosaminoglycans (GAGs) applied to healthy newborns of 2–3 days of age. Methods: The GAGs of urine of 155 healthy newborns having 2– 3 days of age were rapidly treated with HCl after precipitation with ethanol. Generated hexosamines were separated by HPLC equipped with a fluorimetric detector after derivatization with a fluorescence molecule. Results: Both hexosamines, galactosamine (GalN) and glucosamine (GlcN) were rapidly and clearly separated and quantified obtaining a GalN/GlcN ratio of 0.74 with a CV% of ~29 %. By comparing these new data obtained on the urine of newborns of 2–3 days of age with those previously published [Coppa GV et al. Anal. Biochem. 411, 32, 2011], we obtained significant differences of GalN/GlcN ratio in patients affected by MPS I, II, III and VI. Subjects with MPS IV were found borderline and further studies are necessary. No significant differences were observed with 83 heathy subjects of ~4 years old. Discussion: Contrary to other analytical approaches, the present procedure is able to measure total abnormal amounts of urinary GAGs, high-molecular mass and related fragments, as well as specific hexosamines belonging to a group of GAGs. We propose this assay for possible application in MPS early diagnosis. In fact, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method may be suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis and treatment. Partially supported by MIUR-PRIN2012

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Determination of total and single species of all uronic acid-bearing glycosaminoglycans in urine of newborns of 2–3 days of age for a possible early diagnosis of mucopolysaccharidoses Volpi N 2, Maccari F 2, Galeotti F 2, Tomanin R 3, Monachesi C 1, Galeazzi T 1, Catassi C 1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 P-430

Development of a routine LC-MS/MS method for the analysis of underivatized urinary oligosaccharides for the diagnosis of oligosaccharidoses. Piraud M 1, Pettazzoni M 1, Menegaut L 2, Froissart R 1, Vianey-Saban C 1 1 Metab Dis Lab, Hospices Civils, Lyon, France, 2Biochem Dpt, Univ Hospital, Dijon, France

Background: The first step of oligosaccharidoses diagnosis is to evidence abnormal oligosaccharides (OS) excreted in urine, usually performed by thin layer chromatography (TLC, Humbel and Collart, 1975). These OS have been delineated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Bonesso et al., 2014), an apparatus that is not widespread in diagnostic laboratories. This method was suitable for the urinary screening of oligosaccharidoses. Methods: We have adapted this method to the use of a more common triple quadrupole tandem mass spectrometer (API 4500, Applied Biosystems©) coupled to liquid chromatography, for identification of OS. Each underivatized OS could be identified by several transitions (i.e. pairs of parent ion > product ion). Results: A characteristic urinary MS/MS pattern of various specific OS was found in urine from known patients affected with each type of oligosaccharidosis. This method allowed the identification of all the oligosaccharidoses classically identified by TLC: fucosidosis (n = 6), alpha-mannosidosis (n = 10), aspartylglucosaminuria (n = 7), GM1 gangliosidosis (n = 20), sialidosis (n = 11), galactosialidosis (n = 5), and Schindler-Kanzaki disease (n = 4), and extends the field of diagnosis to mucolipidosis type II (n = 9), Sandhoff disease (n = 11), and beta-mannosidosis (n = 1). Discussion: In the absence of specific standard and labelled internal standard for each compound, quantification could not be validated. The method is thus qualitative, but much more sensitive and specific than TLC, and allows the routine screening of oligosaccharidoses, even of moderate or adult forms. It is very rapid and easy to run (LC analysis time of 15 min per sample). No interference was noticed with drugs or other diseases, except for mucopolysaccharidoses, for which the signal corresponding to sialidosis was slightly elevated.

1 Dep Odont Spec Cl Sc, UNIVPM, Ancona, Italy, 2Dep Life Sc, UNIMORE, Modena, Italy, 3Dep Women Child Health, Univ Padova, Padova, Italy

P-431 Background: We developed a high-throughput analytical assay able to measure total and single species of all uronic acid-bearing urinary glycosaminoglycans (GAGs), i.e. hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), heparin/heparan sulfate (HS), applied to healthy newborns of 2–3 days of age. Methods: The GAGs of urine of 331 healthy newborns having 2– 3 days of age were rapidly extracted by precipitation with ethanol and treated with specific enzymes in the order to generate unsaturated disaccharides belonging to single type of GAG. After derivatization with a fluorescence tag, disaccharides were separated by capillary electrophoresis (CE) equipped with a laser induced fluorescence (LIF) detector. Results: In newborns of 2–3 days of age, HA was 1.0 μg/mL creat, CS 31.8 μg/mL, HS 2.6 μg/mL with trace-amounts of DS and heparin. Total GAGs were 35.4 μg/mL. The relative % of HA was 3 %, CS 87.8 % and HS 9.3 %. Moreover, urinary CS was found to be composed of 50 % 4-sulfated disaccharide, 20 % 6-sulfated disaccharide and 29 % disaccharide not-sulfated with a charge density of 0.71. HS was mainly formed of 79 % N-acetyl-groups, 21 % N-sulfated groups, 10 % 2-sulfated uronic acid residues and 15 % 6-sulfated disaccharides for an overall charge density of 0.46. Discussion: The illustrated procedure is able to measure the total content and single species of all urinary known uronic acid-bearing GAGs with possible future application in MPS early diagnosis. In fact, the relative percentages of single species of GAGs as such as its total content are known to change with MPS diseases also in relation to single type of MPS. Partially supported by MIUR-PRIN2012

Spinal Magnetic Resonance Imaging Findings in Mucopolysaccharidoses Type IVA Canda E 1, Eraslan C 2, Yazici H 1, Ucar S K 1, Coker M 1 1 Div Ped Metab Dis, Ege Univ Med Faculty, Izmir, Turkey, 2Div Radiology, Ege Univ Med Faculty, Izmir, Turkey

Background: Mucopolysaccharidosis IVA (MPS IVA), Morquio A, is caused by the deficiency in lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Systemic skeletal symptoms are caused by excessive Keratan sulphate storage. Methods: Our aim is to discuss clinical and laboratory and the spinal MR findings of 12 patients with MPS IVA. Results: Patients’ mean age was 13.5 (4–33) years and mean age at diagnosis was 8 (3–32) years. Male/female was 5/7. Eleven patients have corneal clouding, 5 patients have hearing loss, 8 patients have cardiac valve involvement and one patient has cardiomyopathy. All patients have foramen magnum stenosis, intervertebral disc protrusion, loss of vertebral corpus height, malformations of vertebral bodies, platyspondylia, periodontoid tissue and ligaments thickening. Four patients have cervical myelopathy. Two patients have spinal cord compression and decompressive surgery is planned. 17 years old male was died due to respiratory insufficiency and infection. All patients could walk without support except 3 patients. Eight of the patients have been treated with elosulphase alpha.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Discussion: Spinal MR investigation should be performed during follow up of the patients with MPS IVA. Spinal cord compression can be with or without signs of compressive myelopathy.

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Multidetector computed tomography for the evaluation of the trachea in patients affected by mucopolysaccharidoses Grimaldi M 1, Di Marco D 2, Rigoldi M 3, Piperno A 2 3, Biondi A 2 4, Gasperini S 4, Galimberti C 4, Parini R 4 1

Dep Neuroradiol, Humanitas Research Hosp, Milan, Italy, 2Univ MilanoBicocca,School Med and Surg, Monza, Italy, 3Center Rare Dis,Dep Int Med,S Gerardo H, Monza, Italy, 4Center Metab Dis,MBBM Found, S Gerardo H, Monza, Italy Background: Patients (pts) affected by mucopolysaccharidoses (MPS) frequently require surgery and preoperative airways assessment is crucial due to high frequency of airways abnormalities. Conventional techniques (radiography, fiberoptic bronchoscopy /laryngoscopy, axial computed tomography) are often unable to provide sufficiently detailed information. Multidetector computed tomography (MDCT) has greater anatomical coverage, optimal temporal and spatial resolution, short scanning time. Moreover, two-and three-dimensional (2D and 3D) images can be reconstructed from the original MDCT data set. Methods: We retrospectively processed MDCT data sets of the trachea from 12MPS pts (mean age 10.2y, range 1–21). They were compared with 16 age-matched control pts (mean age 10.2 y, range 3–22 y) who had chest MDCT due to other conditions. The lower section of the true vocal cords was considered the cranial extremity of the trachea; the axial plane through the border of the tracheal carina was considered the caudal extremity. Trachea was divided into 3 segments: proximal (1), medium (2) and distal (3) and the smallest diameter (D) in each segment was measured (D-min 1, 2, and 3, respectively) both in the frontal (coronal) and sagittal (median) sections. Results: MPS pts showed significantly smaller tracheal D as compared with control pts in the proximal and medium segments. The mean coronal D values (D-min 1–3 values) were 8.83 (±2.48) mm in MPS group and 10.89 (±2.52) mm in controls (p < 0.0001). The mean sagittal D-min 1–3 values were 8.73 (±2.49) mm compared to 10.30 (±3.23) mm in controls (p < 0.002). As for morphology, MPS patients showed tracheal deformities at several levels, in comparison to control pts. Discussion: 2D-and-3D images acquired by MDCT allow reliable detections on size and morphology of the trachea and have a potential for preoperative tracheal evaluation in MPS patients. Conflict of Interest declared.

P-433

Short-Term Outcome of Surgical Correction of Genu Valgum in Four Patients with Mucopolysaccharidosis Type IVA Sivri H S 1, Aksoy M C 2, Yilmaz G 2, Yildiz Y 1, Bilginer Gurbuz B 1, Pektas E 1, Dursun A 1, Tokatli A 1, Coskun T 1 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 2Dept Ortho Trauma, Hacettepe Univ Hosp, Ankara, Turkey

Background: Mucopolysaccharidosis type IVA (MPS IV-A) is characterized by skeletal deformities, including progressive genu valgum, which usually becomes severe by the fourth year of life, necessitating surgery. Hemiepiphysiodesis is a newer approach in genu valgum correction in MPS IV-A patients, which was classically corrected by osteotomy. Here, we present short-term outcomes of four MPS IVA

patients with genu valgum who were surgically treated in a single center. Methods: Hospital records of MPS IV-A patients with genu valgum, who were surgically treated in our center were reviewed. Intraoperative findings, complications, short-term outcomes were assessed. Results: Bilateral hemiepiphysiodesis of distal femur and proximal tibia was performed on three MPS IV-A patients aged 5, 6.5 and 7.5. All three could walk independently within 2 days. 6-min walk tests performed 3–6 months later showed improved capacity. One 9-year-old female patient underwent successful correction osteotomy and fixation of bilateral femur and tibia. She is still immobilized 2 months after surgery. Intraoperatively, bones were noticed to be brittle in all four patients. No complications were encountered except for postoperative pain. Discussion: The surgical approach and optimum timing of genu valgum correction in MPS IV-A patients are still debated. Our patients quickly recovered from hemiepiphysiodesis and showed improvement. Compared to osteotomy, hemiepiphysiodesis is less invasive, requires shorter hospital stay, no postoperative casting, but longer correction times and patient growth. Weak bone structure may cause displacement of surgical screws. Since severely affected MPS IV-A patients may have attained 90 % of their adult height by the age of four, poor potential for growth is a limiting factor for this surgical approach. Therefore, hemiepiphysiodesis should be performed early, underlining the importance of early diagnosis of MPS IV-A and rapid management of genu valgum.

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Mucopolysaccharidosis type I due to maternal uniparental disomy of chromosome 4 Gurner M 1, Chin S 1, Owens G 1, Pyragius T 1, Brion K 1, Trinh M 1, Friend K 1, Yu S 1, Ketteridge D 2, Stark S 1, Fletcher J 1 1 Dept Gen and Mol Path, SA Pathology, Adelaide, Australia, 2Metab Unit, SAPath, Women and Child Hosp, Adelaide, Australia

Background: Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder, caused by a deficiency of the enzyme α-L-iduronidase. MPS I results in multisystem dysfunction, particularly within the musculoskeletal, respiratory, ENT and cardiac systems. Neurological involvement is often seen in the most severely affected phenotypes. Case Report: We present a 2.5 year old male who was diagnosed at 13 months with MPS I. The patient’s symptoms included severe early onset respiratory problems, bilateral developmental dysplasia of the hips, bilateral fixed flexion elbow deformities, coarse facial features, bilateral hernia, hearing loss, corneal clouding, bilateral claw hand deformities, hepatosplenomegaly and a gibbus deformity of the spine. Results: A diagnosis of MPS I was confirmed by abnormal urinary GAG electrophoresis and markedly reduced activity of α-Liduronidase in leucocytes. The patient was screened for a number of common mutations within the IDUA gene and was found to be homozygous for the p.Trp402* mutation. However, familial studies revealed that only the mother was a carrier of this mutation. Full IDUA sequencing ruled out the presence of a second mutation. Short tandem repeat (STR) analysis confirmed paternity; however one STR loci revealed that the patient had inherited both alleles from the mother and suggested a maternal UPD of chromosome 4. Single nucleotide polymorphism (SNP) array confirmed that the patient had maternal heterodisomy of chromosome 4 with 2 homozygous (isodisomic) regions. One of the homozygous regions contained the IDUA gene; which was the cause of MPS I in this patient. Discussion: These findings emphasise the importance of segregation analysis for determining the inheritance of mutations. Given that the recurrence risk for future pregnancies is dramatically reduced, this family can receive the appropriate genetic counselling.

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Screening Test II revealed age-appropriate social development and mildly delayed fine and gross motor skills as well as lingual development. No adverse events occurred. Discussion: Early initiation of investigational rhGUS in a 9-month-old infant with MPS VII resulted in stabilization of disease progression and even reversal of some disease manifestations. Conflict of Interest declared.

Do cytokine levels play a role in pathogenesis of mucopolysaccaridosis patients? Inci A 1, Tumer L 1, Demirtas C Y 2, Karaoglu A 1, Okur I 1, Olgac A 1, Ezgu F S 1, Biberoglu G 1 1 Div Ped Metab Dis,Gazi Univ Hosp, Ankara, Turkey, 2 Div Med Biochemistry, Gazi Univ Hosp, Ankara, Turkey

Background: Mucopolysaccaridoses are lysosomal storage diseases with multiorgan disfunction as acid hydrolases,degregading glycosaminoglycans, are deficient in lysosomes. In mucopolysaccaridoses, glycosaminoglycan deposition causes inflammatory cascade activation via the toll-like receptor pathway. In animal models of MPS, studies showed that intralysosomal accumulation of glycosaminoglycans activated oxidative stress and inflammation. Subsequently cytokine, chemokine, proteases secretion were increased and apoptosis was triggered. In this study, the aim was to show that glycosaminoglycans can trigger the inflammatory pathway via cytokines. Methods: In this study we included 41 MPS patients and 29 healthy children as control groups. We studied tumor necrosis alpha, interleukin beta and interleukin 6 levels by ELISA method. Results: Cytokine levels were significantly increased in MPS patients compared to control groups (p ≤ 0.05). Cytokine levels in patients treated with enzyme replacement therapy(ERT) were found higher than control group. Patients treated with ERT and no ERT were compared but there were no significant difference between two groups. Discussion: The inflammatory pathway is activated in MPS patients due to glycosaminoglycan accumulation in lysosomes but ERT has no effect on cytokine levels and anti inflammatory drugs could be assumed to be more effective when given with ERT.

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Early initiation of investigational enzyme replacement therapy in a 9month-old infant with mucopolysaccharidosis type VII Karaoglu A 1, Inci A 1, Biberoglu G 1, Okur I 1, Kilickaya A 1, Tumer L 1, King B 2, Haller C 2, Ezgu F S 1 1 Div Ped Metab Dis, Gazi Uni Hosp, Ankara, Turkey, 2Ultragenyx, California, United States

Background: Mucopolysaccharidosis type VII (MPS VII-Sly Syndrome) is a very rare form of MPS characterized by a deficiency of the lysosomal enzyme β-glucuronidase (GUS), required for the degradation of the glycosaminoglycans (GAGs) dermatan, heparan and chondroitin sulfate. Progressive accumulation of these GAGs leads to dysfunction in numerous tissues and organs. Progression of the disease is inevitable especially in early onset cases. Clinical trials are ongoing with recombinant human GUS (rhGUS) in patients with MPS VII, and compassionate use in a 12-year-old boy with advanced disease resulted in clinical and laboratory improvement. Case Report: A 2-month-old female infant was referred for having phenotypical findings suggestive of MPS. The baby reportedly had generalized oedema at birth that later resolved which could be consistent with hydrops. Skeletal xray showed dysostosis multiplex. An enzyme panel for MPS revealed a decreased GUS level of 0,69 (Normal: 129,9 ± 45,8) nmol/hr/mg.protein consistent with MPS VII. Every other week enzyme replacement therapy (ERT) with investigational rhGUS was started at 9 months of age as a compassionate use provided by Ultragenyx. The first 9 IV infusions were given at a dose of 2 mg/ Kg and the last 8 at a dose of 4 mg/Kg. Results: Significant reduction from baseline was seen in urinary total GAG excretion of 40 % on 2 mg/Kg dose and 73 % on 4 mg/Kg dose, and liver and spleen volumes were normalized. At age 19 months, length and weight are within normal limits and head circumference is at the upper limit of normal. Neurodevelopmental assessment made with the Denver Developmental

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EEG features in patients with Mucopolysaccharidoses III at different disease stages. Barone R 1 2, Cocuzza M D 1, Guida C 1, Miano G 3, Sofia V 3, Fiumara A 2 1 Child Neuropsychiatry, Univ Child Hosp, Catania, Italy, 2Div Metab Dis, Univ Child Hosp, Catania, Italy, 3Div Neurol, Univ Hospital, Catania, Italy

Background: Mucopolysaccharidoses (MPS) III are neurodegenerative disorders characterized by intellectual and motor regression, behavioural and sleep disturbances with death usually in the second decade of life. We retrospectively evaluated electroencephalography (EEG) records of a cohort of patients with MPS III at different disease stages. Methods: Twelve patients (MPS IIIA: 6; IIIB: 4; III C: 2) were studied. They comprised 4 males and 8 females aging from 1 to 31 years. Follow-up studies were obtained in seven patients. Mean duration of follow-up was 5 years (2– 7.2). Results: In one patient repeated EEG between ages 1 and 3 years showed normal background activity in wakefulness and the progressive occurrence of slow-wave activity with superimposed sharp-waves during sleep. Three other patients aged 6 to 9 years showed slowing of occipital-dominant rhythm and background activity during wakefulness. One of them had also focal spike or sharp-wave discharges during wakefulness. Eight subject aged 11 to 31 years had moderate to severe slowing of background activity with dominant theta and delta activity and progressive loss of occipital dominant rhythm. In addition six out of eight had focal discharges and generalized slow-spike-wave patterns. Two patients experienced almost continuous focal slow spike-wave activity during wakefulness featuring non-convulsive status epilepticus. Discussion: EEG shows progressive changes during MPS III course that appear to be related with age and disease course. Ictal EEG activity including non-convulsive status epilepticus occur frequently with disease progression. EEG recording is recommended in patients with MPS and acute worsening of behaviour and/or sleep disturbance.

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Mucopolysaccharidosis type II in 44 Czech, Slovak, Serbian and Croatian patients: clinical manifestation and analysis of mutational spectrum Dvorakova L 1, Vlaskova H 1, Sarajlija A 2, Ramadza D P 3, Poupetova H 1, Hruba E 1, Hlavata A 4, Bzduch V 5, Peskova K 1, Storkanova G 1, Kecman B 2 , Diordjevic M 2, Baric I 6, Fumic K 7, Barisic I 8, Reboun M 1, Kulhanek J 9, Zeman J 9, Magner M 9 1

Inst IMD, GenUnivHosp,FacMed1CharlesUniv, Prague, Czech Republic, DepMetClinGen, MCHCInst Serb, UnivSchMed, Belgrade, Serbia, 3Dep Pediat, Univ Hosp Center, Zagreb, Croatia, 41 Dep Pediat, Comenius Univ Child Hosp, Bratislava, Slovakia, 52 Dep Pediat, Comenius Univ Child Hosp, Bratislava, Slovakia, 6DepPediat, UnivHospCenter, SchoolMed, Zagreb, Croatia, 7Dep Lab Diag, Univ Hosp Center, Zagreb, Croatia, 8Child Hosp, School Med, Zagreb, Croatia, 9 DpPedAdoMed,FMed1CharlesUniv,GenUnivHosp, Prague, Czech Republic 2

Background: Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an X-linked lysosomal storage disorder caused by deficiency of iduronate-2sulphatase (IDS). We analysed the clinical and laboratory data from 44 patients with this disease.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Methods: Sequencing of PCR products was performed using Sanger method and NGS (MiSeq, Illumina). Results: 21 Czech, 7 Slovak, 9 Croatian patients and 7 Serbian patients (43 M/ 1 F) were included in the study (average age 12.8 years, range 1.2–43 years). Prevalence ranged from 1:69,223 in Serbia to 1:263,543 in Czech Rep. Disease manifested already in infancy in the majority of patients (71 %). The leading symptoms at onset included inguinal hernias (in 13 pts) and developmental delay (in 10 pts). Attenuated, intermediate and severe form were observed in 6, 4 and 31 patients, resp. Cognitive functions were normal in 9 pts, 5 pts had mild, 5 moderate and 21 severe developmental delay, typically after developmental regression (60 %). The most common clinical signs were dysostosis multiplex (100 %), joint contractures (98 %), hepatomegaly (93 %), splenomegaly (71 %), hearing impairment (79 %), macrocephaly (76 %), short stature (70 %), inguinal hernias (72 %), and cardiomyopathy (38 %). Enzyme replacement therapy was introduced in 21 pts, hematopoietic stem cell transplantation was carried out in two boys with poor outcome. The excretion of glycosaminoglycans over 70 g/mol creatinine at the onset of the disease predicted the severe form while the level of residual enzyme activity did not correlate with the disease severity. The causative mutation was identified in each of 39 analysed patients. Discussion: Although the genotype phenotype correlation is not straightforward in MPS II, recombinations IDS/IDS2 and gross deletions were always associated with severe phenotype. Moreover, the impact of recurrent mutations (e.g. p.Ser333Leu, p.Arg468Gln or p.Arg88Cys) may be predicted with high probability. Supported by MZ CR—RVO VFN64165, GAUK 580716.

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Increase of serum levels of matrix metalloproteinase-2 in four paediatric MPS II patients Magner M 1, Asfaw B 2, Kulhanek J 1, Poupetova H 2, Langer J 1, Zeman J 1, Svandova I 3 1 Dep Pediatrics, Univ Hosp, Charles Univ, Prague, Czech Republic, 2Inst IMD, Univ Hosp, Charles Univ, Prague, Czech Republic, 3Dep Physiol, Fac Science, Charles Univ, Prague, Czech Republic

Background: Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive metabolic disorder caused by a deficiency of lysosomal enzyme iduronate-2-sulfatase required for the degradation of mucopolysaccharides or glycosaminoglycans (GAGs). Subsequent primary accumulation of different GAGs results in a wide spectrum of clinical features. Matrix metalloproteinases (MMPs) are key enzymes involved in the degradation of extracellular matrix and other processes. The aim of this study was to describe protein levels of matrix metalloproteinase 2 (MMP-2) in the serum of paediatric patients with MPS II. Methods: Four patients (3 M/1 F, none under the enzyme replacement therapy) formed the study group, compared to four healthy age- and sex-matched participants. Whole blood samples were collected by single venipuncture. Serum samples were prepared by centrifugation. Serum samples were diluted 1:100, proteins were fractionated by 10 % SDS-PAGE (25 ug of protein/well). MMP-2 levels were measured by Western blot using rabbit polyclonal IgG MMP-2 antibody (H-76, sc-10736, Santa-Cruz Biotechnology, Inc.). Results: Two MMP-2 bands migrated as proMMP-2 (92 kDa) and the activated MMP-2 (68 kDa) with MMP-2 being the most prominent. We evaluated level of the activated MMP-2. Quantification of the intensity of bands revealed that the level of MMP-2 was significantly increased in the female patient (P = 0.002) and one male patient (P = 0.025) as compared to controls. In the two other male patients we also observed increase of the serum MMP-2 level as compared to healthy controls, but it reached no statistical significance (P = 0.888 and 0.200, respectively). Discussion: This study presents a baseline for our further studies of metalloproteinases role and differential expression in the pathophysiology of MPS II. We demonstrated increase of MMP-2 level in serum of four paediatric MPS II patients. Supported by RVO-VFN 64165/2012.

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Effect of enzyme replacement therapy in a 5-year-old boy with mucopolysaccharidosis IV A Magner M 1, Marik I 3, Kulhanek J 1, Svecova S 1, Tesarova M 1, Asfaw B 2, Poupetova H 2, Bartl J 2, Zeman J 1 1 Dep Pediatrics, Univ Hosp, Charles Univ, Prague, Czech Republic, 2Inst IMD, Univ Hosp, Charles Univ, Prague, Czech Republic, 3Amb Centr Defects Locomotor Apparatus, Prague, Czech Republic

Background: Mucopolysaccharidosis IVA (MPS IVA, Morquio syndrome A, OMIM 253000) is an AR inherited metabolic disorder caused by deficiency of N-acetylgalactoseamine-6-sulfatase, with severe skeletal dysplasia and disproportionate growth disorder. FDA approved enzyme replacement therapy (ERT) with elosulfase alfa (Vimizim, BioMarin Pharmaceutical Inc.) in 2014. Only a single study evaluating effects of the therapy in 15 children younger than 5 years (Jones et al. 2015) has been published yet. Here, we present a case report of 5-year-old boy with the diagnosis of MPS IV A, who was put on the therapy at the age of 48 months. Case Report: The diagnosis was determined upon a clinical suspicion of skeletal dysplasia due to dorsolumbal kyphoscoliosis at the age of 43 months. Growth failure with a fall from the 86th percentile at 1 year of age to the 3rd percentile was already present. The clinical presentation further included macrocephaly, mild facial dysmorphia, flared lower ribs, sternal protrusion, joint hypermobility and hepatomegaly (+2 cm). The boy complained of knee pain and refused to run. The diagnosis was confirmed at both enzymatic and molecular-genetic level, the boy is a combined heterozygote for mutations c.151G>A (p.Glu51Lys) and c.1219A>C (p.Asn407His). Results: The well-tolerated therapy was introduced at the age of 48 months. Upon the treatment, growth velocity raised temporarily from −3.5 SDS to +0.3 SDS with subsequent drop to −1.5 SDS. Lower extremities pain vanished and the boy started to run and even walk the stairs. Cervical lordosis and thoracic kyphosis are less distinct, also with the help of brace treatment. Hepatomegaly resolved. Glycosaminoglycans excretion remained elevated (29.5 g/mol Cr prior to therapy, 24.0 g/mol Cr after 12 month upon therapy, norm < 15.5 g/ mol Cr). Discussion: The earliest feasible ERT start is probably crucial to maximize the possible benefits in MPS IV A treatment. Supported by RVO-VFN 64165/2012.

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Four-years study in 137 Russian patients with mucopolysaccharidosis (MPS) Pushkov A A 1, Savostyanov K V 1, Gevorkyan A K 1, Kuzenkova L M 1, Podkletnova T V 1, Pakhomov AV 1, Namazova-Baranova L S 1, Baranov A A 1 1

Scientific Center of Children’s Health, Moscow, Russian Federation

Background: Mucopolysaccharidosis is a rare genetic disease characterized by deficiencies of lysosomal enzymes, leading to accumulation of glycoaminoglycans (GAG) in many tissues resulting in distorted growth or function. Methods: The study included 312 patients 191 boys and 121 girls at the age of 6 month to 29 years. At first total GAG measured in urine, than the qualitative analysis of GAG by TLC to clarify the type of MPS was done. In the third step the enzyme activities for MPS1, MPS2, MPS4a and MPS6 was measured using HPLC tandem MS/MS. The next step included the molecular genetic analysis. Results: The study identified 137 patients with different types of MPS. Among them, 32(23 %) patients with MPS1, 68(49 %) with the MPS2, 21(16 %) with MPS3, 5(4 %) with MPS4 and 11(8 %) with MPS6. Among these patients, we identified 26 not described earlier mutations in IDS gene, 12 in IDUA gene, 4 in SGSH gene, 3 in NAGLU gene, 2 in GALNS gene and 5 in ARSA gene.

S188 Interestingly, one patient with a clinical manifestations of MPS3 without mutations in SGSH and NAGLU gene was found a novel mutation c.743G > T (p.Gly248Val) in HGSNAT gene and the diagnosis MPS, type 3C was established for the first time in Russia. Discussion: Such a large number of patients we have described in 4 years, as well as a variety of identified mutations may indicate a significant increase in interest among physicians in recent years, as well as a great diversity of populations inhabiting the territory of Russia.

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Mutation analysis for mucopolysaccharidosis gene’s cluster in a southwestern colombian affected population Sanchez A 1, Moreno L M 1, Satizabal J M 1 1

Universidad del Valle, Cali, Colombia

Background: Southwestern region of Colombia has showed an increasing on incidence of mucopolysaccharidosis diseases at recent years. Several authors have proposed different mechanisms to explain this increasing. Our research group has screened gene variants on a set of patients suffering any type of Mucopolysaccharidosis. With the results of this screening we found a pattern of transitional mutations C > T or T > C along gene sequences that leads to proposed a molecular mechanism involved in de novo development of pathological sequence variants. Methods: Gene sequencing was performed on a group of 11 patients diagnosed with any of the Mucopolysaccharidosis described in literature, to characterized variants in sequences. For new SNPs, SIFT and PolyPhen2 analysis were ran to determine pathological effect. Results: A total of 69 gene variants were reported, from those 28 were characterized as transitions C > T and T > C. For those 28, 5 were not registered previously. 3 were associated as pathogenic and explained the symptoms in the patients. Just 6 gene variants were located at intron regions and none of them were associated with alterations on gene splicing or truncated proteins. Finally, 7 variants were synonymous and 17 did not show protein shift. Discussion: Several authors have reported a preference on mutations along CpG islands with a prevalence of nearly 80 % in transitional mutations. A possible explanation of this behavior would be related with epigenetic mechanisms like DNA methylation to bias gene expression on altered sequences to alleviate any pathogenic effect. We consider this finding very valuable to propose a broad screening of methylation patterns along mucopolysaccharidosis genes to elucidate molecular etiopathogenesis.

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Outcome of haematopoietic stem cell transplantation in two mucopolysaccharidosis type II severe patients Parini R 1, Spada M 2, Gasperini S 1, Galimberti C 1, Bertola F 3, Biamino E 2, Pasetti M 1, Nichelli F 1, Biondi A 1, Rovelli A 1 Dept Pediat MBBM Found S Gerardo Hosp, Monza, Italy, 2Dept Pediat University of Torino, Torino, Italy, 3Consorzio Gen Molec Umana, Monza, Italy. 1

Background: Mucopolysaccharidosis type II (Hunter syndrome-MPS II) is a X-linked lysosomal storage disease involving central nervous system in more than 50 % of cases. The available treatment is enzyme replacement therapy (ERT). Haematopoietic stem cell transplantation (HSCT) was attempted in patients (pts) with variable age and progression, who had a poor evidence of neurologic improvement or stabilization. Since HSCT is currently a much safer procedure compared to

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 the past, HSCT was offered to two severe MPS II pts diagnosed quite early at 12 and 16 months. Case Report: Patient(pt)1 was born at term with head circumference 44 cm (>97th centile). Brain MRI showed subaracnoid CSF spaces and 3rd ventricle enlargement. He had a normal development in the first year of life. The diagnosis was reached at 12 months(mo) after reevaluation of the clinical picture (inguinal hernia, facial features). His mental development (Griffiths scale) at 15mo was normal (GQ 100). Pt 2 had uneventful birth and development in the first year of life. The evidence of umbilical hernia, hepatomegaly, kyphoscoliosis and typical facial features led to the diagnosis of MPS II at 16mo. His GQ was 87 at 17mo. Results: Enzymatic and molecular diagnosis was confirmed in both pts who started ERT immediately after diagnosis and were transplanted with an unrelated cord blood unit (UCB) at 18 and 22mo of age respectively. Both engrafted showing stable full donor chimerism and no major transplant-related adverse events. Both had a good somatic and motor development showing improvement of their cognitive skills with GQ slowly worsening and then improving at latest examination (Patient 1 at 3 y6mo: 75; 4y6mo: 87. Patient 2 at 3y10mo: 60; 4y10 mo:69). Discussion: The latest GQ results of these pts seems to indicate that they may have reached a stabilization of their cognitive skillness. This trend is similar to that observed in many Hurler transplanted pts seems to indicate a possible effect of HSCT.

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Hematopoietic stem cell transplantation in a patient with mucopolysaccaridosis II Moey L H 1, Chew H B 1, Winnie Ong P T 1, Leong H Y 1, Muzhirah H 1, Chng G S 1, Keng W T 1, Hishamshah M I 2, Ida S O 2, Ngu L H 1 1

Genetics Department, Kuala Lumpur Hosp, Kuala Lumpur, Malaysia, Paediatric Institute, Kuala Lumpur Hosp, Kuala Lumpur, Malaysia

2

Background: Mucopolysaccaridosis II (MPS II) is the most common type of MPS in Malaysia. Enzyme replacement therapy (ERT) has emerged as the preferred disease-specific treatment. However, its effectiveness in the central nervous system is limited. Hematopoietic stem cell transplantation (HSCT) is an alternative but rarely performed outside Japan due to uncertainty about its efficacy and benefit. Here we report a patient whom underwent HSCT in our center. Case Report: The patient is the younger brother of a known deceased MPS II patient with severe phenotype. He was screened soon after birth and his urinary glycosaminoglycans was elevated with increase dermatan sulphate and heparan sulphate. Iduronate -2- sulphatase activity in leucocytes was markedly below the normal range. Sequencing of IDS gene revealed one heterozygous mutation c.601_602delAG (p.Ser201HisfsX2). Due to the limited accessibility of long-term ERT, parents were agreeable for HSCT. He underwent HSCT at the age of 14 months old. Results: The patient had the full engraftment without any major transplant-related complications. His plasma iduronate-2-sulphatase activity had gradually increased to normal level. We have followed his progress for 30 months post HSCT. His neurocognitive development although delayed but continued to progress forward. He has hyperactivity. He did not have organomegaly, joint contracture, cardiac abnormality or respiratory difficulty. His growth has been normal. Overall his health status and neurocognitive function were much better as compared to his deceased brother whom did not receive any disease-specific treatment. Discussion and Conclusion: Our experience suggest at least short-term benefit in treating MPS II with HSCT, although its effectiveness in the long-term is not clear at the moment. We suggest HSCT may be worthwhile to be explored in early stages of the disease for patients with MPS II especially in countries without sustainable long-term access to ERT.

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Dermatan sulfate and heparan sulfate quantification in CSF, plasma and dried urine spots by UPLC-MS/MS Young S P 1, Zhang H 1, Dickson P I 2, Beasley J A 1, Chen A H 2, Le S 2, Weetall M 3, Millington D S 1 1 Duke University School of Medicine, Durham, United States, 2UCLA Medical Center, Torrance, United States, 3 PTC Therapeutics, South Plainfield, United States

Background: A method established for dermatan sulfate (DS) and heparan sulfate (HS) quantification in urine using ultra-performance liquid chromatography-tandem mass spectrometry was adapted and evaluated for use with cerebrospinal fluid (CSF), dried urine spots (DUS) and plasma. Methods: Methylated dimers of DS and HS were prepared from 25 μL CSF or plasma, or 100 μL urine spot extract, mixed with deuteriumlabeled methylated dimers of DS and HS and analyzed by UPLC-MS/ MS with separation on a BEH® Amide UPLC column and detection by selected reaction monitoring. Peak area ratios to the internal standards were converted to a concentration by means of calibration curves. Results: DS and HS analysis in DUS extracts had acceptable inaccuracy and imprecision over the calibration ranges (≤20 %). DUS have the requisite sensitivity to diagnose patients affected with mucopolysaccharidoses, when appropriate reference ranges established from dried samples are used, and provide a viable option for shipment from remote locations. A method in plasma has shown promising results with DS and HS elevated by one order of magnitude in the plasma from a mouse model of MPS I. DS and HS were elevated in the CSF of untreated patients (n = 4) with MPS I and a decrease was observed after 26 weeks of intravenous (IV) enzyme replacement therapy (ERT), although values did not normalize. No reduction in DS or HS was observed after 3 or 12 months of intrathecal (IT) ERT in patients (n = 6) who were already treated with IV- ERT, except for one patient who had marked elevations of CS and DS prior to ITERT. Discussion: A quantitative method by UPLC-MS/MS has the required sensitivity and specificity to detect elevations of DS and HS in CSF, plasma and DUS from patients with MPS I, and may be useful for diagnosis and/or monitoring response to therapies. Conflict of Interest declared.

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Characteristics of patients aged 5 years and older at first signs and symptoms of mucopolysaccharidosis type II: data from the Hunter Outcome Survey (HOS) Harmatz P 7, Lin S P 1, Muenzer J 2, Giugliani R 3, Guffon N 4, Jego V 5, Burton B 6 1 Dept Ped, Mackay Mem Hosp, Taipei, Taiwan, 2Dept Ped, Univ NC, Chapel Hill, United States, 3Med Genet Service HCPA, Porto Alegre, Brazil, 4CRMR, Child Hosp, HCL, Bron Cedex, France, 5Cytel Inc, Geneva, Switzerland, 6 Lurie Child Hosp, Chicago, United States, 7UCSF Benioff Child Hosp, Oakland, United States

Background: Patients typically show signs and symptoms of MPS II (Hunter syndrome) at 2–4 years of age, but some develop disease manifestations at a later age. Little is known about these individuals. Methods: Using data from patients with MPS II who were followed prospectively in the HOS registry (Shire), we compared clinical characteristics (recorded at any time) of individuals aged ≥5 years at first signs and symptoms (‘late-onset’) with those aged < 5 years (‘early-onset’).

Results: As of January 2016, there were 56 patients aged ≥5 years at first signs and symptoms (6/56 aged ≥10 years) and 689 patients aged < 5 years. Median (P10; P90) age at diagnosis was 8.0 years (5.0; 17.0) for the late-onset group (n = 53) and 3.0 years (1.1; 6.0) for the early-onset group (n = 653). Coarse facial features, joint stiffness, hernia and hepatomegaly were common and of similar prevalence in both groups. Manifestations that were more common in the late-onset group included pain, cardiac murmur, carpal tunnel syndrome and dyspnoea; less common signs and symptoms included behavioural problems (hyperactivity, frequent chewing), constipation, diarrhoea and abnormal dentition. Cognitive impairment was reported in 24 % (13/54) of the late-onset group and 65 % (435/667) of the earlyonset group. Median number of surgical procedures per patient was similar for both groups: 4 (2; 10) and 5 (1; 10) for the late- and early-onset groups (n = 50, n = 599), respectively. Median age at first surgery was 5.5 years (0.1; 21.8) for the late-onset group (n = 49) and 2.4 years (0.2; 8.0) for the early-onset group (n = 574). The late-onset group had more carpal tunnel decompressions, hernia repairs and procedures classified as ‘Other’ and fewer adenoidectomies, tonsillectomies and ear tube insertion procedures than the early-onset group. Discussion: This preliminary analysis showed that patients with relatively late onset of MPS II symptoms nonetheless have significant disease burden. Conflict of Interest declared.

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Bone crisis in atypical localization in GD patients under long-term enzyme replacement therapy Andrade Campos M 1 2, Sancho Val I 3, Garcia I 5, Roca Espiau M 6, Giraldo P 1 2 4 1 Translational Research Unit, IIS Aragon, Zaragoza, Spain, 2CIBER de Enfermedades Raras, CIBERER, Zaragoza, Spain, 3 Department of Hematology, Alcaniz Hosp, Alcaniz, Spain, 4FEETEG, Zaragoza, Spain, 5 Miguel Servet Pediatric Hospital, Zaragoza, Spain, 6MRI Diagnostic Center Dra Roca, Zaragoza, Spain

Background: The pathophysiology of skeletal manifestation in Gaucher disease (GD) is produced in part by chronic inflammation and bone metabolism alterations due to substrate accumulation. Long-term effects of enzyme replacement therapy (ERT) on skeletal involvement are related with the baseline burden disease and therapy regularity and adherence. About 30 % of child patients had bone crisis before the start of ERT. Only 2.5 % of children treated since the early years of life without previous history present a bone crisis during ERT. These are reported rarely in small bones and were normally preceded by crises in long bones. Case Report: We have observed in 3 of 53 GD patients diagnosed in childhood in Spain and under ERT developed bone crisis in small feet bones assessed by MRI. Results: Case 1. Male, diagnosed at 18 months with spleen enlargement, anaemia and thrombocytopenia, genotype [R463C] + [G377S], he started ERT (60 U/Kg) and reverted visceral and haematological manifestations. After 10 years on ERT he developed a bone crisis in right astragalus and calcaneus, the episode was resolved after ERT dose increase. Case 2. Female patient diagnosed at 4 years of GD1, genotype [N370S] + [84GG] due to epistaxis, splenomegaly and thrombocytopenia, She started ERT after diagnosis developing infusion reaction controlled after switching ERT, and after 13 years of ERT, developed a bone crisis in her right calcaneus, resolved with an increase of dose to 60 UI/Kg. Case 3. Male diagnosed at 3.5 years old, genotype [N370S] + [R130W], due to splenomegaly and started ERT at 60 UI/2 weeks, after 10 years on ERT he developed a bone crisis in right tarsus. Apparently without a trigger, but during growth period. Discussion: Bone crisis usually occurs on epiphysis of long bones and predominantly in patients without therapy or splenctomyzed. In small and distant bones as our cases is an uncommon event and would be related to the intensity of physical activity in loading areas.

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independent for 9 of them needing minimal assistance mainly in using the knife. Discussion: HSCT has completely modified the natural history of MPSI Hurler pts. In fact at 6–10 years of age untransplanted pts would have been much more limited in their activities. Our results suggest that, despite the beneficial effect of HSCT, most of them had joint and musculo-skeletal limitations as shown by their difficulties in mobility, dressing and cutting the food. The mild to moderate mental delay of some pts might have a role, but less relevant. We hope that earlier diagnosis and/or new treatments will further improve the prognosis of Hurler pts.

Assesment of microvascular endothelial function in children and adolescents with mucopolysaccharidosis type VI Ozturk-Hismi B 1, Kumru B 2, Kilic T 5, Sezer S 3, Keskin M 4, Bagci C 5 1 Div Metab Dis, Tepecik Research Hosp, Izmir, Turkey, 2Dietetics, Child Hosp, Gaziantep, Turkey, 3Div Ped Cardio, Child Hosp, Gaziantep, Turkey, 4 Div Ped Endocrin, Univ Hosp, Gaziantep, Turkey, 5Depart Physiology, Gaziantep Univ, Gaziantep, Turkey

P-450 Background: Cardiovascular lesions, including coronary artery stenosis, are frequently associated in Mucopolysaccharidosis patients. and can cause sudden death. Early diagnosis of coronary artery lesions is difficult, Endothelial vasodilator function, is an independent predictor of cardiovascular morbidity and morbidity. Endothelial function assesments may detect coronary artery lesions that can be underdiagnosed by the other measures such as coronary angiography.in MPS patients. Methods: Postocclusive reactive hyperemia (PORH) was measured in 15 children and adolescents with MPS-VI (age 7.7 ± 3.6 years old; 9 girls) and 13 age and gender matched healthy controls (8.1 ± 4.2 yil; 8 girls), with Lazer Doppler Flowmetry. All MPS-VI patients were on ERT(2–86 months, mean 28 months). An independent t-test was used to compare PORH between individuals with MPS-VI and controls. Results: Compared with controls (C), patients with MPS-VI showed a significantly lower PORH (MPSVI: 380.08 ± 97.48 vs C: 478.86 ± 141.13 %, p < 0.05). Discussion: Our findings suggest that children and adolescents with treated MPS-VI have significantly poorer endothelial function when compared to healthy controls. Further investigation into the utility of endothelial function for risk stratification and the long term implications of reduced endothelial function in MPS-VI is warranted.

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Health-Related Quality of Life of transplanted MPS I Hurler children Scalone L 2, Ciampichini R 3, Mantovani L G 3, Cesana G 2, Scordo F 2, Gasperini S 1, Galimberti C 1, Biondi A 4, Rovelli A 1, Parini R 1 1 Dept Pediat MBBM Found S Gerardo Hosp, Monza, Italy, 2CESP Res centre Publ Health Bicocca Univ, Milano, Italy, 3CHARTA Foundation, Milano, Italy, 4Dept Pediat MBBM Found Bicocca Univ, Monza, Italy

Clinical and biochemical spectrum of mucopolysaccharidosis type III (Sanfilippo syndrome) in Morocco Talbaoui H 1, Dahri S 1, Kriouile Y 2, Oudghiri F Z 2, Froissart R 3, Saban C 3, Chabraoui L 1 1 Biochemistry laboratory, Univ Hospital, Rabat, Morocco, 2Pediatry service, Univ Child Hosp, Rabat, Morocco, 3Biochemistry Laboratory Univ Hosp Est, Lyon, France

Background: Mucopolysaccharidosis III (Sanfilippo syndrome) is composed of four subtypes (four different enzyme deficiencies): type IIIA (heparan N-sulphatase), type IIIB (α–N- acetylglucosaminidase), type IIIC (acetyl-CoA α-glucosamide acetyltransferase) and type IIID (Nacetyl glucosamine 6-sulphatase). The four enzymes are involved in the lysosomal degradation of heparan sulphate, the glycosaminoglycan excreted in the patient’s urines. Methods: In our laboratory we screen for MPS since 1991 using urinary glycosaminoglycan and leucocyte enzyme determination. This work is a retrospective study concerning 39 MPS III patients: Results: 17 MPS IIIA, 7 MPS IIIB, 15 MPS IIIC and no MPS IIID. The age of diagnosis of patients was 6.93 ± 2.96 years; 7.40 ± 2.07 years and 8.63 ± 4.92 years for types A, B and C respectively. Consanguinity was observed in 83 %, 100 % and 90 % for A, B and C respectively. Psychomotor retardation, coarse facial features and speech delay were reported in all types. However hypertrichosis, hepatosplenomegaly and macrocephaly were present in type A, while severe behavioral problems and bone disorders characterized type B. Discussion: We emphasize the importance of establishing an early enzymatic diagnosis of MPS III, particularly Type IIIA so as to be able to include Moroccan patients in a possible gene therapy that could change the prognosis of the disease.

P-451 Background: Haematopoietic stem cell transplantation (HSCT) is the only treatment for MPSI Hurler patients (pts) preventing central nervous system deterioration. Its safety and long term efficacy are known. Few data are available on Health Related Quality of Life (HRQoL) of transplanted children. We assessed HRQoL in Hurler pts who underwent successful HSCT. Methods: The MPS-Health Assessment Questionnaire (MPS-HAQ) was administered at baseline and yearly to successfully transplanted Hurler pts as part of their long term clinical follow-up. We analysed the answers about assistance required in mobility and self-care at the age between 6 and 10 years. Results: The data refer to 10 pts, 2 males, with a developmental quotient (DQ) at the time of the data analysed ranging from 50 to 110. The answers were provided by the fathers in 3 cases and mothers in 7. The 10 pts were all completely independent in their mobility at home and climbing stairs; 2/10 had the need of minimal assistance while walking outside; 8/10 needed assistance (mostly minimal) in getting into a car. All but one needed assistance in toileting activities (grooming, bathing, tooth brushing, managing toilet seat and toilet paper) and in dressing upper part of the body; all 10 patients needed assistance in dressing lower part of the body. Eating was not

Targeted population screening for mucopolysaccharidoses—an efficient tool for the diagnosis of patients Murko S 3, Nieves Cobos P 3, Gal A 2, Santer R 1 3, Lukacs Z 3 1

Dept Pediatr, Univ Med Cent Eppendorf, Hamburg, Germany, 2Inst Hum Genet, Univ Med Cent Eppendorf, Hamburg, Germany, 3Metab Lab, Univ Med Center Eppendorf, Hamburg, Germany Background: Lysosomal storage diseases present with variable symptoms and, as indicated by different age of onset, with different severity. This may cause a delay in diagnosis which may be further exaggerated by the relatively low incidence of these diseases. For these reasons, they are generally not prime candidates in diagnostic algorithms. Recently, the use of dried blood specimens has been introduced for enzyme activity measurement of a number of lysosomal enzymes which has significantly facilitated testing. Methods: Dried blood spots on Guthrie testcards were shipped from distant health care providers (Middle East, Russia, Southern Europe,

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 South Africa, and others) to the metabolic laboratory of the University Medical Center Hamburg-Eppendorf. Patients with symptoms compatible with mucopolysaccharidoses (MPS), e.g., coarse face, hepatosplenomegaly, and rheuma-like symptoms without inflammation. Samples were tested for MPS I, MPS II (male) and MPS VI using fluorometry or tandem mass spectrometry. Results: Among 4705 samples tested to date, 7.8 % were positive for MPS I, 3.5 % for MPS II, 4.8 % for MPS VI, and 2.7 % showed enzyme activities compatible with mucolipidosis II/III. In addition, ten samples were suspicious for multiple sulfatase deficiency. Among the first 200 samples received in the laboratory, all positives were assessed by molecular genetic methods. Thus, results of enzymatic test for MPS I were confirmed in 85 % of patients, while all MPS II and MPS VI cases were confirmed. Discussion: Targeted population screening of patients with symptoms remotely associated with distinct lysosomal storage diseases proved to be a highly efficient approach in expediting the diagnosis of patients. In addition, this policy is cost effective, especially in comparison to whole population screening. Furthermore, it may circumvent ethical problems that arise in a general screening testing non-symptomatic individual.

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Gen,National Research Cen, Cairo, Egypt, 4Dep Clin Gen,National Research Centre, Cairo, Egypt Background: Morquio A disease (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient activity of the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) encoded by the GALNS gene. This deficiency leads to a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing their accumulation within the lysosomes and consequently prominent skeletal and visceral abnormalities. Methods: Clinical evaluation and biochemical GALNS enzyme activity determination were carried out for the patients from four unrelated Egyptian families. Mutational analysis was performed to PCR products by sequencing of the 14 exons and exon-intron boundaries of GALNS gene for the 4 patients. Results: Sequence analysis revealed four novel mutations; three nonsense mutations (p.Q12X, p.Q220X, p.Y254X) and one missense mutation, p.D40G. All four patients were offspring of consanguineous marriages and were homozygous for the corresponding mutation. The activity of the GALNS enzyme was below normal reference range in all of them. The p.Q12X and p.Y254X were associated with severe MPS IVA phenotype. Discussion: Molecular analysis of GALNS gene revealed four novel mutations in four different Egyptian Morquio A patients.

Using mathematical—structural model in prediction of pubertal spurt in patients with MPS I and MPS II. Rozdzynska-Swiatkowska A 1, Cieslik J 3, Tylki-Szymanska A 2 1 Anthrop Lab, CMHI, Wasaw, Poland, 2Dep of Ped, Nutrit and Metab Dis, CMHI, Warsaw, Poland, 3Dep of Hum Biol Dev, Inst of Anthrop,UAM, Poznan, Poland

Background: Growth spurt in patients with MPS starts later than in general population which is complemental with bone age. For the assessment of the process of physical growth in patients with MPS in time, the following growth spurt parameters were determined: age, height and velocity of growth at the onset of spurt (spurt take off— TO); age, height and velocity of growth at the peak of spurt (peak of height velocity—PHV);, predicted final body height. Methods: The mathematical structural growth model of Bock, Thyssen and du Toit (BTT) in AUXAL software was used to evaluate the structure of body height growth in the studied population and to compare these data with previously estimated growth parameters of healthy peers. The BTT model assumes three phases of logistic growth and is able to detect and locate growth velocity maxima in adolescence and mid childhood as well as their preceding velocity maxima, making it possible to explain growth spurt parameters, even when the longitudinal data are not complete. Analyses were performed only for individual cases of growth in patients with MPS I and MPS II. Results: The values of the expected final body height were similar to the values obtained by our patients. Discussion: Results show that the BTT model may be an appropriate tool for professionals to rate the growth of the individual patient. However, the analysis of development structure, revealed using the BTT model, should be interpreted carefully because of its predictive character.

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Four novel mutations in the N-acetylgalactosamine-6-sulfate sulfatase gene among Egyptian patients with Morquio A disease Fateen E M 1, El Mawgoud H A 2, Essawi M L 3, Aglan M S 4, Ibrahim M M 1, Eissa N R 3 1 Dep Biochem Genetics,National Research C, Cairo, Egypt, 2 Dep Biocem,Facult Pharm,El Azhar Uni, Cairo, Egypt, 3 Dep Med Molec

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Enzyme Replacement Therapy for Mucopolysaccharidosis Type VI: Experience from a Brazilian Reference Center Curiati M A 1, Mendes C S C 1, Rand M H 1, Feliciano P 1, Aranda C S 1, Martins A M 1 1

CREIM - UNIFESP, Sao Paulo, Brazil

Background: Maroteaux-Lamy syndrome was described in 1963 by Maroteaux and coworkers as a Hurler-like syndrome, but with normal intelligence and excretion of dermatan sulfate only. The disease is caused by deficiency of arylsulfatase B. Mental development is usually normal in patients with this syndrome, although physical and visual impairments may impede psychomotor performance. Enzyme replacement therapy (ERT) with galsulfase weekly has shown to improve signs and symptoms and wellbeing in patients with MPS VI. Patients and Methods Retrospective analysis of 12 MPS VI patients and response to the ERT over time. Results The patients, median age of 11,5 years, ranging from 3,5 to 28 years, median age of diagnosis of 3,13 years, ranging from 1,2 to 5,8 years. Median age for starting ERT was 6,6 years, ranging from 1,5 years to 20 years. Median time receiving ERT was 6 years, ranging from 2 years to 13 years. From the total of patients, 6 presented macrocrania, 8 hearing impairment. Nine patients presented cardiac involvement (6 showed improvement, 2 stabilisation and 1 worsening), Eleven with hepatomegaly (10 showed improvement and 1 worsening) and 10 with splenomegaly (100 % improvement), 11 with joint limitation (2 with improvement, 8 stabilisation and 1 worsening), 12 with coarse face (10 stabilisation and 2 worsening), all patients presented recurrent airway infections, and 100 % showed frequency reduction. Seven presented abnormal neurologic exam, being 2 with cognitive impairment and 7 with only motor symptoms. Discussion: A significant number of patients presented marked improvement in organ function, specially cardiac and respiratory, showing that ERT prevents GAGs from maintain its accumulation in these tissues. In our population, ERT showed to be effective in preventing progression of the disease in most organs, reducing the frequency of upper airway infections in all patients. This fact increases quality of life, since it reduces the number of hospitalizations. Conflict of Interest declared.

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are associated. Considering the deleterious systemic effects of chronic inflammation and redox imbalance, our results suggests that the use of antiinflammatory and antioxidant drugs may be an important complementary strategy in MPS II treatment. Financial support: CAPES, CNPq and FIPE/HCPA.

Epileptic seizures profile in patients with Mucopolysaccharidosis (MPS) types I, II and VI Mendes C S C 1, Rand M H 1, Curiati M A 1, Feliciano P 1, Aranda C S 1, Martins A M 1

19. Lysosomal disorders: sphingolipidoses 1

CREIM - UNIFESP, Sao Paulo, Brazil

Background: MPS types I, II and VI, are clinically heterogeneous inherited disorders, affecting various organs. In all types, are described attenuated and severe phenotypes. Seizures may occur during the progression of MPS, and are more frequent in severe phenotypes. Methods: Retrospective analysis of patients with MPS I, II and VI being followed at a reference center for Inborn Errors of Metabolism, regarding the presence of seizures, age of onset and type of seizures. Results: We evaluated 20 patients with MPS I, 12 male and 10 female, with ages ranging from 7 to 30 y, 22 patients with MPS II, 1 female and 11 male, aged between 11 m-28y, and 12 patients with MPS VI, 5 females and 7 males, aged 3–26 y. We did not find any reports of seizures in patients with MPS I. Regarding the MPS II patients, we found 4 (19 %) of patients with seizures, being 3 with severe phenotype and 1 mild phenotype. Age of onset ranged from 2 to 17 y. Patients with severe phenotype had generalized tonic and atonic seizures and 1 patient with unclassified seizures. The patient presented with mild phenotype motor partial seizures with phonoarticulatory deficit. Two (16.6 %) of patients with MPS VI had partial seizures with generalization, both with more severe phenotype, with ages of 7 and 21 to, and one patient developed seizures in presence of intracranial hypertension. Discussion: The frequency of seizures found in our patients were consistent with literature findings. However there are few studies regarding this neurological manifestation in MPS patients, especially in type I and VI. Most of our patients who developed seizures, had severe phenotype. Conflict of Interest declared.

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Nitrative and inflammatory status in long-term idursulfase-treated Mucopolysaccharidosis type II patients Jacques C E D 1, De Souza H M 1, Forest N M M 1, Mathias A M B 1, De Souza C F M 1, Giugliani R 1, Vargas C R 1 1

Serv Gen Med, Hosp Clin POA, Porto Alegre, Brazil

Background: Mucopolysaccharidosis II (MPS II) is an X-linked lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase activity. This leads to an abnormal storage of glycosaminoglycans (GAG) in tissues and body fluids. It is known that GAG and oligosaccharides fragments can act as direct activators of pathological cascades, increasing pro-inflammatory cytokines production. Moreover, reactive nitrogen species (RNS)—such as nitric oxide (NO), nitrogen dioxide radical and peroxynitrite—can cause a variety of cellular damages, and also lead to the activation of inflammatory signaling pathways. In this work, we evaluated the RNS and pro-inflammatory cytokines production in long-term idursulfase-treated MPS II patients. Methods: Urinary nitrate/nitrite, and plasmatic NO, IL-1β and TNF-α were measured in 8 MPS II patients [mean age: 17.1 years-old; mean time on ERT: 5.2 years (range: 1.5–7.0 years)]. Nitrate/nitrite and NO contents were assessed by Griess reaction methods. IL-1β and TNF-α plasmatic concentrations were measured using ELISA assays. Results: Our results showed that treated MPS II patients present higher levels of RNS in both plasma and urine, compared to the control group. Furthermore, pro-inflammatory cytokines concentrations were found increased in these patients. It was also observed a significant positive correlation between plasmatic IL-1β and NO concentrations. Discussion: These results evidence that, despite the long-term treatment with enzyme replacement therapy, MPS II patients present a disruption of inflammatory and nitrative status, and possibly these pathophysiological mechanisms

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The spectrum of Niemann Pick type C disease in Greece. Mavridou I 1, Dimitriou E 1, Vanier M T 4, Vilageliu L 2, Grinberg D 2, Latour P 4, Xaidara A 3, Lycopoulou L 3, Bostantjopoulou S 5, Zafeiriou D 6, Michelakakis H 1 1

Dept Enzym Cellular Fun, In Child Health, ATHENS, Greece, 2Dept de Genetica, Univ Barcelona, Barcelona, Spain, 31st Dept Ped Univ Athens, ATHENS, Greece, 4Lab Gillet-Merieux Neurog Molec, Lyon, France, 53rd Dept Neurol,Aristotle Univ, Thessaloniki, Greece, 61st Dept Pediatrics, Aristotle Univ, Thessaloniki, Greece Background: Niemann Pick type C (NPC) is a rare neurovisceral lysosomal storage disease. So far two genes, NPC1 and NPC2, have been linked to the disorder. We present fourteen cases (including 2 siblings) diagnosed in Greece during the last 28 years. Methods: In all, diagnosis was established through filipin staining; esterification of exogenous lipoprotein-derived cholesterol was studied in 7. Chitotriosidase activity was assayed in plasma of 12 cases and DNA analysis was performed in 12 (including one pair of siblings) cases. Age of diagnosis ranged from 3 1/2 months to 48 years. Systemic manifestations of the disease were present in the neonatal period in 7/14 patients. Neurological involvement was apparent in childhood in 5 and in adulthood in 2 patients. Result and Discussion: Increased chitotriosidase activity was found in 9/12 cases. Classic and variant filipin patterns were observed in 10 and 4 cases, respectively. Reduced esterification of exogenous lipoprotein-derived cholesterol was observed in 7/7 cases studied. Mutation analysis identified in 11 unrelated cases 9 different genotypes owing to 12 different NPC1 mutations of which 8 previously described (p.E1089K; p.A1132P; p.F284Lfs* 26; del promoter and exons 1–10; p.Q92R; p.R1186H,p.P1007A; p.S940L) and 4 novel mutations (p.Asn701Lys fs*13; c.3591 + 3insT; p.K1057R; p.Cys1119*). One mutation in homozygosity was identified in the NPC2 gene in one case (c.190 + 5G > A). All patients were of Greek origin and came from islands of southern Aegean, the Peloponese, Central and North Greece. Conflict of Interest declared.

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Natural course of classical and non-classical Fabry disease: a large multicenter cohort study. Arends M 1, Wanner C 2, Hughes D 3, Mehta A 3, Biegstraaten M 1, Hollak C E M 1 1 Div Endo Metab, Acad Med Cen, Amsterdam, Netherlands, 2Dep Neph, Univ Clin Wuerz, Wuerzburg, Germany, 3Dep Acad Haem, Royal Free Univ Coll, London, United Kingdom

Background: It is widely known that phenotypic differences exist between males and females with Fabry disease (FD), but also between classically affected and non-classically affected patients. Limited reports on the natural disease course of males and females exist and none have distinguished classical and non-classical patients. The aim

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 of this study was to describe the natural history of FD specified for gender and phenotype. Methods: Longitudinal data on major clinical events that occurred before first visit and hence before treatment with ERT were retrospectively collected for all FD patients from 3 international centers of excellence. Transversal data for the eGFR and LVM, assessed at first visit, were used to assess disease course. Results: Data on 499 adult FD patients were available (mean age: 43, 41 % males, 57 % classical FD). The event rate of males with classical FD was higher compared to males with non-classical FD (HR: 5.2, p < 0.001). In females this difference was less pronounced (HR: 2.9, p < 0.001). The eGFR was inversely associated with age, which was similar for all subgroups except for males with classical FD (−1.1 vs −1.8, p < 0.01). Renal disease occurred in non-classical patients at low rate and higher age. The LVM in classical FD males was on average 30 % higher (p < 0.001) than the LVM in non-classical FD males. In females, the difference in LVM was 20 % (p < 0.001). Discussion: This is the first report on disease course from a large group of patients, specified for gender and phenotype. As expected, males with classical have more severe cardiac and renal disease and an increased risk of developing complications compared to nonclassical males and females. Of interest, classical females also have a higher risk to develop complications compared to non-classical females. Renal disease is rare in females and non-classical patients. These data are of high importance to support guidelines for followup and treatment and to study effects of intervention. Conflict of Interest declared.

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Acid Sphingomyelinase Deficiency: diverse clinical manifestations and heterogeneous natural history. McGovern M 1, Avetisyan R 2, Sanson B J 2, Lidove O 3 1

Dept Pediatrics, Stony Brook Univ School, Stony Brook, New York, United States, 2Sanofi Genzyme, Cambridge, Massachusetts, United States, 3Hosp de la Croix Saint Simon, Paris, France Background: Acid sphingomyelinase (ASM) deficiency (ASMD), also referred to as Niemann-Pick disease (NPD) type A and B, is a rare autosomal recessive lysosomal storage disorder caused by SMPD1 mutations. Current treatment is limited to symptom management; however, an enzyme replacement therapy is in clinical development. Disease recognition and timely diagnosis at initial presentation are important and require a thorough understanding of ASMD manifestations and natural history. Methods: Synthesis of published literature. Results: Patients with NPD A have a nearly uniform natural history characterized by early onset and rapid progression of psychomotor degeneration leading to death, usually by 3 years of age from respiratory failure. NPD B is a chronic progressive disease with a heterogeneous natural history. Typical disease manifestations include hepatosplenomegaly, progressive infiltrative lung disease, liver dysfunction, heart disease, growth delay, osteopenia/osteoporosis, atherogenic lipid profile, and cytopenias. Common symptoms include bleeding, shortness of breath, joint/limb pain, abdominal pain, and bruising, which may lead to chronic fatigue, limited physical activity, inability to work, and social isolation. A variant form of NPD B presents with neurologic symptoms, ranging from mild hypotonia to cognitive impairment or progressive loss of motor function. Many patients succumb to pneumonia, respiratory failure, bleeding, or liver failure before or in early adulthood. More research is needed to increase information about ASMD disease progression, genotype-phenotype relationships, prognostic factors, and impact on quality-of-life. Discussion: ASMD is a rare, multi-systemic and heterogeneous disease, which contributes to low disease awareness and delays in diagnosis. More evidence about ASMD natural history and better characterization of subpopulations may facilitate earlier disease recognition. Conflict of Interest declared.

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A novel mutation in NPC1 associated with neonatal cholestasis and neurological deterioration despite normal oxysterol and cholesterol esterification findings. Santra S 1, Vijay S 1, Sreekantam S 1, Simmons L M 1, Hutchin T 1, Blundell J 1 , Wright E 1, Kearney S 1, Raiman J 1 1

Birmingham Children’s Hospital, Birmingham, United Kingdom

Background: Niemann-Pick disease type C (NP-C) patients can develop neonatal cholestasis, hepatosplenomegaly & neurological deterioration. The diagnosis is confirmed by finding 2 mutations in NPC1 or NPC2. Supportive biochemical findings include an abnormal plasma oxysterol profile & abnormal filipin staining/cholesterol esterification in cultured fibroblasts. We report a child compound heterozygous for 1 pathogenic & 1 novel NPC1 variant, with biochemical findings not diagnostic of NP-C, who developed neurological symptoms aged 10. Case Report: A male infant of 1st cousin Pakistani parents presented aged 2 months with jaundice (resolved by 10 months) & hepatosplenomegaly which persisted. NPC1 sequencing when neurologically normal aged 4, showed compound heterozygosity for the c.3020C > T (p.Pro1007Leu) mutation in exon 20 & a novel c.2154A > C (p.Glu718Asp) variant in exon 14. Given the novel variant’s uncertain nature, he was followed closely. Learning difficulties were noted aged 9. By 10, he developed ataxia & fine motor difficulties. Slow vertical saccade inititation was confirmed with eye-tracking studies. Results: Liver biopsy at 7 months showed inflammatory changes with no storage cells. Chitotriosidase was pseudodeficient. Leucocyte beta-glucosidase, sphingomyelinase & acid lipase assays were normal. Cultured fibroblasts showed a non-classical NP-C pattern, a majority of cells showing moderate intensity of perinuclear filipin staining. Cholesterol esterification studies were normal. Parental DNA studies confirmed in trans inheritance. Plasma oxysterol analysis at 9 years was 18.2 ng/ml, below the reported NPC-1 range (35.3–1170). Discussion: This child’s clinical progress supports the pathogenicity of the novel c.2154A > C NPC1 variant. Active neurological monitoring is recommended in patients with novel NPC1 variants even in the absence of abnormal biochemical findings. Conflict of Interest declared.

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High frequency of p.His281Tyr mutation in GLB1 gene in patients with GM1-gangliosidosis in Ukraine. Mytsyk N I 1, Olkhovych N V 1 2, Trofimova N S 1, Pichkur N A 2, Gorovenko N G 1 1 Div Genet Diagn, Inst Genet Regener Med, Kiev, Ukraine, 2Cent Orph Dis, Nat Child Hosp OKHMATDET, Kiev, Ukraine

Background: GM1-gangliosidosis is a severe neurodegenerative disorder with autosomal recessive mode of inheritance, which is referred to sphingolipidoses. The incidence is 1:100,000–1:200,000 live births worldwide. The disorder is caused by GLB1 gene mutations leading to acid betagalactosidase deficiency. The deficiency of acid beta-galactosidase results in the accumulation of GM1-ganglioside in the lysosomes, which leads to the development of a severe neurodegenerative disorder in patients. Methods: The analysis of mutations in GLB1 gene (chr 3p22.3, 16 exons) was conducted in 26 patients from Ukraine, diagnosed with GM1-gangliosidosis. SSCP, PCR, and Sanger exon sequencing of GLB1 gene were used for the analysis. Results: The mutation analysis of GLB1 gene in 26 Ukrainian patients, diagnosed with GM1-gangliosidosis, revealed 12 pathogenic mutations in different exons, mainly in exons 2, 6, 7, 8, 15, and 16. The most frequent mutation was the missense p.His281Tyr in exon 8, which was found in 16 out of 26 patients (pts), in both homozygous state (4 pts) and heterozygous state (12 pts), which amounted to 38.5 % of all the mutations revealed. All patients with this

S194 mutation had the infantile form of GM1-gangliosidosis with severe neurological symptoms. Discussion: The mutation analysis of GLB1 gene in patients from Ukraine with GM1-gangliosidosis demonstrated that, contrary to other populations, missense mutation p.His281Tyr is highly frequent among Ukrainian patients (38.5 %) and may be considered as the major mutation for this population.

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Functional analysis of common splicing mutations detected in HEXB gene causing Sandhoff disease. Mugnaini J 1, Brasil S 2, Desviat L 2, Dodelson de Kremer R 1, Argarana C E 3, Perez B 2, Oller Ramirez A M 1 1 CEMECO, Child Hosp, Nat Univ Cordoba, Cordoba, Argentina, 2CBMSO, Universidad Autonoma Madrid, Madrid, Spain, 3CIQUIBIC, Nat Univ Cordoba, Cordoba, Argentina

Background: Sandhoff Disease (SD) is a fatal neurodegenerative disorder caused by mutations in the HEXB gene. To date, no effective treatment is available. A high incidence of the infantile variant of SD has been described in a delimited region in Argentina where the mutation c.445 + 1G > A was found in 95 % of mutant alleles. This variant affects a donor splice site and abolishes normal mRNA splicing. Another mutation, c.1509-26G > A, has been reported around the world in several patients with the juvenile form of SD.It is associated with the creation a new acceptor splice site which causes intron retention in mRNA. In this work we report the functional analysis of these two mutations in a cellular splicing model. Methods: Target regions of the HEXB gene were amplified by PCR, cloned into pSPL3 vector and mutations were introduced by site-directed mutagenesis. Wild type and mutant minigenes were transfected into COS-7 and HEK293 cells and upon a 24 h incubation period, RNA isolation and RTPCR analysis were performed. RT-PCR products were verified by gel electrophoresis and the introduced mutations confirmed by Sanger sequencing. Results: Wild type and mutant minigenes transfected into COS-7 and HEK293 cells showed expression patterns similar to those reported for control’s and patient’s cells, respectively. The mutant minigene containing c.445 + 1G > A resulted in a complete exon 2 skipping and the vector carrying c.1509-26G > A gave rise to the retention of 24 extra base pair belonging to intron 12. Discussion: Even considering that reporter vectors cannot accurately recapitulate the in vivo situation, the mutant minigenes carrying c.445 + 1G > A and c.1509-26G > A resulted in suitable in vitro SD models and could become important tools for the further study of different molecular therapeutic approaches as antisense mRNA therapy or U1 treatment. *Financial support: Fundación Ramón Areces, SLEIMPN, CONICET, PICTFONCYT and SECyT-UNC.

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Multiplex Ligation-dependent Probe Amplification assay: screening for deletions/duplications in the GBA1 gene in Gaucher disease patients. Siebert M 1 2 3, Basgalupp S P 2 4, Vairo F P 2 5, Schwartz I V D 2 4 5 6 1 Post Grad Progr Sci Gastro Hepat, UFRGS, Porto Alegre, Brazil, 2BRAIN lab, HCPA, Porto Alegre, Brazil, 3Unit Mol Prot Anal (UAMP), HCPA, Porto Alegre, Brazil, 4Post Grad Progr Med Sciences, UFRGS, Porto Alegre, Brazil, 5 Med Genet Serv, HCPA, Porto Alegre, Brazil, 6Depart of Genet, UFRGS, Porto Alegre, Brazil

Background: Gaucher disease (GD), one of the most common lysosomal storage disorder, is inherited as an autosomal recessive trait. It is caused by deficiency of the enzyme glucocerebrosidase due to mutations in the GBA1 gene. Our aims were to validate the P338-X1 GBA kit (MRC-Holland) for Multiplex Ligation-dependent Probe Amplification (MLPA), and to detect large deletions and/or duplications present in GBA1 in GD patients from Southern Brazil.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Methods: The sample group included 36 unrelated GD patients in whom the GBA1 gene was previously analysed by Sanger sequencing, being 31 with both mutations identified, 4 with only one characterized allele and 1 with both still uncharacterized alleles. The MLPA kit contains one probe for each of the following regions of GBA1: 5’UTR, exons 3, 4, 6, 8, 9, 10, and intron 7. The exon 10 probe generates a normal signal at L444, but reduced signal when L444P or L444R is present. Amplified products were analyzed with the ABI3500 equipment and Coffalyser software. Results: Out of 72 alleles, 35 (48.6 %) were found to have L444P and 1 (1.4 %) L444R, confirming our sequencing results; however, we were not able to distinguish if the reduced signal was due to the presence of RecNciI, L444P + A456P or L444P + E326K. Only one patient, heterozygous for RecNciI, presented a heterozygous deletion in intron 7. No other evidence for deletion/duplication was found. Discussion: After MLPA analysis, it is still remaining 5 uncharacterized alleles from 4 GD patients. Additional approaches are necessary to evaluate uncovered regions of GBA1 (exons 1, 2, 5, 11, promoter and 3’UTR).

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Molecular mechanism of autophagic pathway in Gaucher cells Dokmeci (Emre) S 1, Oral O 2, Yuce A 3, Gozuacik D 4 1 Hacettepe University Dept of Med Biol, Ankara, Turkey, 2Sabanci University Nanotech Res Appl, Istanbul, Turkey, 3Hacettepe Univ Gastroent Hepatology, Ankara, Turkey, 4Sabanci Univ Fac of Eng and Nat Sci, Istanbul, Turkey

Background: Lysosomes regulate cellular homeostasis via degradation and recycling of biomolecules. Lysosomal hydrolases and lysosomal membranebound proteins are two main actors for functional lysosome and any mutation on the coding region of these proteins causes accumulation of un-metabolized target substrates in the cells and finally coming out of lysosomal storage disease (LSD). Gaucher’s disease is a LSD resulting from the mutation of the lysosomal membrane-associated glycoprotein glucocerebrosidase (GBA) or the GBA cofactor, saposin C. The disease causes intracellular accumulation of glucosylceramide and other glycolipids. Autophagy is a conserved cellular pathway, leading to the engulfment of portions of cytoplasm and organelles and subsequently delivers the cargo to lysosomes for degradation. Although the relevance of autophagy is shown in different LSDs, the underlying molecular mechanism in Gaucher disease is poorly understood. Methods: Here, we investigated molecular significance of autophagic pathway in fibroblasts cells obtained from Gaucher patients. First, we analyzed the expression of autophagy and/or lysosome-related genes and proteins and then carried out active lysosome staining by using confocal microscopy analyses. In order to test autophagic flux, we used the differential pH sensitivities of RFP and GFP in mRFP-GFP-LC3 probe. Finally, we investigated lysosomes in detail by performing enzymatic activity tests. Results: We observed significant attenuation in the expression of key autophagy-related genes and accumulation of their proteins in mutant cells. We found inhibition of autophagosomes to fuse with lysosomes, that is associated with lysosomal pH and reduced enzyme activity. Discussion: Our data indicate that autophagic pathway is directly affected by mulfunctional lysosomes and may underlie the mechanism of clinical severity of Gaucher patients.

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Consensus recommendation on a diagnostic guideline for acid sphingomyelinase deficiency Giugliani R 1, Dionisi-Vici C 2, Hwu P 3, Lidove O 4, Lukacs Z 5, Mengel E 6, Mistry P K 7, Schuchman E 8, Wasserstein M 9, McGovern M 10 1 Medical Genetics Service, HCPA, Porto Alegre, Brazil, 2Hospital Bambino Gesu, Rome, Italy, 3National Taiwan University Hospital, Taipei, Taiwan, 4 Hopital de la Croix St Simon, Paris, France, 5Univ Medical Center

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Hamburg-Eppendorf, Hamburg, Germany, 6Med Cent of the Johannes Gutenberg Univ, Mainz, Germany, 7Yale University School of Medicine, New Haven, United States, 8Icahn School of Medicine at Mt Sinai, New York, United States, 9Albert Einstein College of Medicine, Bronx, United States, 10Stony Brook Univ School of Medicine, Stony Brook, United States Background: Acid sphingomyelinase deficiency (ASMD) is a rare, progressive, multiorgan lysosomal storage disease historically known as Niemann-Pick disease types A (NPD A) and B (NPD B). ASMD results in the progressive accumulation of sphingomyelin in reticuloendothelial organs (liver, spleen, bone marrow, and lung), hepatocytes, and in severe cases, brain. ASMD manifests as a clinical spectrum ranging from a rapidly progressive infantile neurovisceral form (NPD A) which is uniformly fatal by age 3, to chronic neurovisceral (NPD B variant) and visceral (NPD B) forms that have variable ages of onset, slower progression, and longer lifespans. Disease management is aimed at mitigating symptoms and regular assessments for multisystem involvement. Methods: An international panel of laboratory and clinical ASMD experts gathered to review the evidence base and develop guidelines for the diagnosis, nomenclature, and management of ASMD. Results: During a meeting at which published material and personal experience were reviewed, the panel members discussed the best approach for diagnosis and developed a diagnostic guideline and new recommended nomenclature to better capture the clinical spectrum of ASMD. The diagnosis of ASMD is based on ASM enzyme activity and SMPD1 gene sequencing. Because of symptom overlap between ASMD and Gaucher Disease, another lysosomal storage disorder, it is recommended that, whenever possible, parallel enzyme testing for Gaucher Disease and ASMD is performed. Discussion: Although care of ASMD patients is typically provided by metabolic disease specialists, the guideline is directed at a wide range of providers since it is important that primary care providers (e.g., pediatricians) and specialists (e.g., pulmonologists, hepatologists, and hematologists) be able to recognize ASMD in order to provide the appropriate patient care and referrals. Early diagnosis is essential for effective disease and symptom management. Supported by Sanofi Genzyme. Conflict of Interest declared.

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Technical difficulties in the diagnosis of Krabbe leucodystrophy by enzyme analysis Hutchin T 1, Terry F 1, Santra S 1, Egerton C 2, Church H 2, Preece M A 1

fluorescent artificial substrate (6HMU-gal). The most likely explanation is that the p.(Tyr319Cys) mutation is still able to hydrolyse the artificial substrate but not the natural substrate. Discussion: We are not aware of any published reports of this issue in other cases of Krabbe disease although such problems with other artificial substrates are known, particularly for a particular mutation in sphingomyelinase. This demonstrates the potential difficulties in the diagnosis of Krabbe disease and other lysosomal storage disorders. In suspected patients with high residual activity studies should be repeated with the use of a natural substrate if possible. Alternatively DNA analysis should be undertaken which can also include prosaposin and activator proteins as appropriate.

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International Niemann-Pick Disease Registry Project. Bolton S 1, Hiwot T 1 1

Queen Elizabeth Hospital Birmingham, Birmingham, United Kingdom

Background: Niemann-Pick Diseases (NPD) are a group of lysosomal storage disorders with an extremely low prevalence (type A and B 1:250,000, type C 1:120,000). NPD are lifelong, chronic and fatal diseases. The rarity and severity of the diseases mean that it is crucial for affected patients to be followed to aid the understanding of NPD. The International Niemann-Pick Disease Registry Project aims to establish the largest and most comprehensive single inventory of patients with NPD, to include biochemical and genetic data, natural history data and patient reported data, and to increase awareness of NPD and quality of patient care. Methods: Data is captured in consensus core and extended datasets which provides the foundation of the multifunctional web-based registry. We are achieving high usage of the registry by linking it to laboratory testing. The learning needs of patients and healthcare professionals have been identified through questionnaires and focus groups. Results: 1) The registry is open in 5 countries, with 216 patients enrolled (51 NPA/B patients and 165 NPC patients). 2) A curated list of EU laboratories offering NPD diagnostic testing has been produced. 3) An online SMPD1 genetic mutation database has been produced, with a NPC1/2 mutation database in development. 4) A Patient Reported Dataset registry is currently being developed and piloted. 5) 3 papers linked to the registry have been accepted for publication. Discussion: Ultimately it is hoped that the registry will be an instrumental tool for recruitment to future clinical research to enhance and facilitate better care for NPD patients.

1 Birmingham Childrens Hospital, Birmingham, United Kingdom, 2Willink Unit, St Mary’s Hospital, Manchester, United Kingdom

P-468 Background: Artificial substrates are routinely used in the diagnosis of lysosomal storage disorders. Whilst easier to use than natural substrates they can show different activities with some mutations, potentially complicating or delaying a diagnosis. Case Report: Ex preterm twins, initially felt to have prematurity-associated cerebral palsy, presented with deteriorating school performance and worsening spasticity and mobility. There was radiological evidence of leukodystrophy suggestive of Krabbe disease. Results: At 6 years of age a deficiency of β-galactocerebrosidase enzyme in leucocytes confirmed a diagnosis of Krabbes leucodystrophy. DNA analysis of the GALC gene showed compound heterozygosity for the common 30 kb deletion and a rarer missense mutation p. (Tyr319Cys). Shortly after, both twins were referred to our centre for a haemopoietic stem cell transplant. As part of the work up for this, baseline enzyme levels prior to transplantation were measured. Surprisingly enzyme activity in both twins was at the lower end of normal activity. Initial diagnosis had been made with a radiolabelled natural substrate which showed a small residual activity. The follow up testing however was done using a

Diagnostic utility of chitotriosidase activity, CCL18/PARC and 7ketocholesterol concentrations in Gaucher, Niemann-Pick A/B/C and lysosomal acid lipase deficiency. Irun P 1 2 4, Cebolla J J 2 3 4, Alfonso P 1 2, De Castro-Oros I 4, Lopez de Frutos L 2 3, Giraldo P 1 2 3 1 CIBERER, Instituto de Salud Carlos III, Zaragoza, Spain, 2Instituto Investigacion Sanitaria Aragon, Zaragoza, Spain, 3FEETEG, Zaragoza, Spain, 4Dpto Bioquimica, Universidad Zaragoza, Zaragoza, Spain

Background: Gaucher (GD), Niemann-Pick Type A/B (NP-A/B), NiemannPick Type C (NP-C) and the lysosomal acid lipase deficiency (LALD) are lysosomal storage diseases (LSDs) difficult to diagnose because of the great heterogeneity of signs and symptoms, sometimes common to several pathologies and the consequent alteration of biomarkers.

S196 Methods: In order to assess the diagnostic utility of chitotriosidase activity (ChT), CCL18/PARC and 7-ketocholesterol (7KC) concentrations in the mentioned LSDs these parameters were retrospectively measured in 146 plasma samples from subjects with suspected LSD (32 GD, 7 NP-A/B, 90 NP-C and 17 LALD) received between 2014 and 2015. ChT was evaluated using a fluorogenic substrate, CCL18/PARC concentration by ELISA and 7KC by liquid chromatography followed by tandem mass spectrometry. Results: Of a total of 32 samples received with the suspicion of GD, 9(28 %) showed high ChT and/or high CCL18/PARC, 4/9 were confirmed as GD and the rest as NP-A/B (1), NP-C (1) while two were carriers of a mutation in NPC1. Only 3/7 (43 %) with the suspicion of NP-A/B had altered biomarkers and were confirmed. Among the 20/ 90 (22 %) with the suspicion of NP-C and some elevated biomarker, four samples, showing all biomarkers altered, were confirmed as NP-C while two were carriers and showed some biomarker altered. Of the 7/17 (41 %) referred to as LALD suspect and some elevated biomarker, only five were affected. Discussion: The determination of three biomarkers: ChT activity, CCL18/PARC and 7-ketocholesterol concentrations (the latter not applicable in GD) would have reduced the number of samples that can be subjected to confirmatory diagnostic test from 146 to 39 (27 %). Overall, 18 affected subjects were identified, accounting for 46 % of those with some biomarker altered.

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Evaluation of bone quality in patients with type 1 Gaucher disease with microindentation. Preliminary results. Perez-Lopez J 1, Herrera S 3, Molto M 1, Guerri-Fernandez R 3, Cabezudo E 4, Novelli S 5, Esteve J 2, Hernandez A 6, Roig I 7, Solanich X 8, Prieto-Alhambra D 9, Nogues X 3, Diez-Perez A 3 1

Hospital Vall d’Hebron, Barcelona, Spain, 2 Hospital Clinic, Barcelona, Spain, 3Hospital del Mar, Barcelona, Spain, 4Hospital de Sant Joan de Deu, Manresa, Spain, 5Hospital de Sant Pau, Barcelona, Spain, 6 Hospital Comarcal de Sant Jaume, Barcelona, Spain, 7 Consorci Hospitalari Parc Tauli, Barcelona, Spain, 8 Hospital de Bellvitge, Barcelona, Spain, 9University of Oxford, Oxford, United Kingdom Background: Over 80 % of patients with type 1 Gaucher disease (GD1) shows bone affectation, causing predisposition for bone fractures. Although with significant limitations, the most used techniques for bone assessment are dual energy X-ray absorptiometry (DXA) and magnetic resonance imaging (MRI). However, microindentation has showed a good correlation with bone fractures in patients with osteoporosis. Our goal was to study the bone quality in patients with GD1 using the microindentation and its new measurement parameter called Bone Mineral Strength index (BMSi), as a new and accurate tool for this assessment. Methods: 14 GD1 patients and 29 controls were enrolled. Clinical and laboratory parameters of all individuals were collected, followed by Bone Mineral Density (BMD) in their column and hip and BMSi in tibia. Impact Bone Microindentation measurements were carried out (Osteoprobe, Active Life Scientific, Santa Barbara, CA, USA) in areas not affected by bone infarcts or necrosis. Results: Comparing GD1 patients with controls, there were no significant differences among patients with GD1 and controls regarding age, gender, height and weight. We found a significant difference in BMSi (81.7(7.78) p < 0.05) and BMD in column (0.99(0.14) p < 0.05), but not in BMD in hip (0.76(0.10) p = 0.14). No differences were found between GD1 patients regarding Severity Score Index (SSI) or treatment. Discussion: Our preliminary results suggest that could become microindentation as a novel and accurate technique for the study of the bone quality in GD1 patients. However, more data is needed to find out its role in clinical practice.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 P-470

Assessment of diagnostic parameters and disease biomarkers for detecting early-stage Fabry disease and monitoring its progression—results from the SOPHIA study. Weidemann F 1 2, Beer M 3, Kralewski M 4, Kampmann C 5 1

Dept Intern Med I, Univ Hosp Wuerzburg, Wuerzburg, Germany, Katharinen-Hospital Unna, Unna, Germany, 3Dept Diagn Interv Radiol, Univ Hosp Ulm, Ulm, Germany, 4 Shire Deutschland GmbH, Berlin, Germany, 5Univ Med Center, Univ Mainz, Mainz, Germany 2

Background: Determining optimum combinations of diagnostic parameters and disease biomarkers to detect early-stage Fabry disease (FD) may enable timely therapy initiation. The SOPHIA study (Sophisticated Assessment of Disease Burden in Patients with Fabry Disease) evaluated comprehensive clinical and diagnostic assessments for early detection of Fabry-related organ pathology. Methods: A multicentre, observational, prospective diagnostic study conducted in Belgium, Denmark, Finland, Germany and Sweden. Eligible patients were aged ≥25 years, had confirmed FD with only mild symptoms, and did not receive enzyme replacement therapy prior to or during the study. Diagnostic assessments included cardiac magnetic resonance imaging with late-gadolinium enhancement (LGE; indicating myocardial fibrosis), echo- / electrocardiography techniques and plasma biomarkers at baseline, 12 months and optionally at 24 months. Results: Patients (N = 35; 29 [82.9 %] female) were aged mean (SD) 45.0 (10.2) years at baseline. Of all variables analysed, LGE and elevated globotriaosylsphingosine (lyso-Gb3) were the most prevalent indicators of Fabry-related pathology, found in 19/29 and 22/27 patients, respectfully. A statistically significant positive correlation was found between baseline lysoGb3 and LGE (Spearman’s correlation coefficient 0.41; p = 0.03). Fibrosis progressed from baseline (more basal segments) to last observation (basal and mid segments; LGE volume mean [SD] mL 1.70 [1.53] vs 3.31 [2.70]), especially in the 6 patients with baseline LGE ≥2.5 mL (mean [SD] mL 3.43 [1.06] vs 6.68 [1.03]). Markedly elevated high-sensitivity troponin T (hs-TNT) was associated with myocardial fibrosis (100 % of patients with hs-TNT >14 ng/mL were LGE positive). Discussion: Plasma lyso-Gb3 and cardiac LGE were sensitive tests for detecting early-stage FD and may be warranted in all patients with mild FD to detect early organ involvement, facilitating prompt treatment, which might help prevent FD progression. Conflict of Interest declared.

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Study of comorbidities in a Spanish cohort of Gaucher disease type 1 patients Perez-Lopez J 1, Giner V 9, Torralba-Cabeza M A 10, Perez S J 3, Perez S J 3, Molto-Abad M 1, Roig I 7, Vicente L 11, Luana-Galan A 12, Cabezudo E 4, Solanich X 8, Roig-Espert B 13, Patera E 6, Lozano-Almela M L 14, HurtadoGarcia R 15, Reyes M 10, Revilla N 14, Novelli S 5, Esteve J 2 1 Hospital Vall d’Hebron, Barcelona, Spain, 2Hospital Clinic, Barcelona, Spain, 3Hayward Genetics Center, Tulane Univ SOM, New Orleans, United States, 4Hospital de Sant Joan de Deu, Manresa, Spain, 5Hospital de Sant Pau, Barcelona, Spain, 6Hospital Comarcal de Sant Jaume, Barcelona, Spain, 7 Consorci Hospitalari Parc Tauli, Barcelona, Spain, 8Hospital de Bellvitge, Barcelona, Spain, 9Hospital Virgen de los Lirios, Alcoy, Spain, 10Hospital Lozano Blesa, Zaragoza, Spain, 11Hospital de Terrassa, Terrassa, Spain, 12 Hospital Arnau de Vilanova, Lleida, Spain, 13Hospital de Manises, Valencia, Spain, 14Hospital Morales Meseguer, Murcia, Spain, 15Hospital Vega Baja, Orihuela, Spain

Background: Specific treatment in Gaucher type 1 (GD1) has allowed a lifespan similar to general population. Aging process results in an increase

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 frequency of comorbidities and poly-pharmacy, so that it may affect the outcomes of these patients. We studied the comorbidities and poly-pharmacy in a Spanish cohort of GD1 patients. Methods: 37 GD1 patients were enrolled. Disease complications (including Severity Score Index [SSI]), simultaneous comorbidities (including Charlson Index [ChS]), as well as concomitant drugs were evaluated. Mean and standard deviation were measured in quantitative variables. Results: 20 women and 17 men were studied, with mean of age 46.8(17.4) years. SSI was 7.4(3.7). All patients were under treatment for GD, whose length of treatment was 11.1(6) years. Seventeen (46 %) patients presented bone complications (5 osteonecrosis, 5 osteoporosis, 4 hip replacement and 3 bone fractures), 8(20.5 %) were splenectomized, 7(17.9 %) monoclonal gammapathy, 1(2.7 %) Parkison disease and none with cancer. ChS was 4(1.4), including dyslipemia in 6(16.2 %) patients, diabetes mellitus in 2(5.4 %). Fourteen (37.8 %) patients showed poly-pharmacy with a mean of 4.5(2.8) drug per patient, including psychotropic drugs in 11(30 %), calcium and D vitamin supplements in 10(27 %), pain kills in 7(17.9 %), statins in 6(16.2 %), antiarrhytmics in 2(5.4 %), and biphosphonates, antiagregants and antiparkinsonians in 1(2.7 %) respectively. Discussion: Our data suggests that bone involvement leads to the most important disease complications in GD1 (46 % of patients), and that poly-pharmacy is very frequent among these patients (37.8 %). Psychotropic drugs, calcium/vitamin D supplements and statins are the most common concomitant medication.

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Characteristics of 27 patients with type 3 Gaucher disease: a descriptive analysis from the Gaucher Outcome Survey Schwartz I V D 5 6, Goker-Alpan O 3, Kishnani P 7, Zimran A 2, Renault L 1, Panahloo Z 1, Deegan P 4 1 Shire, Zug, Switzerland, 2Shaare Zedek Med Cent & Hadassah Med Sch, Jerusalem, Israel, 3LSD Unit & Cent Clin Trials, O&O Alpan, Fairfax, Virginia, United States, 4Addenbrooke’s Hospital, Cambridge, United Kingdom, 5Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil, 6Univ Federal do Rio Grande do Sul, Porto Alegre, Brazil, 7Duke University School of Medicine, Durham, North Carolina, United States

Background: The Gaucher Outcome Survey (GOS) is an international Gaucher disease (GD)-specific registry established in 2010 for patients with a confirmed GD diagnosis, regardless of GD type or treatment status. Patients with neuronopathic type 3 GD (GD3) may show non-neurological manifestations similar to those of type 1 GD. We examined real-world data from GOS for insight into the disease characteristics and management of GD3 patients. Methods: The analysis included all GOS patients recorded as GD3. Patient demographics, disease parameters and treatment history were collected. Adverse event (AE) data were collected for patients who received velaglucerase alfa. Results: For GOS patients with GD type data (970/1003), 27/970 (3 %) were recorded as GD3 (median [range] age at GOS entry 17.4 [0.5–56.3] years, 12 [44 %] male, 7 [26 %] splenectomised). Most patients were from the USA (14/ 27, 52 %); others were from the UK, Israel, and Brazil. None were Ashkenazi Jewish. The most common GBA genotype was L444P/L444P (11/19, 58 %). At analysis, 21 patients were on GD treatment (imiglucerase, velaglucerase alfa, or eliglustat). For patients treated with velaglucerase alfa (n = 13), 3 AEs deemed possibly drug related were reported in 1 patient. The median (range) time on treatment before GOS entry was 7.9 (0.6–27.8) years. At entry, mean height was 139 ± 34 cm, and weight was 49 ± 23 kg. Physical examination at entry showed 5/23 (22 %) patients had hepatomegaly and 4/13 (31 %) had splenomegaly. All 21 patients with data (including splenectomised) had normal haemoglobin concentrations, and 19/21 (90 %) had platelet counts >150 x 109/L. Discussion: GOS GD3 patients have control of haematological parameters, and most receive GD treatment. Analysis of patients in Asian and Arab countries where GD3 is more prevalent, will provide a global view. Collected GOS data can provide insight into GD3 patients from different regions and aid management through a better understanding of natural disease progression. Conflict of Interest declared.

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Clinical use of plasma oxysterols for rapid diagnosis of Niemann-Pick type C Lay I 1 2, Ardicli D 3, Samadi A 1, Akbiyik F 1, Serdaroglu E 3, Haliloglu G 3, Yuce A 4, Coskun T 5, Topcu M 3 1 Dep of Med Biochem, Hacettepe Uni Hosp, Ankara, Turkey, 2Clin Lab, Hacettepe Uni Hosp, Ankara, Turkey, 3Div Pediatric Neur, Hacettepe Uni Hosp, Ankara, Turkey, 4Div Pediatric Gastro, Hacettepe Uni Hosp, Ankara, Turkey, 5Div Pediatric Metabo, Hacettepe Uni Hosp, Ankara, Turkey

Background: Niemann-Pick Type C (NP-C) is a rare neurodegenerative disorder caused by impaired intracellular transport of unesterified cholesterol and glycolipids owing to mutations in either NPC1 or NPC2 gene. The diagnosis of NP-C is often delayed due to heterogeneous clinical presentations and the lack of a rapid and a reliable blood test. Accumulation of cholesterol in the cells leads to non-enzymatic oxidation of cholesterol and thus, the formation of oxysterols which can be measured in blood as powerful diagnostic biomarkers for NP-C. Methods: Plasma cholestane-3beta-,5alpha-,6beta-triol and 7-ketocholesterol levels were analyzed in 102 suspicious NP-C patients in the context of clinical, biochemical and molecular data with LC-MS/MS (Shimadzu 8040, Japan) after derivatization with dimethylglycine esters. Eight point calibrators and 3 levels of QC were used. Results: Nineteen patients demonstrated extremely high levels of plasma cholestane-3β,5α,6β-triol (mean ± SD: 101.7 ± 56 ng/mL) and 7ketocholesterol (mean ± SD: 170.0 ± 112.4 ng/mL). Mean cholestane3β,5α,6β-triol levels was 20.9 ± 8.4 ng/mL and 7-ketocholesterol was 31.2 ± 14.5 ng/mL for 70 healthy individuals. NP-C diagnoses were confirmed with molecular analysis in 13 patients (11 NPC1 and 2 NPC2 mutations). Among the 6 suspicious patients whose molecular analyses were not performed yet, 2 patients showed low level of sphingomyelinase activity from DBS. At the cutoff of 39 ng/mL, cholestane-3β,5α,6β-triol demonstrated a specificity of 98.6 % and a sensitivity of 100 %. Both a specificity and a sensitivity of 100 % at a cut-off of 72 ng/mL was observed for 7-ketocholesterol. Discussion: Both oxysterol levels were significantly high in NP-C patients compared to controls, and could be used as rapid and reliable biomarkers for NP-C. Differential diagnosis should always be performed in patients with NPA/B.

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Cardiac dysfunction and troponin I levels in patients with Fabry disease followed at Hospital de Clinicas de Porto Alegre, Brazil Vairo F 1, Angrezani J 1, Teixeira A 1, Giugliani R 1 2 1 Med Gen Serv, Hosp Clin Porto Alegre, Porto Alegre, Brazil, 2Dep Genetics, Univ Fed Rio Grande do Sul, Porto Alegre, Brazil

Background: Fabry disease (FD) is an X-linked inborn error of metabolism characterized by the accumulation of globotriaosylceramide (Gb3) due to deficient activity of the lysosomal enzyme α-galactosidase A. In the heart, Gb3 accumulates in myocites, conduction cells and endotelial cells, leading to hypertrophy, apoptosis, necrosis and fibrosis. Troponin I is a myocardial injury biomarker, used especially for diagnosis of ischemia. Objective: Evaluating troponin I levels and their correlations with clinical, biochemical and cardiac dysfunction parameters in patients with FD. Methods: We evaluated troponin I serum levels, electrocardiographic and echocardiographic findings in 16 males (mean age 35.2 ± 12.3 years) and 17 females (mean age 43.4 ± 15 years). Results: Seven males (43.7 %) and 4 females (23.5 %) had increased troponin I levels. Three males (18.7 %) and 4 females (23.5 %) had conduction abnormalities and 8 males (50 %) and 4 females (23.5 %) had left ventricular hypertrophy in ECG. No patient had electrocardiographic evidence of myocardial ischemia. Regarding echocardiographic findings, 4 males (25 %) and 3

S198 females (17.6 %) had increased interventricular septum wall thickness (>11 mm) and 7 males (43.7 %) and 6 females (35.3 %) had increased left ventricular mass (>215 g). Three males (18.7 %) and 5 females (29.4 %) had diastolic dysfunction. There was association between troponin I alterations and electrocardiographic and echocardiographic abnormalities. There was no association between troponin I alterations and disease severity, renal function and Gb3 levels. Discussion: Our findings indicate that patients with FD without ischemic heart disease have increased troponin I. Furthermore, some patients with increased troponin I had no cardiac dysfunction, evidencing the need of prospective studies to elucidate whether early pharmacological interventions, based on troponin I serum levels, can prevent or decrease the rate of progression of heart disease in patients with FD.

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Metabolic progression to clinical phenotype in classic Fabry disease Spada M 1, Kasper D 2, Pagliardini V 1, Giachero S 1, Biamino E 1, Porta F 1 1 Dept of Pediatrics, University of Torino, Torino, Italy, 2Archimed Life Science GmbH, Vienna, Austria

Background: Fabry disease is an X-linked lysosomal storage disorder due to α-galactosidase A (α-Gal A) deficiency. The enzymatic defect leads to progre ssive acc umulation of globotr iaosylce ramide a nd rela te d glycosphingolipids in the vascular endothelium, particularly in kidney, brain, and heart. Affected males with complete or near-complete α-Gal A deficiency exhibit the classic clinical phenotype of Fabry disease with onset of angiokeratomas, acroparesthesias, hypohidrosis, and corneal opacities in childhood, followed by renal failure, cardiac and cerebrovascular disease. Recently, globotriaosylsphingosine (LysoGb3) was identified as a new pathogenetic effector and hallmark of Fabry disease, representing a promising noninvasive marker for monitoring this disorder. Methods: LysoGb3 assessed on dried blood spot by HPLC-mass spectrometry was compared in two brothers with classic Fabry disease (genotype c. 370–2 A > G) with 12 year age difference. LysoGb3 in the first born was assessed at clinical onset, characterized by acroparesthesias and burning pain at 11 years of age. Based on familiar anamnesis, the second born was diagnosed with Fabry disease in the newborn period, undergoing LysoGb3 analysis 2 days after birth. Results: The comparison of LysoGb3 concentrations in the two brothers with classic Fabry disease revealed a 6-fold increase from the neonatal period to childhood (from 14.5 ng/ml to 94.3 ng/ml). Discussion: Since the clinical phenotype of Fabry disease is invariably preceded by progressive lysosomal storage of glycosphingolipids, the analysis of peripheral LysoGb3 may allow to investigate patients’ metabolic phenotype even in the pre-symptomatic period. Our data depict the extent of glycosphingolipids storage during childhood in classic Fabry disease. Longitudinal LysoGb3 analysis in larger populations could lead to the perspective definition of a metabolic threshold (i.e. a LysoGb3 cut-off) addressing pre-symptomatic treatment of Fabry disease.

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The ENCORE Trial: Outcomes in Adult Patients with Gaucher Disease Type 1 Previously Stabilized on Enzyme Therapy after 4 Years of Treatment with Eliglustat Cox T M 1, Drelichman G 2, Cravo R 3, Balwani M 4, Burrow T A 5, Martins A M 6, Lukina E 7, Rosenbloom B 8, Goker-Alpan O 9, Watman N 10, ElBeshlawy A 11, Kishnani P S 12, Pedroso M L 13, Gaemers S J M 14, Tayag R 15, Peterschmitt M J 14 1

Univ of Cambridge, Addenbrookes Hosp, Cambridge, United Kingdom, Hospital de Ninos Ricardo Gutierrez, Buenos Aires, Argentina, 3State Institute of Haematolgy, Rio de Janeiro, Brazil, 4Mount Sinai Hospital, New 2

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 York, United States, 5Cincinnati Childrens Hosp Medical Center, Cincinnati, United States, 6Universidade Federal de Sao Paulo, Sao Paulo, Brazil, 7 National Research Center for Hematology, Moscow, Russian Federation, 8 Cedars Sinai Tower, Hematology Oncology, Beverly Hills, United States, 9 Lysosomal Disorders Unit, O and O Alpan, Fairfax, United States, 10 Hospital Ramos Mejia, Buenos Aires, Argentina, 11Pediatric Hematology, Cairo University, Cairo, Egypt, 12Duke Univ Sch Med, Dept of Pediatrics, Durham, United States, 13Hosp de Clinicas, Univ Federal do Parana, Curitiba, Brazil, 14Sanofi Genzyme, Cambridge, United States, 15Prometrika, Cambridge, United States Background: Eliglustat is an oral substrate-reduction therapy approved in the US and Europe as first-line treatment for adults with Gaucher disease type 1 (GD1) with compatible CYP2D6 metabolizer phenotypes (>90 % of patients). In the ENCORE trial (NCT00943111, Sanofi Genzyme) primary analysis of GD1 adults (N = 159) previously stabilized on enzyme replacement therapy (ERT), eliglustat was non-inferior to imiglucerase in maintaining stability after 12 months (Cox. Lancet,2015). Methods: During the ENCORE trial and extension, patients (all eliglustattreated) could receive eliglustat for 2.5–4 years, depending on time of enrolment, (which spanned 2 years), trial randomization and geographic location (51 US patients left the trial to receive commercial eliglustat when it became available). We report on long-term safety and efficacy with respect to years on eliglustat therapy for all 157 eliglustat-treated patients in ENCORE; 46 were followed for ≥4 years. Results: Mean values for haemoglobin, platelets, and spleen and liver volumes remained stable over 4 years. Year to year, all 4 measures also remained stable collectively (composite endpoint relative to baseline values) in ≥85 % of patients, and individually in ≥91 %. Year to year, all 4 therapeutic goals for haemoglobin concentration, platelet count, spleen and liver volumes for patients on ERT (Pastores. Semin Hematol,2004) were maintained in ≥92 % of patients; each individual goal was maintained by ≥94 %. Mean bone mineral density Z-scores (lumbar spine and femoral) remained stable and within healthy reference ranges throughout. Eliglustat was well-tolerated over 4 years during both primary and extension phases; 4 patients (2.5 %) withdrew due to adverse events considered related to eliglustat. No new or long-term safety concerns were identified. Discussion: Clinical stability by composite and individual measures was maintained in adults with GD1 treated with eliglustat who remained in the ENCORE trial for up to 4 years. Conflict of Interest declared.

P-477 Combination therapy using miglustat and ketogenic diet for treatment of gangliosidoses: overall survival in 10 infants. Jarnes Utz J R 1, Whitley C B 1 1

University of Minnesota Pediatricsi, Minneapolis, United States

Background: Infantile gangliosidoses (Tay-Sachs disease, Sandhoff disease and GM1 gangliosidosis) are rapidly progressive sphingolipidoses that result in death most often by 3 years of age. Clinical symptoms include hypotonia, seizures, impaired cognitive and motor abilities. To date, no therapies exist to treat these conditions. Miglustat inhibits glucosylceramide synthase in the glycosphingolipid pathway and thereby reduces production of sphingolipids, including GM1 and GM2 ganglioside, but its use is limited by its inhibition of disaccharidases in the gastrointestinal tract, resulting in abdominal cramping, diarrhea, flatulence, nausea and vomiting. These adverse effects may be mitigated by use of a low-carbohydrate diet. Methods: Combination therapy using a ketogenic diet with miglustat (SynerG) was evaluted in infantile gangliosidoses for impact on overall survival, seizure management, and neurodevelopmental parameters evaluated via Bayley Scale of Infant and Toddler Development and Vineland Adaptive Behavior Scale. RESULTS were compared to untreated patients and published natural history data. Results: Syner-G treatment of 10 patients with infantile gangliosidoses (4 with GM1 gangliosidosis, 4 with Tay-Sachs disease and two with Sandhoff disease) was compared to 6 untreated patients (3 with GM1 gangliosidosis and 3 with

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Tay-Sachs disease). Five of 6 untreated patients died by age 3 years, and 1 died at age 4 years. To date, 6 of the 10 patients in the treated patients are alive, with longest survival observed to date of 5.5 years in Tay-Sachs disease and 4.5 years in GM1 gangliosidsis. All treated patients experienced improvement in seizure management and expressive and receptive communication. Discussion: Treatment with Syner-G in infants with gangliosidoses improved overall survival, seizure management and receptive and expressive communication. Syner-G demonstrated palliative results in this population and holds promise as an adjunctive therapy to future treatments for gangliosidoses.

P-478

Development of a Suspicion Index tool to help Diagnosis of Gaucher Disease Bulut D F 1, Yilmaz B S 1, Kor D 1, Seydaoglu G 4, Ozbek M N 5, Sasmaz H I 3 , Soyupak S 6, Ceylaner S 2, Mungan H N O 1 1 Cukurova Univ, Div Ped Metab and Nutr, Adana, Turkey, 2Intergen Genetic Cent, Ankara, Turkey, 3Cukurova Univ, Div Ped Hemato, Adana, Turkey, 4Cukurova Univ, Dep Biostatistics, Adana, Turkey, 5 Diyarbakir Child Hosp, Diyarbakir, Turkey, 6Cukurova Univ, Dep Radiology, Adana, Turkey

Background: Gaucher disease (GD) is a relatively frequent lysosomal storage disease, presenting with multiorgan involvement. Clinical spectrum is heterogeneous and diagnosis may be delayed due to the suspicion of more common hematologic and infectious diseases. With a useful suspicion index (SI), invasive diagnostic methods such as bone marrow aspiration and liver biopsy can be avoided. Also, early diagnosis and enzyme replacement treatment (ERT) can prevent irreversible hematologic and skeletal complications. Methods: 62 GD and 36 non-GD patients were retrospectively analyzed. To build up a useful suspicion index, symptoms, family history, visceral, neurologic and skeletal involvement and laboratory findings of GD and non-GD patients were grouped and scored. Results: Anemia, thrombocytopenia, splenomegaly/splenectomy, Erlenmeyer flask deformity, osteoporosis and serum acid phosphatase levels were found as the strong indicators of GD. Parental consanguinity and family history were also valid predictors of GD. Leucopenia, elevated levels of transaminases, neurological and pulmonary involvement were poor diagnostic parameters. But, these parameters were not valuable to discriminate GD from Niemann-Pick (NP) type A/B or C. Discussion: As early diagnosis and ERT administration decreases the severe and irreversible complications, early awareness is important. SI will aid to leave invasive diagnostic procedures aside and put forward reliable and noninvasive methods like enzyme assay and mutation analysis.

20. Lysosomal disorders: others

P-479

Novel LIPA mutations resulting in lysosomal acid lipase deficiency Reynders J 1, Burton B 2, Del Angel G 1 1 Alexion Pharmaceuticals, Inc., New Haven, United States, 2Northwestern Univ, Feinberg Sch Med, Chicago, United States

Background: Lysosomal Acid Lipase Deficiency (LAL-D) is a lifethreatening genetic disease caused by mutations in the lysosomal acid lipase (LIPA) gene with a clinical spectrum from infancy to adulthood, similar to other lysosomal storage disorders. Disease awareness is low

and the number of known pathogenic mutations is small. Additional analysis is needed to better understand the underlying genetic architecture of the lysosomal acid lipase (LIPA) enzyme and associated diminished activity introduced by the combined genetic mutations on each LIPA allele. Methods: As part of the ARISE study (NCT 01757184), a multi-center, randomized placebo-controlled study of sebelipase alfa, 66 children (>4 yrs) and adults with LAL-D had genetic analysis of the LIPA gene, in addition to undergoing dried blood spot testing to demonstrate reduced LAL enzyme activity consistent with LAL-D. Isolation of DNA and genetic mutation analysis for allelic variants of the LIPA gene was performed (Prevention Genetics, Marshfield, WI, US). Results: Overall, 85 % of subjects had at least one copy of the previously described c.894G > A common exon 8 splice junction mutation (E8SJM), 32 % homozygotes and 53 % compound heterozygotes, resulting in an E8SJM allele frequency of 58.3 % in this study. Seven novel LIPA mutations paired with c.894G > A were discovered (c.1033G > A, c.256C > T, c.309C > A, C.445 T > C, c.526G > A, c.538 + 5G > A, and c.931G > A) and six novel LIPA mutations were discovered in combination with pathogenic non-E8SJM LIPA mutations (c.417C > A, c.607G > C, c.253C > A, c.974C > T, c.791 T > C, and c.294C > G). Discussion: These 13 novel mutations from the ARISE study have been identified in diagnosed patients with LAL-D and represent a ~22 % increase over the ~60 published pathogenic LIPA mutations which will assist in accurate diagnosis. Conflict of Interest declared.

P-480

Identification of 14 novel mutations in 45 Iranian Niemann-Pick Type C (NP-C) patients Houshmand M 4, Tonekaboni S H 1, Aryani O 2, Karimzadeh P 1, Rahmanifar K 3 , Tavasoli A R 3 , Zaman T 3 , Ashrafi M 3 , Salehpour S H 1 , DehghanManshadi M 2, Ghodsinejad V 2, Khalili E 2, KamaliDehghan B 4 1

ShahidBeheshti University of Medical Sci, Tehran, Iran, Islamic Republic of, Medical Genetic Center, Tehran, Iran, Islamic Republic of, 3Medical School of Tehran University, Tehran, Iran, Islamic Republic of, 4NIGEB, Tehran, Iran, Islamic Republic of 2

Background :Niemann-Pick disease type C (NP-C) is a rare lysosomal neurovisceral storage disease due to mutations in NPC1 (95 %) or NPC2 (5 %) genes. Both NPC1 and NPC2 gene products are intimately involved in cholesterol and glycolipid trafficking and/or transport. NP-C disease has a variable phenotype, whereby an alteration in cholesterol and glycolipid homeostasis leads to a broad spectrum of symptoms that include hepatosplenomegaly, liver dysfunction, and neurological abnormalities such as progressive ataxia, cataplexy, vertical supranuclear gaze palsy, seizures, and impairment of swallowing reflexes. Methods: We performed mutation analysis of 45 unrelated Iranian patients diagnosed as NPC by filipin staining, including 22 males and 23 females. Reverse-transcription PCR (RT-PCR) was used to amplify all 25 exons of NPC1 gene and DNA sequencing analysis was performed with Finch TV. Results: DNA sequence analysis identified 31 different variations in 28 patients, 10 of which were not previously reported and predicted to be deleterious using in silico prediction programs (SIFT and PolyPhen2). These unreported novel mutations included 6 missense mutations; p.G657D, p. L472P, p.C184Y, p. N478T, p.Y394H and p.N169I, one nonsense mutation; p.Q136* and 3 small deletions; c.3126 del C, c.2920-2923del CCTG and c.2037del G that were found in 14 patients. Additionally, five previously reported mutations including p. A926T, p.V664M, p.G1034R, c.3591 + 1G > A and c.536 + 1 G > A were found in 8 of the 28 patients. Discussion: Our study further expands the antenatal clinical spectrum of NP-C and provides clues to its prenatal diagnosis.

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P-481

We investigated cardiac manifestations and cardiovascular autonomic function of Fabry patients by using electrocardiography and ultrasonography. Methods: Sixty four Fabry patients (male 27, female 37) were enrolled and performed ECG and UCG. We compared with Fabry patients and sex- and age- matched controls. Fabry patients were divided into 2 groups with and without left ventricular hypertrophy (LVH). We investigated Coefficient of Variation of R-R intervals (CVRR) of ECG as cardiac autonomic function. Results: Male and female Fabry patients were significantly higher than control subjects in terms of QRS duration and Sokolow-Lyon index. Female Fabry patients were significantly longer than control subjects in terms of QTc duration. Fabry patietns with and without LVH were significantly higher than control subjects in terms of sokolow-Lyon index. CVRR was not different between Fabry patients and control subjects. Female Fabry patients tended to decrease LV mass after 24 months treated ERT. There was significant correlation between Sokolow-Lyon index and a change of LV mass after 12-24months in Fabry male patients without LVH (correlation coefficient 0.709, p < 0.05). Discussion: Fabry male patients tended to have high voltage in electrocardiography before becoming left ventricular hypertrophy. The high voltage in electrocardiography may be early cardiac observations of Fabry disease. Fabry male patients who had high voltage in electrocardiography tended to improve left ventricular hypertrophy. In our study the autonomic function of Fabry patiets was as same as healthy controls.

Expert opinion on the management of CLN2 disease Specchio N 10, Williams R 3, Adams H 4, Blohm M 2, Cohen-Pfeffer J 1, De los Reyes E 5, Denecke J 8, Drago K 6, Fairhurst C 3, Frazier M 7, Guelbert N 8, Kiss S 9, Kofler A 10, Lawson J 11, Lehwald L 5, Leung M 3, Mikhailova S 12, Mink J 4, Nickel M 2, Shediac R 1, Sims K 13, Topcu M 14, Von Lobbecke I 15, West A 16, Schulz A 2 1 BioMarin Pharmaceutical Inc, Novato, CA, United States, 2University Medical Ctr Hamburg-Eppendorf, Hamburg, Germany, 3Evelina London Childrens Hospital, London, United Kingdom, 4University of Rochester School of Med, Rochester, NY, United States, 5Nationwide Childrens Hospital, Columbus, OH, United States, 6Journeycare for Children, Chicago, IL, United States, 7BDSRA, Columbus, OH, United States, 8Childrens Hospital of Cordoba, Cordoba, Argentina, 9Weill Cornell Medical College, New York, NY, United States, 10Bambino Gesu Childrens Hospital, IRCCS, Rome, Italy, 11Sydney Childrens Hospital, Syndey, Australia, 12Russian Regional Pediatric Hospital, Moscow, Russian Federation, 13Massachusetts General Hospital, Boston, MA, United States, 14Hacettepe University, Ankara, Turkey, 15Practice Paediatric Physiotherapy, Hamburg, Germany, 16 Batten Disease Family Association, BDFA, Farnborough, United Kingdom

Background: CLN2 disease, an inherited, rare, pediatric-onset, rapidly progressive neurodegenerative lysosomal storage disorder caused by TPP1 enzyme deficiency, is characterized by language delay, seizures, movement disorder, motor deterioration, dementia, blindness and early death. No management guidelines exist for this condition. Our aim is to gain insight into current management strategies. Methods: 24 disease experts (healthcare professionals and patient advocates) completed an online survey with a smaller group participating in a discussion of management practices. Results: Experts share common goals in the management of patients and their families. Goals and interventions evolve as the disease progresses, with a shift in focus from maintenance of function early in the disease to maintenance of quality of life (QoL). The goal of antiepileptic medication is to achieve sufficient seizure control to support function balancing the side effects. Antiepileptic medications may have unique response in patients with CLN2. Carbamazepine and phenytoin should be used with caution. School and home environments should be adapted to accommodate physical and cognitive/ behavioral impairments for ongoing benefit of maintaining social interactions. Physical, occupational and speech therapies should be initiated early and assessed frequently, including use of adaptive devices to support function and independence. Palliative care team engagement is essential soon after diagnosis is made. Discussion: CLN2 management practices are consistent among experts worldwide. A multidisciplinary approach is critical for optimizing care and QoL of patients and families throughout the disease course. This effort to identify common management practices represents an initial step towards development of consensus-based management recommendations. Conflict of Interest declared.

P-482

Cardiac manifestations and cardiovascular autonomic functions of 64 Fabry patients Goto H 1, Tsuboi K 1, Yamamoto H 1 1

LSD Center, Nagoya Central Hospital, Nagoya, Japan

Background: Fabry disease is an X-linked lysosomal strage disorder associated with cardiomyopathy and cardiac arrhythmia in middle age. Fabry patients also have symptoms and signs compatible with autonomic dysfunction. These symptoms and signs are considered to be due to impairment of the peripheral nervous system. Less is known about cardiac manifestations and autonomic function of Fabry patients.

P-483

Severe dilated cardiomyopathy as an unusual clinical presentation in an infant with mucolipidosis type 1. Eyskens F J M 1 2, Marchau F 3, De Sain M 4, Ferdinandusse S 5, Van Kuilenburg A B P 5 1 Div Metab Dis, PCMA, Antwerp, Belgium, 2Div Metab Dis, Univ Mother Child Hosp, Antwerp, Belgium, 3Div Ped Card, Univ Child Hosp, Antwerp, Belgium, 4Metab Lab, UMC, Utrecht, Netherlands, 5Lab Genet Met Dis, AMC, Amsterdam, Netherlands

Background: Dilated cardiomyopathies are the most common type of cardiomyopathy in early childhood and about 50 % have to be classified as idiopathic variants. Hypertrophic cardiomyopathy and/or valvular thickening and/or prolapse has been described in mucolipidosis type 2 and 3. Sporadic cases of dilated cardiomyopathy in young infants affected by mucolipidosis type 2 have been published. Case report: A female neonate, second child of consanguineous Turkish parents and born after an uneventful pregnancy and delivery, presented with mucopolysaccharidosis-like phenotypic ckinical manifestations: coarse face, hepatosplenomegaly, dysostosis multiplex. Echocardiography showed a dilated left ventricle and normal aspect of heart valves. At the age of 5 weeks she became dyspneic, cyanotic and developed feeding difficulties. At 11 weeks of age it was decided to start continous feeding by a nasogastric tube to prevent risk of aspiration pneumonia. She manifested a severe failure to thrive and motoric development was absent. At the age of 12 weeks under maximal inotropic medication and diuretics she developed a cardiac failure with ejection fraction of the left ventricle of 8–10 % and LVEDD of 32 mm (severe dilated cardiomyopathy). She died of cardiac failure at the age of 14 weeks. Results: Metabolic findings: GAG excretion in urine was normal; oligosaccharides in urine showed a pattern compatible with sialidosis or galactosialidosis. Enzymatic acivity of N-acetyl-a-D-neuraminidase turned out to be very deficient: 0.3 (RV: 15.0–45.0) nmol/mg.h. Cathepsin A (galactosialidose) activity was normal. The diagnosis om mucolipidosis type 1 was made. Discussion: To our knowledge this is the first patient affected by mucolipidosis type 1 who developed a severe dilated cardiomyopathy.

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Monitoring oculomotor abnormalities in children with Niemann Pick type C Blundell J 1, Frisson S 1, Chakrapani A 2, Gissen P 2, Vijay S 3, Santra S 3, Olson A 1 1 University of Birmingham, Birmingham, United Kingdom, 2Great Ormond Street Hospital, London, United Kingdom, 3Birmingham Children’s Hospital, Birmingham, United Kingdom

Background: Niemann-Pick type C (NP-C) is a rare recessive disorder associated with progressive supranuclear gaze palsy. Degeneration occurs initially for vertical saccades and later for horizontal saccades. There are studies of oculomotor degeneration in adult NP-C patients [1, 2] but no comparable studies in children. Our aim was to measure eye movements in children with NP-C who had no neurological signs (pre-neurological NP-C) and children with neurological decline (neurological NP-C). Methods: Vertical and horizontal saccades of 3 neurological and 3 preneurological NP-C patients were compared to healthy controls (N = 77) for saccade onset latency, saccade peak velocity and saccade curvature. Results: NP-C patients exhibited clear selective impairments of vertical saccade peak velocity and vertical saccade curvature, with slower peak velocities and greater curvature. Changes were more pronounced in neurological than pre-neurological patients, but curvature abnormality was apparent in both groups. Onset latencies were not different from healthy controls. Longitudinal data, tracking oculomotor change over time, is also presented. Discussion: Vertical saccade peak velocity and curvature are promising measures of disease severity and onset of neurological decline in NPC. They may also help evaluate responses to therapeutic interventions. Conflict of Interest declared.

P-485 Withdrawn

Classic late-infantile CLN2 has pediatric onset with initial symptoms of seizures and language delay followed by progressive dementia, motor and visual deterioration and early death. Variant phenotypes occur more rarely. CLN2 diagnosis is based on laboratory testing following clinical suspicion. Early diagnosis is key to optimizing clinical care and future therapies outcomes, yet delays in diagnosis are common due to low disease awareness, nonspecific initial symptoms and limited diagnostic testing access in some regions. Methods: In May 2015, international experts met to recommend best laboratory practices for early CLN2 diagnosis. Results: When clinical signs suggest NCLs, TPP1 activity should be the first test performed (along with palmitoyl-protein-thioesterase-1 to exclude CLN1). However, since reaching initial suspicion of CLN2 and NCLs is challenging, where available, use of epilepsy gene panels to investigate unexplained seizures in childhood is endorsed. These panels should include TPP1/CLN2 besides genes for other NCLs lacking biochemical tests. Diagnostic TPP1 enzyme test in leucocytes is well established and robust and in DBS is considered diagnostic if followed by molecular testing. Future methods to measure TPP1 activity via MS/MS may improve DBS-based TPP1 testing sensitivity allowing also future newborn screening. Discussion: To confirm clinical suspicion of CLN2, the recommended gold standard for laboratory diagnosis is demonstrating deficient TPP1 activity and detecting causative mutations in each allele of TPP1/CLN2 gene. Conflict of Interest declared.

P-487

A rare lysosomal storage disease: neuronal ceroid lipofuscinosis type 14 Zubarioglu T 1, Yesil G 4, Kiykim E 1, Cansever M S 2, Aktuglu-Zeybek A C 1, Yalcinkaya C 3 1 Div Lab, Med Fac,

Nutr Metab Dis, Ist Univ Cer Med Fac, Istanbul, Turkey, 2Cent Ist Univ Cer Med Fac, Istanbul, Turkey, 3Div Neur, Ist Univ Cer Fac, Istanbul, Turkey, 4Div Med Genet, Bezm Vak Univ Med Istanbul, Turkey

P-486

Expert recommendations for the laboratory diagnosis of neuronal ceroid lipofuscinosis type 2 (CLN2 disease): diagnostic algorithm and best practice guidelines for a timely diagnosis Izzo E 1, AlSayed M 2, Burke D G 3, Cohen-Pfeffer J 1, Cooper J D 4, Dvorakova L 5, Fietz M 6, Giugliani R 7, Jahnova H 5, Lukacs Z 8, Mole S 9 , Noher de Halac I 10, Pearce D 11, Schulz A 12, Specchio N 13, Xin W 14, Miller N J 1 1 BioMarin Pharmaceutical Inc, Novato, CA, United States, 2Dep Med Gen, Alfaisal Univ King Faisal H, Riyadh, Saudi Arabia, 3Chemical Pathology, Camelia Botnar Lab,, London, United Kingdom, 4Inst of Psychiatry, Psychology, and Neur, London, United Kingdom, 5 Inst of Inherited Metabolic Disorders, F, Prague, Czech Republic, 6Dep of Diagnostic Genomics, PathWest Lab, Nedlands, Australia, 7Servico de Genetica Medica HCPA, Dep de, Porto Alegre, RS, Brazil, 8Newborn Screening and Metabolic Diagnost, Hamburg, Germany, 9Molecular Cell Biology, MRC Lab for Mol, London, United Kingdom, 10Univ Nacional de Cordoba, Fac de Ciencia, Cordoba, Argentina, 11Sanford Children Health Res Center, Sioux Falls, United States, 12Dep of Pediatrics, Univ Medical Center H, Hamburg, Germany, 13Dep of Neuroscience, Bambino Gesu Childr, Rome, Italy, 14 Neurogenetics DNA Diagnostic Lab, Massac, Boston, United States

Background: Neuronal ceroid lipofuscinoses (NCLs), a heterogeneous group of lysosomal storage disorders, include the rare autosomal recessive neurodegenerative disorder CLN2 disease (CLN2). CLN2 is due to mutations in TPP1/CLN2 gene causing tripeptidyl-peptidase-1 (TPP1) enzyme deficiency.

Background: The neuronal ceroid lipofuscinoses (CLN) are a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by the intracellular accumulation of lipopigment storage material in lysosomes. Mutations in the KCTD7 gene are very rare and cause a severe neurodegenerative phenotype characterized by intractable myoclonic seizures before 2 years of age and developmental regression. Case report: Two siblings who were 8 and 24 years old respectively were admitted to our outpatients’ clinic with complaints of intractable seizures. They achieved appropriate developmental stages until the age of 12 months. Following the onset of intractable myoclonic seizures between 16 and 24 months of age, neurologic regression started. The 24 years old patient’s physical examination revealed mental retardation, dysarthria ad truncal ataxia. Other sibling was severely neuromotor retarded and he could not sit without support. Diagnostic investigations including complete blood count, liver and kidney function tests, blood lactate and ammonia levels, acylcarnitine profile by tandem mass spectrometry, blood quantitative amino acid chromatography, urine organic acid analysis were normal. Cerebrocerebellar atrophy was noted in brain MRI of the patients. Results: Whole exome sequencing identified the c.818A > T p.Asn273 Ile homozygote mutation in KCTD7 gene and patients were diagnosed as NCL type 14. Discussion: Neuronal ceroid lipofuscinoses are one of the most frequent neurodegenerative disorders in childhood period but especially the rare forms as NCL type 14 could be lately recognized because of diagnostic difficulties. Diagnosis should be considered in patienst with therapy resistant epilepsy, cerebellar atrophy and neurodevelopmental regression.

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c.1288G > A (p.Glu430Lys). The second allele carried a new exonic variant causing an amino acid substitution in a highly conserved protein region, c.368G > A (p.Gly123Glu). This variant was not present in either parent. Paternity was confirmed with further investigations. The de novo variant could have occurred during early embryogenesis or in paternal germline cells. Despite the severe enzyme defect the patient was CRIM positive. The patient showed a good response to ERT but at sixth months a pulmonary infection resulted in further worsening of the cardiomyopathy and hypotonia. We decided to increase the Myozyme dose to 40 mg/kg/week. In 3 weeks the patient’s cardiomyopathy stabilized with the subsequent reappearance of spontaneous limbs motility and tone. Discussion: This case underlines how difficult it can be to predict future clinical presentation and outcomes in patients with mutations which are not as yet clearly defined or described.

Mesenteric lymphadenopathy and hepatic gaucheroma in algerian GD children Hadji A 1, Benali Khoudja N 1, Sokhal S 1, Belbouab R 1, Hassoun F 1, Boukari R 1 1

Div Ped Dis, Univ Med, Algiers, Algeria

Background: Gaucher disease (GD) is a lysosomal storage disorder divided phenotypically into non-neuronopathic, type 1 and neuronopathic, type 2 and 3. There are few reports of mesenteric lymphadenopathy and hepatic gaucheroma in children. Gaucheroma is a rare complication in GD. Case report: We report 6 algerian GD children from 5 families of consanguineous marriage who developed mesenteric lymphadenopathy and in one case an hepatic gaucheroma despite receiving enzyme replacement therapy. Results: Diagnosis of GD was confirmed by decreased leukocyte βglucocerebrosidase activity and molecular study was done with full sequencing of the glucocerebrosidase gene. Two children had type 1 GD (one N370S/L444P and one unknown genotype) and 4 type 3 GD (L444P/L444P) with age ranging from 3 to 14 years. All patients were started on imiglucerase at a dose of 60 U/kg/2 weeks with routine laboratory every 3 months and sonographic follow up every year. Ultrasound of the abdomen showed mesenteric lymphadenopathy in all of them and in one case hepatomegaly with two hyperechoic mass. Liver magnetic resonance imaging (MRI) demonstrated two well delineated mass. Following enzyme replacement therapy (ERT), many of the manifestations of the condition are ameliorated, but persistent mesenteric lymphadenopathy and gaucheroma has been reported. Discussion: Infiltration of lymph nodes with Gaucher cells is one of the complications of the disease with both types 1 and 3 GD, but gaucheroma is very rare, it can mimic malignancy and need to be looked for in the pediatric age group. Studies showed that the response of lymphadenopathy and gaucheroma to treatment is not known.

P-489

GAA de novo mutation in Infantile Pompe disease Fiumara A 1, Arena A C 1, Raudino F 1, Balistreri M C 1, Del Campo G 1, Catarzi S 2, Morrone A 2 3 1 CRRMET, Pediatric Clinic University, Catania, Italy, 2Ped Neurol Unit and Lab Meyer,Child Hosp, Florence, Italy, 3Dep NEUROFARBA, University of Florence, florence, Italy

Background: Pompe disease (GSDII), is an autosomal recessive disorder caused by a deficiency of the enzyme alpha-glucosidase (GAA). De novo mutations occur much more frequently in autosomal dominant disorders than in autosomal recessive disorders. We report an infantile onset GSDII with a newly identified maternally inherited mutation and a de novo uncommon variant. Case report: The male patient is the second child of non consanguineous healthy parents. At birth echocardiography revealed hypertrophic cardiomyopathy. Physical examination showed hypotonia and hyporeflexia. When he was 3 months old and presented worsening hypertrophic cardiomyopathy and hypotonia. No hepatomegaly was present. Chemical investigations showed increased serum GOT, GPT, CK, LDH. GAA activity on DBS was absent. Enzyme replacement therapy (ERT) was started. Results: Molecular analysis of the GAA gene revealed that the maternally derived allele carried a new deleterious splicing mutation c.6932 A > C (IVS3-2 A > C) and an uncommon exonic variant,

P-490

A cost-effective case finding study in Fabry Disease Dursun O 1, Bulbul S F 1 1

Kirikkale Univ, Div Metab Dis,, Kirikkale, Turkey

Background: Identification of Fabry Disease (FD) patients is usually difficult as the changing nature of the disease and the low awareness of the physicians. Though, therapeutic outcomes with enzyme replacement therapy are more favourable in early stages, the diagnosis is often delayed. Thus, there are numarious studies on screening programmes and/or case-finding studies among the high-risk patient groups. This study aims to explore a cost-effective case finding study on FD. Methods: The study was conducted at the waiting halls of four (Internal Medicine, dermatology, neurology, cardiology) outpatient clinics. Patient waiting for examination and accepted to attend the study were asked to fill the preprepared questionnaire. The questionnaire was based on The Mayo Clinic’s Fabry Disease Testing Algorithm and the Faby Disease Practise Guidelines of Laney et al. The selection criteria for further analyses (mutation and enzyme analysis) were; a family history of a renal and/or cardiac disease additional to two symptoms of FD. Results: 681 blood samples were collected out of 5160 patients. Five index FD patients were identified both by mutation and enzyme analyses. Family screening of these cases identified ten additional cases (total 15 FD cases). Heat/cold intolerance (61,5 %), exercise intolerance (50,8 %), acroparethesia (49,8 %) and gastrointestinal problems (49,2 %) were the most prevalent symptoms in the study group, whereas in FD patients, acroparethesia (%100), impaired sweating (%80), gastrointestinal problems (%80) were more prevalent. The family history revealed the existence of renal disease, heart disease and both in 4, 3 and 2 index cases respectively. Discussion: Our study seems to be the most cost effective and selective case-finding study for identifiying the FD patients. Those patients with FD symptoms should be asked for having a family member with hearth or renal disease, in order not to skip the diagnosis. Conflict of Interest declared.

P-491

Clinical presentation and Molecular characterization of Children with Neuronal ceroid lipofuscinosis (NCL I & II) from India Bhavsar R 1, Mistri M 1, Kamate M 2, Shah R 3, Mehta S 4, Shah H 5, Sheth F 1, Sheth J 1

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Institute of Human Genetics, FRIGE House, Ahmedabad, India, 2Child Development Centre, KLES Hospital, Karnataka, India, 3Ankur Neonatal Nursery, Ahmedabad, India, 4Usha Deep Neurology Hospital, Ahmedabad, India, 5Rajvee Child Neurology Hospital, Ahmedabad, India Background: Neuronal Ceroid Lipofusionosis (NCLs) are a group of rare neurodegenerative disorders with autosomal recessive inheritance. Of two common forms, Infantile NCL (INCL) with deficient activity of the enzyme palmitoyl-protein thioesterase 1 (PPT1) occurs due to mutation in PPT1 gene (CLN1); while late infantile NCL (LINCL) with deficient activity of Tripeptidyl Peptidase (TPP1) enzyme occurs due to mutations in TPP1 gene (CLN2). Limited study on clinical aspects and no study on molecular aspects is available from India. Methods: Present study consists of 14 children presented at the age of 3 months to 7 years with impaired motor function, cerebellar and cerebral atrophy, progressive vision loss and seizures. All children were processed for enzyme analysis (PPT1 and TPP1) followed by bi-directional sequencing of PPT1 and TPP1 gene. Results: Based on lysosomal enzymes study from leucocytes seven children were diagnosed with INCL due to deficient activity of PPT1 and seven with LINCL due to deficient activity of TPP1. Bidirectional Sanger sequencing of TPP1 gene revealed 8 pathogenic variants: 4 homozygous missense mutations (2 known-p.V181M, p.N110S and 2 novel-p.V236E, p.C45R) each in one children, 1 homozygous novel missense mutation (p.P238L) in 2 children and 1 compound heterozygous with known and novel allele (p.W186X and E178Nfs*13 respectively) in 1 child. Sequencing of TPP1 gene in 7 children identified seven homoallelic mutations (5 known and 2 novel). Known pathogenic mutation p.R206C observed in 3 children from Gujarat, novel mutation p.Y459S was seen in 2 children from Karnataka and remaining 2 included known pathogenic p.R208X and p.Fs516* homozygous mutant allele. Discussion: Our study demonstrates that children with NCL1 are more likely to have novel mutation while those with NCL2 from Gujarati ethnicity seems to have founder mutation p.R206C and from Southern ethnicity seems to have p.Y459S.

P-492

Pathophysiology of M. Niemann-Pick Type C revisited: Altered Protein Trafficking is Mutation-specific Shammas H 1 2, Kuech E M 1, Das A M 2, Naim H Y 1 1 Dept Phys Chem, Univ Vet Med, Hannover, Germany, 2Dept PED, MHH, Hannover, Germany

Background: Niemann-Pick Type C disease is an inherited neurovisceral lysosomal storage disease characterized by accumulation of cholesterol and sphingolipids in the lysosomes/late endosomes. In 95 % of patients it is due to mutations in the gene encoding NPC1. There is wide variety in the severity of clinical symptoms and in the response to N-butyldeoxynojirimycin (miglustat)-treatment, considered as a substrate reduction therapy by blocking sphingolipid glycosylation. In this study, we investigated the effect of different mutations on NPC1 localization and trafficking of the mutated protein. Methods: A cDNA construct was generated harbouring target amino acid changes, using site-directed mutagenesis PCR. Expression, post translational modification and cellular localization were analysed in overexpressing COS-1 cells. Results: We could classify the mutations into three groups. G1162V, N1156S, L1244P, M1142T, Q774P, R404Q and H510P -mutated proteins showed sensitivity to endoglycosidase H treatment, indicating that they are not complex-glycosylated and thus not trafficked beyond the ER. Confocal microscopy proved localisation of these proteins in the ER. M631R, G1162A and D874V mutated proteins showed the mannose-rich and the complex-glycosylated form of the protein, implicating that the protein is blocked in the ER and

partially trafficked to the lysosomes. This was confirmed by colocalisation with Lamp2. Interestingly, the L1213F, P1007A and D948Y -mutated proteins showed a biochemical pattern similar to the WT and colocalisation with Lamp2 was shown. This suggests that this mutation does not affect the localisation but the function of the protein. Discussion: We showed that the trafficking of the NPC1 mutants along the secretory pathway and their biological activities differs depending on the mutation, which may explain differences in response to miglustat. A mutation-based personalized approach may be necessary when considering substrate reduction therapy by miglustat.

P-493

Intracranial Hypertension in pediatric patients with Cystinosis Del Toro M 1, Martin-Begue N 3, Felipe A 1, Lara E 2, Arranz J A 4, Ariceta G 2 1 Ped Neurol Unit, Hosp Vall d’Hebron, Barcelona, Spain, 2Ped Nephrol Unit,Hosp Vall d’Hebron, Barcelona, Spain, 3Dep Ophthalmology, Hosp Vall d’Hebron, Barcelona, Spain, 4 Metab Lab,Hosp Vall d’Hebron, Barcelona, Spain

Background: Cystinosis is a lysosomal disease due to a deficient transport of cystine out of lysosomes. The accumulation of cystine inside the lysosomes causes damage in different organs and tissues, particularly kidneys and eyes. Neuro-ophthalmologic manifestations are considered a rare and late complication in these patients. We report the association of intracranial hypertension in children with cystinosis in our center. Methods: Retrospective review of the clinical history of eight children with cystinosis followed during the last 5 years. Results: Three girls and five boys, mean age: 9.6 years (range: 5– 14 years) were studied. During follow-up, 4 out of 8 developed papilledema. One of the patients complained of headache and visual loss whereas the other three were all asymptomatic and diagnosed by scheduled funduscopy. MRI in the symptomatic patient showed an Arnold-Chiari anomaly with enlarged ventricles. In the other 3 patients MRI was normal. Cranial hypertension was confirmed by CSF pressure measured by lumbar puncture o intraparenquimal catheter. All four patients had at least one known risk factor for developing intracranial hypertension added to their disease. Two of them required a ventriculoperitoneal shunt and one of them has a residual severe visual defect. Discussion: Intracranial hypertension can develop in patients with cystinosis and lead to visual impairment if it’s not diagnosed and treated early. In most cases it can be assymptomatic so a multidisciplinary approach is necessary with periodic eye examinations in all pediatric patients with cystinosis whether or not they present symptoms.

P-494

New method for molecular genetic diagnosis of glycogen storage disease in Russian patients using next-generation sequencing (NGS). Savostyanov K V 1, Pushkov A A 1, Surkov A N 1, Polyakova S I 2, Nikitin A G 1, Namazova-Baranova L S 1, Baranov A A 1 1 Scientific Center of Children’s Health, Moscow, Russian Federation, 2Inst of Emerg Child Surgery and Trauma, Moscow, Russian Federation

Background: Glycogen storage disease (GSD) is a group of inherited diseases characterized by impared carbohydrate metabolism and manifested by hepatomegaly, hypoglycemia, hyperlactatemia, hyperlipidemia and hyperuricemia. In total, to date, 15 types of glycogen disease are known. The aim of our work was to identify the causes of GSD in 48 children by examining 22 gene using NGS.

S204 Methods: Most of the patients at the time of inclusion in the study between the ages of 6 months to 12 years (31 boys, 17 girls) were clinically diagnosed GSD without specifying the type. The coding and adjacent intronic region genes G6PC, SLC37A4, GAA, AGL, GBE1, PYGM, PYGL, PFKM, PHKA2, PHKB, PHKG2, PHKA1, PGAM2, LDHA, ALDOA, ENO3, PGM1, GYG1, GYS2, GYS1, LDHB, LAMP2 were collected in a panel, according to the information for mutations obtained from international databases and were examined by NGS. Results: In 45 patients (94 %) we revealed the mutations which can lead to different type of GSD. It was 11 (24,5 %) patients with mutations in PHKA2 gene, 10 (22 %) in AGL gene, 10 (22 %) in SLC37A4 gene, 5 (11 %) in G6PC gene, 3(7 %) in PYGL gene, 2 (4,5 %) in LAMP2 gene and one (2 %) patients with mutation in GBE1, PFKM, PHKG2 and PHKA1 gene accordingly. In total we revealed 46 different mutations, among them 16 mutations not described earlier, including 3 in PHKA2 gene (c.2077A > G, c.133C > T, c.9192A > G), 6 in AGL gene (c.643G > C, c.1423 + 1G > A, c.3758G > A, c.1735 + 5G > A,c.2390A > G), 3 in PYGL gene (c.841 T > A,c.176C > G/ c.2416A > T), 2 in GBE1 (c.427A > G, c.1583A > G), LAMP2 (c.733C > T, c.893_923del) and SLC37A4 (?.413G > A, c.1016G > T) gene and one in PFKM (c.496C > G) and PHKA1(c.776 T > C) gene accordingly. Discussion: Our method with high accuracy allows molecular genetic diagnosis of 15 different type GSD.

P-495

The activity of chitotriosidase in blood of patients with lysosomal diseases: a 10 years of laboratory experience Tozzi G 1, D’Amico J 1, Dionisi-Vici C 1, Bertini E 1, Piermarini E 1, Petrillo S 1 , Piemonte F 1 1

Children’s Hospital Bambino Gesu, Rome, Italy

Background: Human chitotriosidase is an enzyme activated in tissue macrophages. It belongs to the family of glycosyl hydrolases, and it can be used as a screening marker in lysosomal storage disorders where tissue macrophages activation is due to accumulation of substrate in various organs. It has been demonstrated that chitotriosidase activity is highly increased in the plasma of patients with Gaucher disease; a mild increase in chitotriosidase activity is also found in Niemann-Pick disease and in GM1-gangliosidosis and Krabbe disease. In this study, plasma chitotriosidase activity was evaluated in blood of 67 patients having 16 lysosomal storage diseases. These specimens were from Division of Metabolism, Children’s Hospital Bambino Gesù. Methods: One ml of peripheral blood was collected in a EDTA vacutainer and plasma was separated and preserved at −20 °C. The activity of chitotriosidase was determined measuring the 4-Methylumbelliferone released after 15 min of reaction at a temperature of 37 °C using the fluorigenic substrate 4Methylumbelliferyl-β-D-triacetylchitotrioside; the fluorescence was detected by a fluorometer (λexc = 365, λem = 448). Controls were plasma specimens from 1000 children enrolled for routine diagnostic testing. Results: In 1000 healthy children, the activity of CT was3.7 ± 1.7 nmol/ml/h. The MPS and CLN hadn’t elevated plasma chitotriosidase (7.60 ± 5.5 nmol/ ml/h, p = 0.04); glycoproteinoses and sphingolipidoses showed a significant increased, respectively 58.5 ± 3.5 nmol/ml/h, p < 0.0001, 280 ± 24 nmol/ml/h, p < 0.0001. Discussion: Chitotriosidase activity can be considered as a primary screening test in children with lysosomal storage disorders. Our experience indicates that it would be important to assess chitotriosidase activity in patients with a suspicion of lysosomal storage disorders since it can be useful to assist the clinician in the diagnosis.

P-496

Plasma metabolomic profile in Spanish patients with lysosomal acid lipase deficiency Yahyaoui R 1 2, Rodriguez-Garcia E 1 2, Garcia-Jimenez M C 7, Dayaldasani A , Munoz-Hernandez C 8, Ruiz-Sala P 3 4 5 6, Garcia-Martin M L 8

1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 1

Clin Lab, Malaga Regional Hospital, Malaga, Spain, 2IBIMA, Malaga, Spain, Centro de Diag de Enferm Molec CEDEM, Madrid, Spain, 4UAM, Madrid, Spain, 5 IDIPAZ, Madrid, Spain, 6CIBERER, Madrid, Spain, 7Dep of Pediatrics, Miguel Servet Hosp, Zaragoza, Spain, 8BIONAND, Malaga, Spain 3

Background: Lysosomal acid lipase deficiency (LAL-D) is a rare lysosomal storage disease characterized by progressive liver disease and dyslipidaemia. The aim of this study was to investigate the metabolic profile of LAL-D using a metabolomic approach. Methods: Two 9-year-old patients with LAL-D treated solely with statins were included. The plasma metabolomic profile was analyzed by a novel advanced test based on 1D 1H-NMR spectroscopy using two different sequences for spectral editing: the first, CPMG (Carr-Purcell-Meiboom-Gill), optimized for the detection of small molecules; and the second, diffusion-weighted spectroscopy, optimized for the detection of large molecules, lipids and macromolecules. The plasma total fatty acids quantification (C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:3ω9, C20:3ω6, C20:4, C20:5, C22:0, C22:1, C22:4ω6, C22:5, C24:0, C24:1ω9 and C22:6) was determined derivatizing fatty acids to fatty acid methyl esters and measured by GC/FID. Results: The metabolomic profile showed an increase in the concentration of fatty acids, especially at signals 2.04 and 2.76 ppm, corresponding to the allyl protons (CH-CH=) and bis-allylic protons (=CH-CH2-CH=) of the unsaturated fatty acids. An increase at signals 4.08/4.29 ppm and 5.22 ppm, corresponding, respectively, to the protons of the CH2 and CH groups of glycerol triglycerides, was also observed. Mean plasma total fatty acids concentrations were markedly increased with respect to controls, especially C18:1 (3756 vs 1628 μmol/L) and the polyunsaturated species: C18:2 (4138 vs 2177 μmol/ L), C18:3 (43 vs 19 μmol/L) and C20:4 (939 vs 602 μmol/L). Discussion: A plasma metabolomic profile has been described in LAL-D for the first time. Increased signals mainly correspond to unsaturated fatty acids and a subsequent quantification of total fatty acids has demonstrated these findings. Further studies are needed to understand better the cellular fatty acid metabolism in LAL-D.

P-497

Wolman disease - a misdiagnosis case with hemophagocytic lymphohistiocytosis Caseiro C 1, Silva E 1, Ribeiro I 1, Laranjeira F 1, Pinto E 1, Oliva T 2, Lacerda L 1 1 Centro Genetica Medica-CHP, Porto, Portugal, 2 Instituto Portugues Oncologia, Porto, Portugal

Background: Wolman disease is an autossomal recessive lysosomal storage disease caused by lipase acid deficiency (LAL) responsible for the hydrolysis of cholesteryl esters and triglycerides within lysosomes. LAL is encoded by LIPA gene and its deficiency produces two well defined inborn disorders, Wolman disease (WD) and Cholesteryl ester storage disease (CESD). WD is the more severe form of the disease. In infancy it presents with prominent hepatosplenomegaly, failure to thrive, hypertrigliceridemia, hypercholesterolaemia and adrenal calcifications, with an estimated incidence of 1:500000 live births, with death usually occurring before 1 year of life. CESD is less severe and most commonly diagnosed in childhood or adolescence. Some inborn errors of metabolism have been described with secondary associations with Hemophagocytic Lymphohistiocytosis (HLH). Here we report a WD patient admitted in the oncology service with a clinical HLH diagnosis. Methods: WD diagnosis underlies two approaches: biochemical assay by LAL activity and molecular assay by LIPA gene analysis. Results: This work reports clinical, biochemical and molecular data of a patient diagnosed with HLH. Bone marrow aspirate showed numerous lipid-laden histyocytes that raised the suspicion of WD. Absence of lipase acid activity in DBS (dried blood spot) confirmed WD diagnosis and mutated alleles in LIPA gene corroborate its existence. Discussion: HLH may arise secondary to WD, so metabolic diseases should be considered and screened when this situation is present, otherwise this disease may be underdiagnosed with patients receiving no or incorrect diagnosis. DBS test for LAL is an easily accessible, accurate and low-cost assay applied in non- transfused patients. WD could be a potential diagnosis in patients

S205

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 presenting with signs and symptoms associated with more common diseases like liver and cardiac diseases or HLH, so clinicians should consider the existence of this severe metabolic disease.

P-498

(p.Glu888X) nonsense mutation was the most frequent. In two cases we identified polymorphisms c.1726G > A and c.2065G > A in compoundheterozygous state, described in patients with decreased activity of lysosomal α-1,4-glucosidase and without clinical manifestations of Pompe disease. Discussion: We showed a high percentage of detection Pompe disease among high-risk patients.

Interpreting cognitive function in children with rapid loss of vision - lessons from the early phase of CLN3 disease.

P-500

Kuper W F E 1, Van Genderen M M 2, Van Hasselt P M 1

Global consensus on barriers to early diagnosis of Gaucher disease

1

Mehta A 1, Salek S 2, Kuter D 3

Dept Met Dis, Wilhelmina Child Hosp,UMCU, Utrecht, Netherlands, Bartimeus Inst, Zeist, Netherlands

2

Background: CLN3 disease is a severe neurodegenerative disorder of childhood, classically presenting with rapid visual decline later followed by psychomotor deterioration. Learning difficulties around diagnosis are often attributed to rapid loss of vision. We aimed to separate the impact of rapid visual loss from neurodegeneration. Methods: We performed a retrospective chart review on patients with either confirmed CLN3 or early onset Stargardt disease, a pure retinal disorder. We collected neuropsychological assessments performed around diagnosis, and evaluated clinical history at the time of diagnosis with regards to school performance and behavioral concerns. Results: 18 CLN3 patients and 13 Stargardt patients diagnosed from 1990 to 2014 in the Dutch NCL Expertise Center Bartiméus were included. Age at presentation was at 6.2 years in CLN3 versus 7.0 years in Stargardt. In 11/12 CLN3 patients in which early school history was described, an extra year in kindergarten (4-5y) was decided due to academic underachievement, versus in 0/10 Stargardt patients. Additionally, behavioral problems were reported in 6 CLN3 patients, but not in patients with Stargardt disease. All Stargardt patients pursued regular education successfully and the 3 Stargardt patients who underwent neuropsychological assessment had IQs above 100. In the 7 patients with CLN3 IQ around diagnosis (age 5–8 years) was decreased (median 73, range 57 = 91), and correlated with age (r = 0.7114, p0.02). Qualitative descriptions suggested that CLN3 patients scored particularly poor on subtests regarding word finding (noted in 5) and short term memory span (noted in 3). Discussion: Early learning and behavioral difficulties in patients with CLN3 disease around diagnosis cannot be attributed to rapid loss of vision.

P-499

1 Royal Free Hosp, UCL School of Medic, London, United Kingdom, 2School of Life and Med Sci Univ of Herts, Hatfield, United Kingdom, 3Center for Hematology, MA Gen Hosp, Boston, United States

Background: Diagnostic delay in patients with Gaucher disease (GD) is common and can lead to disease complications, increased patient morbidity and reduced quality of life. Evidence indicates that the heterogeneous nature of GD impedes differential diagnosis. As part of a broader consensus initiative to define a diagnostic algorithm for the detection of GD, we aimed to determine the most important barriers to early diagnosis in clinical practice. Methods: As part of the anonymous, multi-stage, iterative Delphi process, a panel of 22 expert physicians, from 16 countries, were asked to provide freetext answers to a series of open questions, including: ‘In your experience, what are the greatest barriers to diagnosis in patients with early GD?’ In round 1, an independent facilitator categorized responses as themes, and created a set of summary statements. In round 2, the panel rated the importance of statements using a 5-point Likert scale (1 = not important to 5 = extremely important). To achieve consensus, statements rated most highly (defined as >75 % of respondents assigning a statement an importance score of ≥3) were reissued to the panel in round 3 for rating of their agreement on a 5-point Likert scale (strongly agree; agree; neither agree nor disagree; disagree; strongly disagree). Results: The panel (95 % response rate) provided 30 phrases. These were grouped into nine themes, of which 6 were rated most highly (e.g. “Early signs can be mild and may be overlooked”). Discussion: The literature suggests that the main barrier to early diagnosis is the heterogeneous nature of the disease; however, research also indicates that GD is not commonly considered by many consulting physicians, even in the presence of typical signs and symptoms. Expert consensus on the greatest barriers to diagnosing GD early will be established in this international Delphi initiative which could potentially lead to reduction in misdiagnosis of this rare but disabling condition. Conflict of Interest declared.

Selective screening for Pompe disease in high-risk Russian patients. Savostyanov K V 1, Pushkov A A 1, Basargina E N 1, Zhurkova N V 1, Vashakhmadze N D 1, Namazova-Baranova L S 1, Baranov A A 1

P-501

1

Early diagnosis in Gaucher disease: findings from a global consensus initiative

Scientific Center of Children’s Health, Moscow, Russian Federation

Background: Pompe disease (type II glycogen storage disease) is an autosomal recessive disorder. It is caused by mutations in the gene encoding lysosomal acid alpha-1,4-glucosidase (GAA). Depending on the level of residual GAA activity Pompe disease can manifest with different disease phenotypes from severe early onset form to milder late-onset. Methods: We screened more than 2500 DBS samples from high risk population patients (presenting with cardiomyopathy, muscular hypotonia and other cardinal features) received from various medical organizations of the country. The α-1,4glucosidase activity was measured in all samples using tandem MS/MS. 18 samples with GAA activity below the cut-off value (1.62 micromoles/liter/hour) and normal levels of reference enzyme activity were sequenced by Sanger method. Results: The study identified 16 patients with homozygous or compound heterozygous mutations in the GAA gene. Among identified mutations 6 were novel: four missense c.1292 T > C (p.Leu431Pro), c.2853G > T (p.Trp951Cys), c.1198G > A (p.Val400Ile), c.1448G > A (p.Gly483Glu), one nonsense: c.1961C > G (p.Ser654X), and one small deletion c.1030_1039del (p.Gly344Profs*45). Among other identified mutations, the c.2662G > T

Mehta A 1, Salek S 2, Kuter D 3 1

Royal Free Hosp, UCL School of Medic, London, United Kingdom, 2School of Life and Med Sci Univ of Herts, Hatfield, United Kingdom, 3Center for Hematology, MA Gen Hosp, Boston, United States Background: Gaucher disease (GD) has a broad spectrum of signs and symptoms, with phenotypes ranging from fatal perinatal to asymptomatic adult forms. Given the rarity and the heterogeneous nature of the disease, diagnosis is commonly delayed or missed. In this global consensus initiative, we aimed to define a point scoring system (PSS) to assist with diagnosis of GD early in the disease course. Methods: In an anonymous, multi-stage, iterative Delphi process, a panel of 22 expert physicians, from 16 countries, were asked to provide free-text answers to a series of open questions, including: ‘Which unexplained signs and covariables may be important to consider in early type 1 GD?’. An independent facilitator categorized responses from round 1 into themes, and created a set of

S206 summary factors. In round 2, the panel rated the importance of factors using a 5-point Likert scale (1 = not important to 5 = extremely important). To achieve consensus, factors rated most highly (defined as >75 % of respondents assigning a factor an importance score of ≥3) were reissued to the panel in round 3 for rating of their agreement on a 5-point Likert scale (strongly agree; agree; neither agree nor disagree; disagree; strongly disagree). Results: The panel (95 % response rate) provided 101 phrases, consolidated as 38 themes. These were grouped into 21 factors, of which 10 were rated most highly (including splenomegaly, anaemia, hepatomegaly, thrombocytopenia, family history of Gaucher disease). Those rated most highly were grouped as potential major factors, and the remaining ones as potential minor factors. In each group, factors were ranked by importance and taken forwards for consensus in round 3. Consensus on a final PSS will be presented. Discussion: Published algorithms for GD diagnosis are rare. Here, for the first time, we present a PSS to assist with the early diagnosis of GD, based on an international, multidisciplinary expert consensus. Conflict of Interest declared.

P-502

Alpha Glucosidase on Dried Blood Spot: simple assay in Pompe Disease Sacchini M 1, Procopio E 1, Pasquini E 1, Pochiero F 1, Daniotti M 1, Ombrone D 2, La Marca G 2 4, Catarzi S 2, Morrone A 2 3, Donati M A 1 1 Metab and Musc Unit, Meyer Child Hosp, Florence, Italy, 2Ped Neurol Unit and Lab Meyer Child Hosp, Florence, Italy, 3Dept of NEUROFARBA, Univ Florence, Florence, Italy, 4Dept of Exper and Biomed Scienc, Uni Flo, Florence, Italy

Background: Infantile-onset Pompe disease (IOPD) presents with hypotonia, muscle weakness, motor delay, cardiomyopathy, feeding problems and respiratory insufficiency. Early diagnosis is important to start early EnzymeReplacement-Therapy (ERT). Methods: Alpha-glucosidase (GAA) enzyme assay on dried blood spots (DBS) allows a diagnosis of Pompe disease (PD) simpler and faster. GAA assay on DBS is stable at room temperature. DBS is easy to deliver. It’s a quick and cheap test, requiring only a blood spot. Results: In last 7 years we identified 12 children affected by PD assaying GAA on DBS in tandem-mass spectrometry: 3 were diagnosed by neonatal screening. 9/12 presented classic IOPD, 1 presented non-classic IOPD, 2 presented late-Onset PD (LOPD). At diagnosis 10 showed cardiomyopathy. 8/10 were diagnosed in the first month of life and 2 of these presented respiratory distress. 4 patients diagnosed in the infantile age presented hypertrophic cardiomyopathy and hypotonia. All patients had absent GAA activity on DBS, ranging 0–0.2 micromol/L/h (n.v.2.31– 27.4), confirmed low in lymphocytes (0–0.8 nmol/mg/h, n.v.11–32). We obtained diagnoses confirmation by GAA gene molecular analysis. We have on follow up 8/12: they started ERT at standard dose of 20 mg/Kg every 2 week. All these patients presented a progressive improvement of cardiomyopathy. Discussion: The individual response to ERT has been different due to CRIM-status, development of rhGAA specific antibodies, age of presentation and age of diagnosis, according with literature. PD’s early diagnosis is important to start early ERT that has been shown to be more effective in IOPD and LOPD patient treatment: GAA assay on DBS is reliable, non-invasive, sensitive and specific in newborn and child with PD signs or symptoms. Conflict of Interest declared.

P-503

Evaluation of different approaches to Lysosomal Acid Lipase Deficiency screening Cebolla J J 1 4 5, Irun P 1 3, Pocovi M 1 4, Giraldo P 1 2 3 5 1 Instituto Investigacion Sanitaria Aragon, Zaragoza, Spain, 2 Instituto Aragones Ciencias Salud, Zaragoza, Spain, 3CIBERER, Instituto de Salud Carlos III, Zaragoza, Spain, 4Dept Bioquimica, Universidad de Zaragoza, Zaragoza, Spain, 5FEETEG, Zaragoza, Spain

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Lysosomal acid lipase deficiency (LALD) leads to accumulation of cholesteryl esters and/or triglycerides (TG) in lysosomes. Most common f i n di n g s a r e h y pe r l i p i de m i a , h y p o a l ph a l i p op r o t e i n e mi a a n d hypertransaminasemia. LALD may be misleading with other lysosomal storage disorders (LSD) due to sharing manifestations such as visceromegalies, elevation of LSD biomarkers [Chitotriosidase activity (ChT), CCL18/PARC concentration] etc. Alteration of lipid/liver profile (LLP) and/or histopathological findings are applying as criteria for LALD screening, however biomarkers are not being evaluated. The purpose was evaluate the utility of ChT and CCL18/PARC biomarkers in addition to LLP, in order to identify LALD patients. Methods: Collection 1 [Primary hypercholesterolemia suspicion (n = 2040)] was refined and included 79 individuals (Set 1) with elevated Total Cholesterol (TChol) and no mutations in LDLR, APOB and PCSK9 genes. Collection 2 [LSD suspicion (n = 1295)] was refined and included 64 individuals (Set 2) with elevated ChT activity and/or CCL18/PARC concentration. TChol, HDLChol, LDL-Chol, TG concentration, ALT and AST activity were assessed in both sets by automatic analyser. Plasma LSD biomarkers were evaluated by fluorometric assay (ChT) and ELISA (CCL18/PARC). Leukocyte LAL activity was measured using fluorescence based enzyme assay with slightly modifications on previously published. Probands with anomalous LLP and/or biomarkers, underwent LIPA gene sequencing. Results: 8 subjects from Set 1 and 20 from Set 2 showed altered LLP and/or biomarkers and/or null/reduced LAL activity. LIPA sequencing identified in 3 subjects (Set 2) 1 heterozygous novel mutation in promoter region, 1 homozygous p.delS275_Q298 and 1 complex heterozygous. In Set 1 we had not identified any mutation. Discussion: The measurement of plasma ChT and CCL18/PARC, combined with LLP is an useful approach to identify LALD patients who should undergo testing for enzymatic/genetic diagnostic.

P-504

Dried blood spot screening of Lysosomal acid lipase deficiency (LALD) and confirmatory studies in Spanish LALD suspected patients Cebolla J J 1 4 5, Irun P 1 3, Gonzalez-Dieguez L 6, Del Valle Loarte P 7, BarbaRomero M A 8, Garcia-Jimenez I 9, Ros Arnal I 9, Ortega Gil D 10, Tomasini R 11 , Giraldo P 1 2 3 5 1 Instituto Investigacion Sanitaria Aragon, Zaragoza, Spain, 2 Instituto Aragones Ciencias Salud, Zaragoza, Spain, 3CIBERER, Instituto de Salud Carlos III, Zaragoza, Spain, 4Dept Bioquimica, Universidad de Zaragoza, Zaragoza, Spain, 5FEETEG, Zaragoza, Spain, 6Hosp Univ Central de Asturias, Oviedo, Spain, 7Hosp Univ Severo Ochoa, Leganes, Spain, 8Hosp Gral Univ de Albacete, Albacete, Spain, 9Hosp Univ Materno-Infantil Miguel Servet, Zaragoza, Spain, 10Hosp Univ Virgen de la Arrixaca, Murcia, Spain, 11 Hosp Univ Mutua de Terrasa, Terrasa, Spain

Background: Lysosomal acid lipase deficiency (LALD) is lysosomal storage disorder, triggering accumulation of cholesteryl esters and/or triglycerides. Dyslipidemia, liver alterations, cardiovascular disease and gastrointestinal disturbances (GID) are prevalent. Screening is performed from dried blood spots samples (DBS), however confirmatory studies must be mandatory to assess LALD status. The purposes were performedDBS screening in LALD clinically compatible probands; to identify potentially affected; to confirm LALD status through characterization of biomarkers and LIPA gene mutations in those and to accomplish family studies. Methods: Probands were recruited according to clinical findings. LAL activity were assessed in DBS in all probands using a fluorescence based enzyme assay with slightly modifications; Null/almost null activity were considered LALD potentially patients and required deeper studies. LALD status confirmation was carried out by measuring plasma biomarkers (Chitotriosidase activity, CCL18/PARC and 7-Ketocholesterol concentration) and by LIPA gene sequencing. Results: 134 subjects were referred: 79 non-overweighed pediatrics (PED), hepatomegaly (62.3 %) and GID (48.0 %) are the most common findings; 55 non-overweighed adults (AD), steatosis (62.5 %) and splenomegaly (40.5 %) are prevalent. LAL DBS [median(IQR) nmol/punch/h] in pediatrics

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284

P-505

ALAT, which initially remained normal. Ferritin and ASAT were still elevated at the moment of writing, whereas ALAT and chitotriosidase increased and seem to reach a steady state. Ferritin, ASAT and ALAT seemed to best predict need for ICU. Urinary DCA were positively correlated with LDH, ferritine and CRP; and chitotriosidase with ALAT. Discussion: High urinary DCA were described earlier as artifact from plastic urine containers. The clear elevation, treatment response and relation to other biomarkers indicate that these DCA are also biomarkers of Wolman disease. It is unknown if DCA are related to increased synthesis or defective breakdown, because of (sub) cellular damage from neutral-lipid accumulation. In our patient, ferritin and ASAT seem to correlate best to clinical response. The chitotriosidase increase is puzzling and may reflect macrophage activation. Also in relation with ALAT, it is yet unknown if it is a matter of concern.

Familial analysis of clinical consequences of type I gaucher disease

P-507

were 0.97(0.56–1.23) whereas 0.66(0.31–0.97) in adults; 7 PED and 4 AD showed activity under 0.05 nmol/punch/h. In those, biomarkers levels were higher than cut-off values and LIPA sequencing identified 7 p.delS275_Q298 (E8SJM) homozygous, 3 p.H129R homozygous and 1 complex heterozygous for E8SJM and novel nonsense mutation. Family studies identified 10 carriers and 2 affected. Discussion:These results support the efficiency of this screening approach, allowing us to identify 11 LALD patients. Furthermore, confirmatory studies assess biomarkers and mutations, getting identify 2 patients more and orientate family studies.

Mahmood U 3, Cheema H A 2, Suleman M 2, Bukhari S 3, Mahmood S 1 Limb-girdle muscular dystrophy mimicking Pompe disease 1

University of Health Sciences, Lahore, Pakistan, 2The Childrens Hospital and ICH, Lahore, Pakistan, 3GC University, Faisalabad, Pakistan Background: Gaucher disease is a rare, autosomally inherited, metabolic disorder which affects an individual’s ability to properly metabolize glucocerebroside into glucose and ceramide due to deficiency of βglucocerebroidase. It occurs with a frequency of ≈ 1:50,000 liveborn infants. Case report: Fifty cases have been characterized with hepatosplenomegaly, pancytopenia, and manifestations of bone marrow infiltration by characteristic Gaucher cells in their childhood together with other features including ocular pingueculae, or nodules and dermal hyperpigmentation. These cases showed a wide spectrum of clinical severity. There was no neurologic involvement in these patrients with type I Gaucher disease. Results: Familial pedigrees indicated autosomal recessive inheritance and clinical or radiographic evidence of bone disease occured in 70 %–100 % of the individuals with type 1 GD. Bone disease ranged from asymptomatic osteopenia to focal lytic or sclerotic lesions and osteonecrosis. Discussion: In Pakistan, there is no reported data available for type I Gaucher disease and routine newborn screening for the disease is not practiced at all. GD can be treated by enzyme replacement therapy/ partial or total splenctomy. Furthermore, genotyping of patients with type I Gaucher disease will help in assessing genotype phenotype correlation.

P-506

Urine dicarboxylic acids and other biomarkers for diagnosis and followup of Wolman disease Heiner-Fokkema M R 1, Niezen-Koning K E 1, Van der Sluijs F J 1, Bontekoe A B 1, Janssens-Puister J 1, De Koning T J 2, Derks T G J 2, Van der Doef H P J 2 , Van Spronsen F J 2 1 Univ Med Center Groningen, Dept Lab Med, Groningen, Netherlands, 2Uni Med Ctr Groningen, Beatrix Child Hos, Groningen, Netherlands

Background: A 2-month old boy presented with fever, abdominal pain, hepatosplenomegaly and calcifications of adrenal glands. Wolman disease was expected and confirmed by deficient lysosomal acid lipase activity (24 (3 %), ref 287–855 nmol/h/mg protein). Urine analysis revealed high excretions of three dicarboxylic acids (DCA), i.e. pimelic, suberic and azelaic acids (and their carnitine esters). We evaluated the usefulness of urinary DCA as biomarker for Wolman disease and compared them with other biochemical markers measured during treatment with Sebelipase alpha (Alexion). Methods: Urinary organic acids (17 samples) were measured by GC-MS. Plasma chitotriosidase activity (24 samples) was measured with a fluorimetric assay, using 4-methylumbelliferyl-ß-D-N,N’N”-triacetylchitotriose as substrate. Results: Ferritin, CRP, triglycerides, LDH, ASAT, chitotriosidase and (urinary) DCA were clearly elevated with slightly elevated cholesterol and normal ALAT prior to treatment. All biomarkers decreased upon treatment except

Zakharova E Y 1, Baydakova G V 1 1

Research Centre for Medical Genetics, Moscow, Russian Federation

Background: Limb girdle muscular dystrophies (LGMD) are heterogeneous hereditary neuromuscular disorders. More than 21 autosomal dominant and recessive subtypes are reported. Clinical presentation of adult forms are similar to Pompe disease. Case report: Patient G. is a 46-years-old female presented with a 26-years disease progression that started with limb weakness. During last 16 years she presents the weakness of the respiratory muscles. Blood spot test for Pompe disease was performed. Alpha-glucosidase activity was 0.53 μmol/l (reference: > 1.5). Gene sequencing GAA - heterozygous variant (c.1198G > A p.V400I). This variant has not been describe in HGMD. Based on this laboratory and clinical data diagnosis of Pompe disease was suspected, but additional investigations were recommended. Enzyme assays in several distinct laboratories did not confirm the first finding and the value of alpha-glucosidase activity was slightly decreased or normal (leukocytes – 20.6 nM/mg/h, reference: > 13; blood spot – 15.6 nM/ml/h, reference: > 1.0; fibroblasts – 26 nM/mg/h, reference: > 40). Results: Whole exome sequencing (WES) for exclusion of other cases of muscular dystrophy was provided. Two mutations were found in the fukutinrelated protein gene (FKRP): c.T1219G:p.Y407D and c.C826A:p.L276I. The 826C > A nucleotide change was a common mutation in Limb-girdle muscular dystrophy (LGMD2I), the second mutation was not described previously. The LGMD 2I type is caused by mutation in the FKRP and characterized high phenotype diversity—from severe neonatal to extremely mild adult forms. Discussion: There are many similarities between Pompe disease, and limb girdle muscular dystrophies. In case of controversial results of biochemical and DNA testing for Pompe disease additional investigations to exclude other forms of muscular dystrophy should be recommended before starting of ERT.

P-508

Spectrum of mutations and biochemical characteristics of 21 Russian patients with lysosomal lipase deficiency Kamenets E A 1, Baydakova G V 1, Proshlyakova T Y 1, Mikhaylova S V 2, Strokova T V 4, Maevskaya M V 3, Zharkova M S 3, Zakharova E Y 1 1 FSBI Res Cent for Med Genet, Moscow, Russian Federation, 2Dep of Med Genet Chil Clin Hosp, Moscow, Russian Federation, 3First MSMU Clinic of Inter Med Propead, Moscow, Russian Federation, 4FSBIS Fed Res Cent for Nutr and Biotechn, Moscow, Russian Federation

Background: LAL deficiency (LALD) is a rare autosomal recessive lysosomal storage disease (LSD) presenting in 2 forms: Wolman disease (WD) and Cholesteryl Ester Storage Disease (CESD) relatively mutations in LIPA gene. We aimed to detect the spectrum of mutations in pts with LALD and to

S208 describe the secondary LSD markers: chitotriosidase activity and plasma oxysterols (CT and 7-KC) in them. Methods: Chitotriosidase and LAL activity was measured in dried blood spots by fluorometric analysis. In LAL measurement inhibitor Lalistat 2 was used. 21 pts were selected for molecular study. Coding sequences of LIPA were analyzed by direct sequencing. In 7 of these pts CT and 7-KC were analyzed in plasma by MS/MS. Results: In 21 pts LAL activity was undetected. 10 of them (47,6 %) show a 1,5 – 12-fold chitotriosidase activity increase over reference values. In 2 out of 7 plasma samples levels of CT were high and 7-KC concentration 1,3 –8,5-fold increased over reference values. 15 pts have a recurrent mutation c.894G > A in homozygosity (7) or in compound heterozygosity (8) and show CESD symptoms. Another alleles were: c.796G > T (3), c.894 + 1G > A (1) and 5 novel mutations: c.348G > A, c.817_818delAA, c.398C > A, c.348G > A, c.309C > A. In 3 pts with WD nonsense mutations were found: c.348G > A/c.796G > T, c.348G > A/ c.398delC and c.193C > T/c.193C > T. We have also identified 3 patients with biochemically confirmed CESD and without genetic findings. Discussion: We identified 5 novel LIPA mutations and defined mutation c.894G > A is the most frequent, allelic frequency 52,4 % that seems likely in other European populations. Some mutations of LIPA can’t be detected by sequencing, so the first step of diagnostics is the LAL activity assay with specific inhibiter. High chitotriosidase is observed with a probability near of 50 %. With high level of 7-KC it is an accessory sign of LALD. Also slight increase of CT level is conspicuous.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Glycogen storage disease type II or Pompe disease is an autosomal recessive disorder due to mutations in the GAA gene,encoding the lysosomal enzyme acid alpha-1,4-glucosidase. Proximal limb muscle weakness and diaphragmatic involvement are the main clinical features in adults. Dilative arteriopathy involving cerebral and cervical arteries has also been reported, with rare but potentially life-threatening complications. Brain and kidney infarcts have been recently reported in a patient, extending the spectrum of vessels’ involvement in Pompe disease. Case report: We report the case of a 42 years old man who presented at the emergency department at the age of 38 with acute low-back pain. Abdominal Computed Tomography (CT) scan showed a right renal infarct and further investigations led to the diagnosis of renal artery fibrodysplasia. Antiaggregation and anticoagulation therapy was administered. A week later,he presented with hypertensive encephalopathy and a generalised tonic clonic seizure with postictal coma. The cerebral CT scan revealed a right caudate nucleus hematoma and no intracerebral vessels abnormalities. During the follow-up the patient developed overt axial and pelvic girdle weakness, and severe dyspnoea. Thorough inquiry brought to light that exercise intolerance was present since adolescence. Results: Muscle biopsy demonstrating a vacuolar myopathy and reduced GAA enzyme activity on dried blood spot led us to the diagnosis of Pompe disease and enzyme replacement therapy was initiated. Discussion: Pompe disease results in glycogen accumulation not only in skeletal muscles and diaphragm, but also in the media of arteries. Involvement of cerebral vessels is well-known. This case report demonstrates that renal arteries may also be involved, potentially leading to kidney infarct and that haemorrhagic stroke may occur in the absence of intracranial vessels’ malformation. Thus, increased awareness of non-cerebral arteries fibromuscular dysplasia is warranted.

A rare presentation of Gaucher type 2 disease in a neonate Zdraveska N 1, Kostovski A 1

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1

Univ Child Hosp, Medical Faculty, Skopje, The F.Y.R of Macedonia CLN8p is involved in spatial distribution of lysosomes

Background: Gaucher disease type 2 is a rare lysosomal storage disorder with usual onset between 3 and 6 months of age leading to progressive neurodegeneration and death within the first 2 years of life. Very rare Gaucher disease can occur in neonates and is associated with poor prognosis. Case report: Case study of a 3-day old newborn with an extremely rare presentation of Gaucher disease type 2 will be presented with the clinical complications of liver disease occurring in Gaucher disease. Results: The newborn was admitted because of cholestasis associated with severe thrombocytopenia and hepatosplenomegaly since birth. It was third child in the family; the older sibling died at age of 9 months with suspected, but not confirmed Gaucher disease. Consanguinity was not reported. Laboratory analysis showed thrombocytopenia (20 x109/l), elevated conjugated bilirubin (183 μmol/l) and serum transaminases (AST-749 U/l, ALT-331 U/l). All investigations for structural, viral and metabolic causes of cholestatic jaundice were negative. Bone marrow aspirate did not reveal any engorged cells. The diagnosis was made with enzymatic and molecular studies. Sequence analysis of GBA gene revealed two homozygous mutations p.[H294Q];[D448H]. The clinical course was marked by progression of hepatosplenomegaly, severe cholestasis, portal hypertension and ascites, followed by neurological deterioration (hypertonus, feeding difficulties). The patient died at age of 2.5 months because of liver failure. Only symptomatic treatment was provided. Discussion: Although extremely rare neonatal cholestasis can be the first sign of Gaucher disease; thus making the diagnosis very difficult because the focus is on more prevalent neonatal illnesses. Gaucher disease should be considered in the diagnostic approach of infants with cholestatic jaundice even if, initially the bone marrow aspirate do not reveal typical cells.

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Renal arteries’ involvement in Pompe disease: a case report. Pappa E 1, Papadopoulos C 1, Grimbert P 2, Bassez G 1, Laforet P 1 Pitie Salpetriere Hospital, Paris, France, 2Henri Mondor Hospital, Paris, France

1

Pesaola F 1, Quassollo G 2, Remedi M 2, Noher de Halac I 1, Bisbal M 2 1 Inst de Inv en Cs de la Salud - CONICET, Cordoba, Argentina, 2Inst de Inv Medicas M y M F - CONICET, Cordoba, Argentina

Background: CLN8 disease (one of 13 diseases grouped as Neuronal Ceroid Lipofuscinosis, NCL) is caused by mutations in CLN8, which encodes for a putative 286 aa, transmembrane protein CLN8p. This protein shuttles between Endoplasmic Reticulum (ER) and the ERGolgi Intermediate Compartment (ERGIC). As in all NCL, its malfunction causes aggregates of lipofuscin-like compounds into lysosomes of different cell types, affecting in most extent neurons, causing neurodegeneration. CLN8p role as well as how its mutations trigger lysosomal storage disorder, is still unknown. Here, we evaluate how CLN8 expression is involved in the spatial distribution of lysosomes. Methods: HeLa cells were transfected with one of following constructs: soluble GFP (control), GFP-CLN8wt, shCLN8 or shLuc (sh control). Lysosomes were marked with anti-LAMP1 antibody by immunofluorescence. Images were taken with a Disk Scanning Unit (DSU) microscope and analyzed using ImageJ-Fiji and SpatTrack softwares. Results: Four indexes were calculated to analyze spatial pattern of lysosomes. Nearest Neighborhood (NN) and Clark’s Aggregate Index (CAI) refer to distances between particles. Both indexes showed a significant difference (p < 0.05) between CLN8wt and shCLN8 or control. Radial Distribution Function (RDF) is calculated by SpatTrack and refers to the number of particles surrounding each particle within a certain radius. This index showed differences (p < 0.0001) among all treatments. Finally, we measured distances of each particle to the nucleus that showed difference (p < 0.01) between control and shCLN8 but not among the other treatments. Discussion: Ddifferent levels of CLN8 expression cause changes in the spatial distribution of lysosomes in HeLa cells. Evidences in CLN3 and CLN5 showed altered lysosomal pattern in HeLa cells when these non-lysosomal proteins were mutated. A common modified lysosomal distribution pathway may be related with the physiopathology of CLN3-, CLN5- and CLN8 diseases.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 21. Lysosomal disorders: treatment, enzyme replacement therapy

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Expert opinion on the management of intracerebroventricular (ICV) drug delivery Slavc I 1, Cohen-Pfeffer J 2, Gururangan S 3, Jurecki E 2, Krauser J 3, Lester T 2 , Lim D 4, Maldaun M 5, Schwering C 6, Shaywitz A 2, Westphal M 6 1 Medical University of Vienna, Vienna, Austria, 2BioMarin Pharmaceutical Inc, Novato, CA, United States, 3Duke University Medical Center, Durham, NC, United States, 4University of California, San Francisco, CA, United States, 5IEP-Hospital Sirio-Libanes, Sao Paulo, Brazil, 6University Medical Ctr Hamburg-Eppendorf, Hamburg, Germany

Background: The intracerebroventricular (ICV) route of administration has been used for many decades to treat pediatric and adult patients with a broad range of central nervous system (CNS) disorders. There is no consensus in management of ICV devices and associated rates of reported complications are highly variable. A systematic literature review revealed that noninfectious complication rates per patient range from 1 to 33 %, while infectious complication rates range from 0 to 27 %. Methods: 7 healthcare professionals (neurosurgeons, neuro-oncologists, pediatricians, nurse practitioners) with expertise in ICV delivery met to discuss best practices in management of ICV devices and drug administration and to provide guidance on prevention of complications. Results: Experts share common practices in the management of ICV devices. Most are experienced in delivering drugs through a bolus injection, though one center has had clinical trial experience with an infusion. In either case, extreme care must be taken to follow strict aseptic/sterile techniques. Waiting a minimum of 5 days after device implantation before first use of device is recommended to allow proper wound healing and to reduce risk of backflow of the administered drug through the catheter tract. Experts differ in practice of hair removal over the device, on the type of skin prep solution used, and in use of gown and cap. All experts recommend use of sterile gloves and mask as well as skin disinfection with multiple separate swabs. Discussion: ICV drug delivery is an effective way to deliver drugs into the CNS when stringent measures are taken to prevent complications. Conflict of Interest declared.

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Long-term galsulfase enzyme replacement therapy in Taiwanese mucopolysaccharidosis VI patients: a case series Lin H Y 1 2 3 4, Lin S P 1 2 3, Chuang C K 2, Wang C H 6, Chien Y H 7, Wang Y M 8 , Tsai F J 6, Chou Y Y 9, Lin S J 10, Pan H P 11, Niu D M 5, Hwu W L 7, Ke Y Y 12 1 Dep Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan, 2Dep Med Res, Mackay Memorial Hospital, Taipei, Taiwan, 3Dep Medicine, Mackay Medical College, New Taipei City, Taiwan, 4Mackay Junior College Med, Nurs, Manage, Taipei, Taiwan, 5Dep Ped, Taipei Veterans General Hosp, Taipei, Taiwan, 6Dep Ped, China Medical University Hosp, Taichung, Taiwan, 7Dep Ped, National Taiwan University Hosp, Taipei, Taiwan, 8Dep Ped, Changhua Christian Hospital, Changhua, Taiwan, 9Dep Ped, National Cheng Kung Univ Hosp, Tainan, Taiwan, 10Dep Ped, Chi Mei Medical Center, Tainan, Taiwan, 11 Genetic Center, Nat Cheng Kung Univ Hosp, Tainan, Taiwan, 12Dep Ped, Taichung Veterans General Hosp, Taichung, Taiwan

Background: Information regarding the long-term outcome of enzyme replacement therapy (ERT) with recombinant human N-acetylgalactosamine 4-sulfatase (rhASB, galsulfase, Naglazyme®, BioMarin Pharmaceutical Inc) for Taiwanese patients with mucopolysaccharidosis (MPS) VI is limited. Methods: Nine Taiwanese patients with MPS VI (4 males and 5 females; age range, 1.4 to 21.1 years) treated with weekly intravenous infusions of galsulfase (1.0 mg/kg) in 5 medical centers in Taiwan were reviewed. A set of biochemical and clinical assessments were evaluated annually.

Results: After 6.2 to 11.2 years of galsulfase treatment, 6 patients experienced improvement over baseline in the 6-min walk test by a mean of 150 meters (59 % change over time), and 3 patients also increased the 3-min stair climb test by a mean of 60 steps (46 %). In a manual dexterity test, 3 patients decreased the time required to pick up 10 coins and put the coins into a cup by 15 seconds (33 %). Shoulder range of motion in all 9 patients improved, and Joint Pain and Stiffness Questionnaire scores improved by 0.42 points (21 %). Four patients showed improved pulmonary function. Five patients had positive effects on cardiac-wall diameters. Four patients had improved cardiac diastolic function. Liver and spleen sizes as measured by abdominal ultrasonography remained the same or decreased in all 9 patients. However, the severity degree of valvular stenosis or regurgitation did not show improvement despite ERT. A mean overall 69 % decrease in urinary glycosaminoglycan (GAG) excretion indicated a satisfactory biomarker response. Discussion: Long-term ERT was beneficial and safe for Taiwanese patients with MPS VI. This treatment reduced urinary GAG and had positive effects on a wide range of clinical functional assessments including endurance, mobility, joint function, pulmonary function, liver and spleen size, cardiac hypertrophy and diastolic dysfunction.

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A national pilot study to investigate the effects of sub-maximal aerobic exercise in adults with late-onset Pompe disease: Salford Royal NHS Foundation Trust (SRFT) cohort results. Silk E J S 1, Roberts M E 1, Meehan M 1, Hendriksz C J 1 1

Mark Holland Metabolic Unit, SRFT, Salford, United Kingdom

Background: Late-onset Pompe disease (LOPD) is characterised by progressive skeletal and respiratory muscle weakness. Sub-maximal aerobic exercise ( C mutation and a novel mutation, c.791 T > C, whose main finding was massive hepatomegaly at baseline of trial. The pt’s liver reached to his pelvis, and his spleen was 2 cm below the costal margin. Pt 2: a 21 year old female also with the c.607G > C mutation and a novel mutation, c.153C > A, had hypercholesterolemia and high alanine aminotransferase (ALT) levels at baseline of trial. Results: In pt 1, the baseline level of ALT (91 U/L) and triglyceride (207 mg/ dL) had reductions at Wk 20 (48 U/L, 149 mg/dL) and Wk 50 (68 U/L, 115 mg/dL). Low-density lipoprotein cholesterol (LDL-C), which was 239 mg/dL at baseline, had an increase at Wk 20 (348 mg/dL) and a reduction at Wk 50 (212 mg/dL). Reduction in hepatic fat content (−26.7 %), liver volume (−21.3 %), and spleen volume (−26.6 %) were observed from baseline to Wk 52.In Pt 2, the baseline level of ALT (116 U/L), LDL-C (222 mg/dL), and triglyceride (109 mg/dL), had also changed at Wk 20 (51 U/L, 114 mg/dL and 119 mg/dL) and Wk 50 (47 U/L, 156 mg/dL and 105 mg/dL). Reduction in hepatic fat content (−28.1 %), liver volume (−9.6 %), and spleen volume (−17.1 %) were observed from baseline to Wk 52. Neither pt reported serious adverse events even though pt 2 had positive anti-drug antibodies. Discussion: SA resulted in clinically significant reductions in hepatic and lipid abnormalities in pts with LAL-D up to 1 year post-treatment; we await longer term data on the treatment effects and outcomes in Japanese patients. Conflict of Interest declared.

P-524

Olipudase alfa for the treatment of acid sphingomyelinase deficiency (ASMD): 18-month safety and efficacy data Lachmann R 1, Wasserstein M 2 4, Jouvin M H 3, Nandy I 3, Ji A J 3, Inguilizian H 3, Puga A C 3 1 Nat Hosp for Neurology and Neurosurgery, London, United Kingdom, 2Icahn School of Medicine at Mt Sinai, New York, United States, 3Sanofi Genzyme, Cambridge, United States, 4Currently at Albert Einstein Coll of Med, Bronx, United States

Background: Olipudase alfa (recombinant human acid sphingomyelinase) is an investigational enzyme replacement therapy in development for the treatment of patients with nonneurological manifestations of acid sphingomyelinase deficiency (Niemann-Pick disease B; NPD B). Methods: Five adults with NPD B who completed a Ph 1b clinical trial of olipudase alfa were enrolled in an ongoing extension study (NCT02004704; EudraCT: 2013-000051-40) to evaluate the long-term safety and efficacy of olipudase alfa administered every 2 weeks. We present data for up to 18 months of treatment. Results: Olipudase alfa was well tolerated. There were no deaths, serious adverse events, or adverse events that led to discontinuation reported through 18 months. No patient developed anti-drug antibodies. Ceramide levels were measured in plasma and dried blood spots (DBS). In plasma, they remained normal and stable; DBS ceramide was slightly higher than normal at study start but quickly reduced to normal range, suggesting substrate debulking in cells. Mean spleen and liver volumes decreased from 12.8 MN at baseline to 7.7 MN at 18 months (p < 0.05), and from 1.7 MN at baseline to 1.2 MN at 18 months (p < 0.05), respectively. Mean percent predicted DLCO increased from 58.3 % predicted at baseline to 76.4 % predicted at 18 months (p < 0.05). Mean scores for components of infiltrative lung disease on HRCT scan progressively decreased from baseline to 18 months. At baseline, patients had elevated mean triglycerides, total cholesterol, LDL-C, and a low mean HDL-C. By 18 months, triglycerides decreased by 42.6 %, total cholesterol by 12.4 %, LDL-C by

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 15.4 %, and HDL-C increased by 78.9 %. Chitotriosidase levels steadily decreased from 735 nmol/h/mL at baseline to 286.8 nmol/h/mL at 18 months. Discussion: Albeit derived from a limited number of patients, these data on longer-term effects of olipudase alfa show a sustained safety profile and improvements in multiple clinically relevant parameters. Funded by Genzyme Corp. Conflict of Interest declared.

P-525

Long-term efficacy and safety of reveglucosidase alfa in subjects with lateonset Pompe disease: 144-week follow-up of the POM-001/002 studies Geberhiwot T 10, Byrne B 1, Barshop B A 3, Barohn R 2, Hughes D 11, Bratkovic D 4, Desnuelle C 5, Laforet P 6, Mengel E 7, Roberts M 8, Haroldsen P 9, Smith L 9, Yang K 9, Walsh L 9 1 Univ of Florida School of Med, Gainesville, FL, United States, 2Kansas Univ Med Center, Kansas City, KS, United States, 3Rady Child Hosp, San Diego UCSD, La Jolla, CA, United States, 4IMVS Pathology, Adelaide, SA, Australia, 5Le Centre Hospitalier de Nice, Nice, France, 6Hopital Pitie Salpetriere, Paris, France, 7Johannes Gutenberg Univ Mainz, Mainz, Germany, 8Salford Royal NHS Foun Trust, Salford, United Kingdom, 9 BioMarin Pharmaceutical Inc, Novato, CA, United States, 10Univ Hosp Birmingham, Birmingham, United Kingdom, 11 Royal Free and Univ College Med, London, United Kingdom

Background: Late-onset Pompe disease (LOPD) is a rare neuromuscular disorder caused by a deficiency in lysosomal alpha-glucosidase (GAA). The resulting accumulation of glycogen in skeletal muscle leads to reduced mobility, progressive weakness of respiratory muscles, and respiratory failure in over 70 % of patients. Although currently available treatment for LOPD (recombinant human GAA [rhGAA]) provides some benefits on mobility and respiratory function, patients continue to have disease progression despite long-term therapy. Furthermore, rhGAA is delivered to lysosomes inefficiently. To address these limitations, reveglucosidase alfa (BMN-701), a novel chimeric rhGAA fused with an insulin-like growth factor 2 (GILT)derived peptide, was developed. The GILT peptide enhances the uptake of GAA into the lysosomes of muscle cells. In the first human study of reveglucosidase alfa (POM-001/002), subjects demonstrated improvements in respiratory muscle strength and walking endurance over 72 weeks of treatment. This report examines efficacy and safety of reveglucosidase alfa over 144 weeks of follow-up in POM-001/002. Methods: Ambulatory LOPD subjects naïve to rhGAA were enrolled in an open-label dose escalation study (POM-001; 5, 10, and 20 mg/kg intravenous reveglucosidase alfa for 24 weeks) followed by an extension study for up to 480 weeks (POM-002). Efficacy evaluations included measures of respiratory function (% predicted maximum inspiratory pressure [MIP], maximum expiratory pressure [MEP], upright forced vital capacity [FVC]), and endurance test (6-min walk test [6MWT]). Adverse events were monitored throughout the study. Results: This study enrolled 22 LOPD subjects, of which 15 patients completed 144 weeks of treatment with BMN-701. Data through 144 weeks is currently being analyzed and will be presented at the conference. Discussion: The results of this report will provide long-term efficacy and safety data for reveglucosidase alfa Conflict of Interest declared.

P-526

Morquio A Registry Study (MARS): Design and baseline characteristics of enrolled patients Guffon N 1, Baujat G 2, Bober M B 3, Burton B K 4, Clarke L 5, Garcia P 6, Giugliani R 7, Hendriksz C J 8, Lavery C 9, Raiman J 10, Jurecki E 11, Sisic Z 11, Waite A 11

1

Hosp Femme Mere Enfant, CERLYMM, HCL, Lyon, France, 2Hosp Necker Enfants Malades (AP-HP), Paris, France, 3Al duPont Hosp for Children, Wilmington, DE, United States, 4Lurie Child Hosp and NWU Feinberg, Chicago, IL, United States, 5 Childrens and Womens Centre of BC, Vancouver, BC, Canada, 6Hosp Pediatrico de Coimbra, Coimbra, Portugal, 7 MedGenetServHCPA, DepGenet UFRGS INAGEMP, Porto Alegre, Brazil, 8 Salford Royal Foundation NHS Trust, Salford, United Kingdom, 9MPS Society, Amersham, United Kingdom, 10 Birmingham Child Hosp, Birmingham, United Kingdom, 11BioMarin Pharmaceutical Inc, Novato, CA, United States Background: The Morquio A Registry Study (MARS; NCT02294877) is a voluntary, multicenter, multinational, observational registry for patients with a confirmed diagnosis of Morquio A syndrome (mucopolysaccharidosis IVA; MPS IVA). The purpose of MARS is to characterize and describe the Morquio A population as a whole, including the heterogeneity, progression, and natural history of Morquio A, study safety and long-term effectiveness of elosulfase alfa, and help the Morquio A medical community with the development of recommendations for monitoring subjects and reporting on subject outcomes to optimize care. Methods: Demographic, clinical, laboratory, and safety data are collected upon entry into the registry and at 6 and 12 month intervals thereafter from all or some of the assessments dependent upon the individual’s standard care for up to 10 years. Additional data are collected in an ERT pregnancy and lactation sub-study and a separate clinical trial follow-up sub-study. The goal of the clinical trial follow-up sub-study is to assess the long-term safety and efficacy of elosulfase alfa ERT in patients who previously participated in either the extension of the Phase 3 pivotal trial of elosulfase alfa (NCT01415427) or the pediatric clinical trial of elosulfase alfa for patients less than 5 years of age (NCT01515956). Results: As of February 2016, 72 patients have enrolled in MARS, 59 of whom are reported to have received at least one dose of elosulfase alfa. Median age at enrollment is 13.3 years. Median (min, max) age at diagnosis is 3.3 (0.4, 49.0) years in ERT-treated patients and 9.4 (1.1, 20.2) years in treatment-naïve patients. Discussion: Through standardized collection of data, MARS will assist in systematically filling the current knowledge gaps around Morquio A disease progression and characterizing clinical outcomes in both the presence and absence of ERT, ultimately facilitating optimal monitoring and care for individuals with Morquio A syndrome. Conflict of Interest declared.

P-527

Switch of enzyme replacement therapy (ERT) in the Canadian Fabry Disease Initiative Study (CFDI): Intermediate follow-up at 3 and a half years Morel C F 1, Bichet D 2, Casey R 3, Clarke J T 4, Iwanochko M 1, Khan A 3, Sirrs S 5, Auray-Blais C 6, Doucette S 7, LeMoine K 8, West M L 9 1

Dept Med, Univ Toronto, Toronto, Canada, 2Dept Med, Univ Montreal, Montreal, Canada, 3Med Gen, Dept Ped, Univ Calgary, Calgary, Canada, 4 Med Gen, Dept Ped, Univ Toronto, Toronto, Canada, 5Dept Med, Univ British Columbia, Vancouver, Canada, 6Ped, Biochem, Univ Sherbrooke, Sherbrooke, Canada, 7Dept Com Health Epi, Dalhousie Univ, Halifax, Canada, 8Dept Nurs, QEII Health Sci Centre, Halifax, Canada, 9Dept Med, Dalhousie Univ, Halifax, Canada Background: To determine the effect of switching from agalsidase beta 1.0 mg/kg to agalsidase alfa 0.2 mg/kg every other week (EOW) in patients with Fabry disease. Methods: The CFDI is a prospective multicenter study of ERT in Fabry disease in Canada. Due to shortage of agal-beta, 37 Fabry patients were switched to agal-alfa in May 2010. We report their follow up and compare their outcomes during treatment with agal-beta and alfa, defined as clinical events: renal, cardiac, neurologic and death. The first 6 months post switch were censored because of possible carry over effect from the prior agal-beta treatment.

S214 Results: 37 patients (25 M, 12 F), mean age 47.8 ± 12.6 years, MSSI 32.6 ± 11.5, ESRD 32.4 %, HTN 56.8 %, pacemaker 46 % and stroke/TIA 27 % were switched over 4 weeks from agal-beta to alfa. Time on ERT pre switch was 32.6 ± 11.5 vs. 42.5 ± 7.8 months post. There was no significant change in clinical parameters pre switch to last follow up post switch. Drug use (antiplatelet agents, RAS inhibitors, anti-hypertensives, statins and analgesics) did not differ between the 2 periods. While there were 19 clinical events (prevalence 27 %) in the beta period, there were 29 (47.2 %) in the alfa period. With censoring of the first 6 m on alfa, there were 14 events (39 %). Clinical event rate was identical at 1 per 61 patient months whether on agal-beta or alfa after 6 months. Cardiac events were most common in all time periods. Discussion: The CFDI study is the only prospective randomized controlled study of ERT in Fabry disease. Switch from agal-beta to alfa both in standard dose was associated with a transient increase in clinical events for 6 months but thereafter the clinical event rate returned to baseline for the next 3 years. This finding is unexplained but has been recently reported by Ortiz et al. (J Med Genet 2016;0:1–8) at the start of agal-beta therapy. Alternately this could be associated with the acute dose reduction from 1.0 to 0.2 mg/kg EOW. Conflict of Interest declared.

P-528

Prompt agalsidase alfa therapy initiation is associated with improved renal and cardiovascular outcomes in the Fabry Outcome Survey Hughes D 2, Linhart A 3, Gurevich A 1, Joseph A 1, Thakur M 1, Feriozzi S 4 1 Shire, Zug, Switzerland, 2Royal Free NHS Found Trust, Univ Coll, London, United Kingdom, 3Univ Hosp, Prague, Czech Republic, 4Belcolle Hosp, Viterbo, Italy

Background: Over the past 15 years, the Fabry Outcome Survey has collected data from patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. This post-hoc analysis examined potential benefits of prompt initiation of agalsidase alfa enzyme replacement therapy (ERT) on patient outcomes.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 1

Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Div Ped Cardiol, Univ Cukurova, Adana, Turkey

Background: Infantile Pompe Disease (IPD) is an autosomal recessive storage disease caused by a deficiency of α-glucosidase due to the mutations in GAA gene. It results in glycogen accumulation in cardiac and skeletal muscle. Hypertrophic cardiomyopathy and hypotonia are the main clinical findings. After the introduction of enzyme replacement treatment (ERT) survival has been improved. The prognosis depends on the dose, initiation time of ERT, and the CRIM status. Methods: 30 patients with hypertrophic cardiomyopathy were diagnosed as IPD through α-glucosidase activity measurements and mutation analysis in Çukurova University. Subsequently, 40 mg/kg/biweekly ERT was initiated. The CRIM status could not be determinedNo severe infusion-associated reactions were detected. Results: The initial admission symptoms were family history, hypotonia, recurrent respiratory infections, cyanosis, and supraventricular tachycardia. The median age of diagnosis and the initiation of ERT were 109,5 ± 92,6 and 116,1 ± 93,2 days, respectively. Mutation analysis revealed 4 novel mutations out of fourteen. p.L299P (c.896 T > C) was the most frequent mutation (10 patients). The duration of ERT was 27,3 ± 22,6 months (0–80 months). The difference between the basal and last intraventricular septum thickness and left ventricle posterior wall thickness after ERT were statistically important (p < 0,05). The current age of the patients is 30,78 ± 22,05 months (1–80 months), the overall survival rate is 76,66 % (23 patients) and the ventilator-free survival is 70 % (21 patients). The frequency and the severity of lower respiratory infections and hospitalization were decreased. From 12 patients older than 24 months of age, 11 can able to walk. Discussion: This is one of the largest ventilation-free survival IPD groups. Our study also put forward that irrespective of CRIM status, 40 mg/kg/biweekly ERT is impressingly effective on cardiac function, ventilator-free survival, respiratory infection frequency, and ambulation in IPD.

P-530

Autophagy induction as a potential treatment for lysosomal diseases Methods: All patients with FD diagnosis and ERT start dates were included. Renal outcomes included dialysis, transplantation, renal failure, and proteinuria. Cardiovascular outcomes included myocardial infarction, left ventricular hypertrophy, and heart failure. Time to first renal or cardiovascular event from ERT start (censored at 120 months) was compared between prompt (within 24 months of diagnosis) vs. delayed (>24 months after diagnosis) ERT initiation. Patients with events reported at day of ERT start were excluded. Kaplan-Meier curves were compared with the log-rank test. Cox regression analysis with age at time of diagnosis as a covariate was used to estimate hazard ratios (HR) between groups. Results: This study included 1836 patients: 893 with prompt and 943 with delayed ERT initiation after diagnosis. Groups were comparable by gender, race, height, weight, glomerular filtration rate, and left ventricular mass index. Mean ages at ERT initiation were similar, but mean age at time of diagnosis for prompt and delayed groups was 28 and 40 years of age, respectively. Median time between diagnosis and ERT onset for prompt and delayed groups was 7 and 94 months, respectively. Prompt ERT initiation was associated with significant reduction in risk of renal events (HR = 0.768; p < 0.01) and cardiovascular events (HR = 0.756; p < 0.001). Discussion: This analysis showed prompt agalsidase alfa initiation (within 24 months after diagnosis of FD) was associated with significantly better renal and cardiovascular outcomes vs. delayed initiation (>24 months after diagnosis). This suggests significant benefits with timely initiation of FD therapy. Conflict of Interest declared.

P-529

30 Infantile Pompe patients with 40 mg/kg/biweekly enzyme replacement treatment and 23 survival: a single center experience from Turkey Onenli Mungan N 1, Kor D 1, Seker Yilmaz B 1, Bulut F D 1, Erdem S 2, Demir F 2, Eren H 1, Ozbarlas N 2

Matalonga L 1, Farrera-Sinfreu J 2, Pascual R 3, Arias A 1, Tort F 1, GarciaVilloria J 1, Ferrer Montiel A 3, Ponsati B 2, Gort L 1, Ribes A 1 1 Hosp Clinic, IDIBAPS, CIBERER, Barcelona, Spain, 2BCN Peptides SA, Sant Quinti de Mediona, Spain, 3Ins Bio Mol Cel, Univ Miguel Hernandez, Elche, Spain

Background: Lysosomal storage disorders (LSDs) are genetic diseases caused by the abnormal accumulation of non-degraded macromolecules into lysosomes leading to a biochemical cascade that results in the impairment of the autophagy flux and the prevention of lysosomal clearance. Recent studies have demonstrated that the induction of autophagy in LSDs could decrease the abnormally stored material by enhancing lysosomal exocytosis. Bicalutamide (N-[4-cyano3-(trifluoromethyl)phenyl]-3-[(4-fluorophenyl)sulfonyl]-2-hydroxy-2methylpropan-amide) is a synthetic non-steroidal anti-androgen molecule reported to be involved in the induction of autophagy in human prostate cancer cells. The aim of our work was to evaluate the potential benefits of treatment with R and S enantiomers of bicalutamide in fibroblasts of patients affected by differents LSDs. Methods: Treatment response was evaluated in cultured fibroblasts of patients affected by seven different LSDs by monitoring lysosomal exocytosis, substrate accumulation and cell viability. Results and Discussion: Treatment with (S)-Bicalutamide enantiomer was able to ameliorate significantly the altered biochemical parameters in all the cell lines, while the response to (R)-Bicalutamide, or Ciclodextrin (a previously described autophagy inductor in LSDs) was less effective. Moreover, we have studied the molecular mechanism underlying Bicalutamide’s action and found that S-Bicalutamide acts through the activation of the transcription factor TFEB. This transcription factor enhances the transcription of genes involved in autophagy and lysosomal biogenesis, leading to the subsequent increase of the autophagy flux and the lysosomal exocytosis. Conflict of Interest declared.

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Estimating the value of treatment for Fabry disease: A discrete choice experiment Lloyd A 1, Gallop K 1, MacCulloch A 2, Hughes D 3 1 Bladon Associates Ltd, Oxford, United Kingdom, 2Amicus Therapeutics Ltd, London, United Kingdom, 3Royal Free Hospital, London, United Kingdom

Background: Fabry disease is a rare inherited lysosomal storage disorder caused by deficiency of α-galactosidase A. Effective intravenous enzyme replacement therapies are available, and a new oral treatment is being developed. This study was designed to estimate how important different aspects of treatment for Fabry disease are and whether people will trade effectiveness against other improvements. Methods: A stated preference survey method called a discrete choice experiment (DCE) was designed to assess the importance of different attributes of treatments for Fabry disease. These included overall survival, mode of administration, treatment-related reactions, treatment-related headaches and risk of antibody formation. A UK general public sample was recruited to give a societal perspective. The mixed logit model was used to estimate strength of preference for the attributes. Results: The sample (n = 506) was broadly representative of UK demographics (mean age = 46.9). Participants preferred treatments that were associated with longer life expectancy (each additional year OR = 1.574; 95%CI: 1.504– 1.647). Participants preferred an every-other-day tablet compared to an infusion (OR = 2.262; 95%CI: 2.075–2.463). Participants also chose to avoid treatments with headaches and treatment reactions. Discussion: Participants recognised the seriousness of the condition and preferred the most effective treatments. However, if we assumed two treatments had equal efficacy then participants had a strong preference for the oral treatment, and for the avoidance of treatment-related reactions and treatmentrelated headaches. Assuming normal life expectancy, the public would trade 1.88 (of 34.6 remaining) years of life to avoid intravenous medicine administered every 2 weeks compared to an oral treatment for Fabry disease. Conflict of Interest declared.

P-532

Survival in idursulfase-treated and untreated patients with mucopolysaccharidosis type II: data from the Hunter Outcome Survey (HOS) Burton B K 1, Jego V 4, Mikl J 3, Jones S A 2 1 Children’s Memorial Hospital, Chicago, United States, 2Manchester Centre for Genomic Medicine, Manchester, United Kingdom, 3Shire, Lexington, United States, 4Cytel Inc, Geneva, Switzerland

Background: Idursulfase therapy (Shire) for the life-limiting disorder MPS II (Hunter syndrome) has been available since 2006; its impact on survival has not been evaluated Methods: Kaplan–Meier (K–M) estimates and Cox regression modelling were used to compare survival in idursulfase-treated and untreated male patients followed prospectively in HOS, a global, multicentre registry that collects real-world data on MPS II. Median values are shown unless otherwise specified Results: As of January 2016, data were available for 768 idursulfase-treated (treatment duration, 55.8 months) and 96 untreated patients. Age at symptom onset was 1.5 yrs in both groups; age at diagnosis was 3.3 and 3.2 yrs in treated and untreated patients, respectively. Prevalence of cognitive impairment was also similar (treated 57.2 %; untreated 58.3 %; p = 0.536). 15.2 % of treated and 26.0 % of untreated patients had died. K–M survival estimates (95 % CI) were 33.0 (30.6–42.2) yrs in treated and 21.2 (18.0–not reached [NR]) yrs in untreated patients (log rank p = 0.016); follow-up was 12.7 and 13.9 yrs. In those with cognitive impairment, survival was 20.9 (19.4–23.9) and 18.0 (15.4–21.2) yrs in treated and untreated patients; in those without cognitive impairment, values were 43.6 (34.6–NR) yrs in treated patients and NR in

untreated patients (only 2 events). A Cox model adjusted for cognitive impairment, region, and ages at diagnosis and symptom onset indicated a 49 % lower risk of death in treated patients: adjusted death hazard ratio 0.51 (95 % CI: 0.31–0.84). Conversely, risk of death was 1.96-fold higher in untreated patients Discussion: The model will be further refined by exploring additional potential confounding factors to obtain improved estimates of survival in treated and untreated patients with MPS II. This first report of improved survival with long-term idursulfase therapy is a valuable addition to our evolving understanding of patient management Conflict of Interest declared.

P-533

Real-world treatment patterns from 647 patients with Gaucher disease: an analysis from the Gaucher Outcome Survey Deegan P 4, Fernandez-Sasso D 5, Giraldo P 6 7, Lau H 3, Panahloo Z 1, Zimran A 2 1 Shire, Zug, Switzerland, 2Shaare Zedek Med Cent & Hadassah Med Sch, Jerusalem, Israel, 3NYU Langone Medical Center, New York, New York, United States, 4Addenbrooke’s Hospital, Cambridge, United Kingdom, 5 Instituto William Osler, Buenos Aires, Argentina, 6 CIBER de Enfermedades Raras, IIS Aragon, Zaragoza, Spain, 7Translational Research Unit, IIS Aragon, Zaragoza, Spain

Background: The Gaucher Outcome Survey (GOS) is an international Gaucher disease (GD)-specific registry established in 2010 for patients with a confirmed GD diagnosis, regardless of their treatment status. GOS collects real-world data from GD patients, including information on disease manifestations and treatment history. Here we describe treatment patterns from sites in 10 countries. Methods: For patients who had received any GD-specific treatment at any time, data on the treatment received (drug name), dose, and frequency of administration were collected. GD-specific treatments included enzyme replacement therapies (alglucerase, imiglucerase, velaglucerase alfa, taliglucerase), substrate reduction therapies (miglustat, eliglustat), and ambroxol. Results: GD-specific treatment data were available for 647 patients. 573 were receiving treatment at the time of analysis, with velaglucerase alfa (316/573, 55 %) and imiglucerase (184/573, 32 %) most widely used. 446/647 patients with treatment data (69 %) had been treated for >5 years and 368/647 (57 %) had only received one GD drug. In the remaining patients, the most common treatment sequence was a switch from imiglucerase to velaglucerase alfa (114/ 279, 41 %). For the 377 patients treated with velaglucerase alfa at any time, 474/500 dosing schedules (95 %) were administered biweekly and the most widely used doses were ≥60 U/kg (166/492, 34 %) and < 20 U/kg (117/492, 24 %). There were apparent differences in velaglucerase alfa dosing between the 3 highest-enrolling countries (>100 patients enrolled in each), with most patients receiving < 20 U/kg in Israel, 20–40 U/kg in the UK and ≥60 U/kg in the USA. Discussion: Many patients received velaglucerase alfa at the recommended starting dose of 60 U/kg every other week, although there were differences in dosing between countries. This analysis provides a basis upon which to examine outcomes data, for insight into the real-life effectiveness of treatment regimens. Conflict of Interest declared.

P-534

Non-neuronopathic Gaucher Disease: A Retrospective Review Comparing Clinical Outcomes of 2 Weekly and 4 Weekly Enzyme Replacement Therapy. Heales H 1, Ramaswami U 1, Hughes D 2, Mckie M 1, Mehta A 1 1 Royal Free London NHS Foundation Trust, London, United Kingdom, 2 University College London, London, United Kingdom

S216 Background: Current approved therapy for non-neuronopathic Gaucher Disease (GD; OMIMM 230800) includes every other week (EOW) enzyme replacement therapy (ERT). We compared achievement of therapeutic goals of EOW ERT with a 4 weekly dosing regimen of ERT (Imiglucerase or Velaglucerase alfa). Methods: Single centre, retrospective review of 19 patients stable on standard EOW ERT, switched to a 4 weekly regimen.14/19 with complete data were analysed. 9/14 doubled their EOW dose, maintaining same total 4 weekly doses (group I). In 5/14 in whom clinical manifestations were considered mild, 4 weekly total doses had a reduction by 25–50 % (group II). ERT dose in units/ kg/EOW pre switch: median (range): 16.6 (6.5–25.6); 21.6 (17.8–30.8) in Groups I and II respectively. Platelets, serum ferritin, haemoglobin (Hb) and chitotriosidase were measured (a) pre switch; (b) 9 months (±3 months) post switch; (c) 2–3 years post switch. 8/14 on Imiglucerase and 6/14 on Velaglucerase alfa, median age at start of therapy 34.4 years (6.5–54.2) and 54 years (19.2–69.30) respectively. All patients remained on the same ERT preparation post switch. Pre-switch ERT duration was 9.7 years (0.8– 18.8 years). 5/14 patients were splenectomised. Results: Haematological parameters showed no significant change pre and post switch. In group I, chitotriosidase increased variably post switch: (a): 516 (99–2484); (b): 572 (172–3956); (c) 1057 (96–10105) respectively (p values ns). In group II, chitotriosidase remained stable post switch. No significant differences noted according to splenectomy or ERT preparation. Discussion: Following switch to 4 weekly ERT, in group I, chitotriosidase variably increased. In group II, in whom chitotriosidase was stable post switch, an individualised approach with lowering of total daily dose was possible. Regular assessment of Hb, platelets and chitotriosidase in conjunction with patients’ needs and quality of life is recommended when switching from EOW ERT.

P-535

Early treatment with Sebelipase-alfa of two young LAL-D siblings: first outcome data Tummolo A 1, Ortolani F 1, Masciopinto M 1, Melpignano L 2, Di Mauro A M , Piccinno E 1, Papadia F 1

1

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Long-term data from patients with Gaucher disease: a descriptive analysis from a single center in the Gaucher Outcome Survey Elstein D 1, Zimran A 2 1 Shire, Zug, Switzerland, 2Shaare Zedek Med Cent & Hadassah Med Sch, Jerusalem, Israel

Background: The Gaucher Outcome Survey (GOS) is an international, multicenter, observational, open-ended disease registry designed to document the clinical outcome of patients with Gaucher disease (GD) over time. This analysis evaluates long-term data collected from GOS patients in one site in Israel. Methods: This was a retrospective analysis of patients with ≥6 years of followup data enrolled at the Shaare Zedek Medical Center. Treatment details (drug names, dosing schedules) and clinical parameters were recorded. A subset of patients who switched from alglucerase (ALG) or imiglucerase (IMI) to velaglucerase alfa (VEL, n = 28) with no more than 4 months separating the different treatment regimens was analysed. Results: 127 patients had ≥6 years follow-up data (52 [41 %] male; mean ± SD age: 32.6 ± 18.2 years at treatment start, 45.9 ± 17.9 years at GOS entry). Before first visit, 24 patients had been splenectomized; another 6 were splenectomized during follow-up. The most prevalent GBA genotype was N370S/N370S (n = 62). Follow-up data were available for 18.3 ± 4.8 years for laboratory data and 18.1 ± 5.0 years for abdominal imaging. 98 patients received IMI and 89 received VEL at any time. Change (median [range]) in hemoglobin concentration, platelet count and multiples of normal for liver and spleen size were (2.3 % [−24.8, 68.2], 14.2 % [−52.5, 82.9], −22.6 % [−75.7, 73.2] and −25.1 % [−78.7, 25.1]) in the ALG/IMI and (3.5 % [−14.5, 15.7], −0.4 % [−35.9, 51.5], 5.3 % [−32.7, 61.4] and −3.1 % [−53.6, 27.1]) in the VEL treatment periods, respectively. Discussion: This single-center experience demonstrates that long-term GD treatment is associated with sustained improvements in disease-specific parameters, and treatment with velaglucerase alfa even in patients with complex medical histories can result in stable outcomes with some additional benefits. Further analysis may provide insights into the effectiveness of the treatment regimens used over a period of almost two decades. Conflict of Interest declared.

1 Dep of Metabolic Dis, Child Hosp, Bari, Italy, 2Hospital Medical Head Office, Bari, Italy

P-537 Background: Lysosomal acid lipase deficiency (LAL-D) is a rare lysosomal storage disease caused by deficiency of LAL responsible for the hydrolysis of cholesterol esters and triglycerides. Its partial deficiency leads to Cholesteryl Ester Storage Disease (CESD) resulting in dyslipidaemia, hepatomegaly, elevated transaminases, liver damage with progression to failure. Therapeutic options have recently included enzyme-replacement therapy (ERT) with Sebelipase-alfa, a recombinant human LAL enzyme. Most of existing data come from adult patients, treated with ERT after liver transplantation. We report here the first outcome data of two young siblings, commenced with ERT with a preserved liver function. Case report: the older sibling was diagnosed at the age of 14 years with combined hyperlipidaemia, mild elevation of transaminase levels, and mild hepatomegaly. Treatment with simvastatin obtained a partial reduction in LDL-C. Liver biopsy showed portal and periportal fibrosis. His brother was detected at 7 years with severe hypercolesterolaemia, hypertriglyceridemia, hypertransaminasemia and liver steatosis. Genetic molecular analysis revealed compound heterozigosity for c.1033G > A (p.D345N)/ c.894G > A(p.S275_Q298del). Results: They started ERT at the age of 21 and 10 years respectively, at the dosage of 1 mg/Kg every-other-week. After the first 9 weeks, therapy was well tolerated and resulted in a decrease of liver transaminases in both patients (ALT: −38 %/−47 %, AST −55 %/−49 %, increase of LDL-C (+54 %/+ 38 %). TGLs reduced in the older patient (−25 %), remained almost stable in the younger one (+3 %). Discussion: First treatment data confirm rapid reduction of transaminases, rise of LDL-C concentrations, and a precocious effect on TGLs levels, with no significant adverse effects. Ongoing evidences may increase our knowledge on differences in biochemical response to therapy and multiorgan involvement of young patients as compared to adult and/or post-transplanted patients.

Galactosialidosis: moving a step closer towards the development of enzyme replacement therapy with recombinant human protective protein/ cathepsin a Koppaka V 1, Cadaoas J 1, Cullen S 1, Gomero E 2, Guzman C 1, Haller C 1, Hu H 2, Jayashankar K 1, Machado M 1, Morris G 1, Mosca R 2, Natesan A 1, Schatz A 1, Vellard M 1, D’Azzo A 2 1 Ultragenyx Pharmaceutical, Novato, United States, 2St Jude Child Research Hospital, Memphis, United States

Background: Galactosialidosis (GS) is a lysosomal glycoproteinosis caused by genetic deficiency of the serine carboxypeptidase Protective Protein/Cathepsin A (PPCA) that secondarily affects the activity and stability of the glycosidases, Neuraminidase 1 (NEU1) and β-galactosidase (β-GAL). A recombinant human PPCA (rhPPCA) is under investigation as a potential therapeutic for the treatment of GS patients. Administration of rhPPCA in GS patients is predicted to restore NEU1 and β-GAL activities and reduce the buildup of oversialylated substrates in tissues and body fluids. We have conducted a proofof-concept study in the GS mouse model that verified the potency of rhPPCA by reducing oligosacchariduria and substrate storage in tissues. Methods: GS mice were treated with rhPPCA via intravenous bolus injection for 8 weeks, followed by assessment of PPCA-NEU1-β-GAL activities, tissue distribution, and reduction of storage substrates in target tissues. Results: A dose-dependent increase in cathepsin A activity was detected in multiple affected tissues of treated mice. This was accompanied by rescue of

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 NEU1 activity, normalization of β-GAL levels, diminished cytoplasmic vacuolization and reduction of urinary sialyloligosaccharides. Surprisingly, in the brain, a clear dose-dependent effect was observed in the choroid plexus epithelium and the mononuclear leucocytes in the meninges with improvement in morphology and complete reversal of the disease phenotype at higher dose. No histopathology was associated with rhPPCA treatment. Discussion: This study demonstrated a clear dose-dependent efficacy and no toxicity. The next steps are to conduct IND-enabling toxicity and safety studies and to identify patients globally for a phase I/II clinical trial. Interviews with physicians and patients will be conducted to gain insight into clinical problems experienced by individuals with GS to inform the clinical trial. Conflict of Interest declared.

P-538

Abnormalities of cellular membranes can be reversed by substrate reduction in Fabry disease

Background: severe allergic reaction to enzyme replacement therapy (ERT) can occur in patients affected by lysosomal storage disorders, even after many years of infusions. This fact compromises the continuation of the ERT, which often has not a valid therapeutic alternative. Case report: a 21 years old man affected by late-onset Pompe disease, developed a severe allergic reaction during an infusion of the specific ERT with recombinant alfa glucosidase (rhGAA), after 8 years of regular injections. The reaction consisted in urticaria, swelling of the lips, and breathing difficulty. Specific immunoglobulin E (IgE) versus rhGAA and the skin allergy testing with rhGAA resulted positive. Desensitization was tried first with a protocol consisting in the administration of increased doses of rhGAA, which failed to induce immune tolerance. Thereafter, treatment with omalizumab, a monoclonal antibody that specifically binds human IgE, administered subcutaneously, was started. Results: after 2 months of treatment, omalizumab was able to counteract the allergic manifestations and the patient could restart regular ERT infusions every other week. Discussion: to our knowledge, this is the first reported case of a severe allergy to the ERT successfully treated with omalizumab in an adult patient with a lysosomal storage disorder.

Brogden G 1, Shammas H 1 2, Maalouf K 1 2, Naim S L 3, Wetzel G 1, Amiri M 1 , Von koeckritz blickwede M 1, Das A M 2, Naim H Y 1 1

Dept Phys Chem, Univ Vet Med, Hannover, Germany, 2Dept PED, MHH, Hannover, Germany, 3Faculty of Science, Fribourg, Switzerland Background: It is still not entirely clear how a-galactosidase (GAA) deficiency translates into clinical symptoms of Fabry disease (FD). The current communication investigates the effects of a GAA-mutation on the trafficking and processing of lysosomal GAA and their potential association with alterations in the membrane lipid composition. Methods: To address the consequences of the N215S mutation on maturation and trafficking of the GAA, endoglycosidase H was utilised to differentiate between the mature complex glycosylated protein form of GAA and the immature mannose-rich ER-located protein species. Furthermore, the trafficking of a lipid raft associated protein, DPPIV, was assessed by a biotinylation assay to determine if DPPIV trafficking to the cell surface is compromised. Moreover, FD and wild type fibroblasts were cultured with or without Nbutyldeoxynojirimycin (NB-DNJ) for 3 days. Lipid rafts were isolated via sucrose density gradient and the distribution of flotillin 2 and cholesterol was determined by Western blotting and HPLC respectively. Additionally, the concentration of Gb3 and sphingomyelin was determined by TLC and HPTLC respectively. Results: Abnormalities in lipid rafts were observed in fibroblasts isolated from a male patient with Fabry disease bearing the mutation N215S. Interestingly, lipid raft analysis revealed that the distribution of cholesterol and flotillin 2 were distinctly altered in the FD fibroblasts when compared to wild type cells. Furthermore, increased levels of the glycolipid Gb3 and sphingomyelin were observed in non-raft membrane fractions of FD cells. Substrate reduction with NB-DNJ in vitro was capable of reversing these abnormalities. Discussion: These data indicate that alterations of lipid rafts may contribute to the pathophysiology of Morbus Fabry. Our results suggest substrate reduction therapy with N-butyldeoxynojirimycin as a promising treatment option in GAA-deficiency. Conflict of Interest declared.

P-539

Successful desensitization to enzyme replacement therapy using omalizumab in a patient with late-onset Pompe disease. Sechi A 1, De Carli M 2, Macor D 1, Bianchi K 1, Dardis A 1, Zampieri S 1, Ciana G 1, Tripodi S 3, Galosi S 4, Bembi B 1 1

Reg Cent of Rare Dis, Univ Hosp of Udine, Udine, Italy, 2Dep of Med, Univ Hosp of Udine, Udine, Italy, 3Ped Dep, Pertini Hosp, Rome, Italy, 4Ped Dep, Umberto I Hosp, Rome, Italy

P-540

Efficacy and safety of migalastat, an oral pharmacological chaperone for fabry disease: results from two randomized phase 3 studies Feldt-Rasmussen U 1, Giugliani R 2, Germain D P 3, Hughes D 4, Wilcox W R , Schiffmann R 6, Bichet D G 7, Jovanovic A 8, Bratkovic D 9, Castelli J 10, Skuban N 10, Barth J 10 5

1 Rigshospitalet, Univ of Copehagen, Copenhagen, Denmark, 2Med Gen Serv, HCPA-UFRGS Porto Alegre, Porto Alegre, Brazil, 3Univ Versailles St Quentin en Yvelines, Montigny, France, 4Royal Free Hospital, Univ College London, London, United Kingdom, 5Dept of Human Genetics, Emory Univ, Atlanta, United States, 6Baylor Research Institute, Dallas, United States, 7 Hopital du Sacre-Coeur, Univ of Montreal, Montreal, Canada, 8Salford Royal NHS Foundation Trust, Salford, United Kingdom, 9Women and Children’s Hospital, North Adelaide, Australia, 10Amicus Therapeutics, Cranbury, United States

Background: Fabry disease is an X-linked disorder of lysosomal αgalactosidase A (α-Gal A) deficiency, leading to substrate accumulation and multiorgan disease. Migalastat, an oral pharmacological chaperone, stabilizes specific mutant forms of α-Gal A, increasing trafficking to lysosomes. Methods: Two randomized phase 3 studies were conducted (migalastat 150 mg QOD). Study 011 (FACETS, N = 67) was a double-blind, placebocontrolled, 24-month trial in ERT-naïve patients. Study 012 (ATTRACT, N = 56) was an active-controlled, 18-month trial in ERT-experienced patients with a 12-month open-label extension (OLE). Efficacy analyses for both studies focused on patients with amenable mutations. Results: In Study 011, post-hoc Stage 1 (6 months, placebo-controlled; P = 0.008) and prespecified Stage 2 (months 6–12; P = 0.014) analyses revealed statistically significant reductions in kidney interstitial capillary GL-3 and plasma lyso-Gb3. Over 24 months, the annualized rate of change in eGFRCKD-EPI ± SEM with migalastat was −0.3 ± 0.7 mL/min/1.73 m2. Left ventricular mass index (LVMi, [95 % CI]) decreased by 7.7 g/m2 (−15.4, −0.009; P < 0.05). Diarrhea, reflux, and indigestion improved (P < 0.05). During the 18-month controlled period of Study 012, migalastat and ERT had comparable effects on renal function. LVMi was significantly decreased with migalastat (−6.6 g/m2 [−11.0, −2.2]); there was no significant change with ERT. Predefined renal, cardiac, or cerebrovascular events occurred in 29 % and 44 % of patients on migalastat and ERT, respectively. During the OLE, GFR remained stable and LVMi reductions were maintained. Migalastat was well tolerated and effective across patient subgroups in both studies. Discussion: In both Studies 011 and 012, renal function remained stable and LVMi decreased with migalastat. In Study 011, GI symptoms also improved. Migalastat has promise as a first-in-class oral chaperone for patients with Fabry disease with amenable mutations. Conflict of Interest declared.

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both lysosomal and extralysosomal glycogen. VAL-1221 has further demonstrated safety in both single- and multi-dose studies of toxicity. Discussion: VAL-1221 represents a novel enzyme replacement candidate with improved targeting to skeletal muscle and enhanced efficacy in mouse models of glycogen storage disease. The ability of VAL-1221 to penetrate muscle fibers via both mannose-6-phosphate receptors and antibody-directed pathways allows for glycogen clearance in both lysosomes and extralysosmal spaces; including, low pH autophagic vacuoles. Efficacy of VAL-1221 supports the muscle-delivery potential of the VAL-1205 antibody-delivery platform; therefore, Valerion is developing clinical-ready material of both the VAL-1221 enzyme-replacement candidate and the VAL-1205 antibody capable of being conjugated to proteins or enzymes deficient in other indications of genetic muscle disease. Conflict of Interest declared.

Migalastat improves gastrointestinal symptoms in patients with Fabry disease: results from a double-blind, placebo-controlled phase 3 trial (FACETS) Schiffmann R 1, Bichet D G 2, Hughes D 3, Giugliani R 4, Wilcox W 5, Shankar S P 5, Germain D P 6, Viereck C 7, Castelli J 7, Yao M 7, Skuban N 7 , Barth J 7 1 Baylor Research Inst, Dallas, United States, 2Hopital du Sacre-Coeur, Univ of Montreal, Montreal, Canada, 3Royal Free Hospital, Univ College London, London, United Kingdom, 4Med Gen Serv, HCPA-UFRGS Porto Alegre, Porto Alegre, Brazil, 5Emory Univ School of Medicine, Atlanta, United States, 6Univ Versailles St Quentin en Yvelines, Montigny, France, 7Amicus Therapeutics, Cranbury, United States

Background: Gastrointestinal (GI) symptoms are a prominent manifestation of Fabry disease, with profound and often debilitating negative effects on patients. Migalastat is a first-in-class, oral pharmacological chaperone for patients with Fabry disease with amenable GLA mutations. Methods: FACETS was a 24-month trial with a 6-month double-blind, placebo-controlled (DBPC) stage, conducted in male and female patients with Fabry disease who were naïve to enzyme replacement therapy. The Gastrointestinal Symptoms Rating Scale (GSRS) was used to assess patientreported GI symptoms in patients with amenable mutations (n = 50) who received migalastat HCl 150 mg every other day or placebo. Results: During FACETS, GI symptoms improved with migalastat in 3 of 5 domains (diarrhea, reflux, indigestion) assessed by GSRS. During the DBPC stage, a statistically significant decrease from baseline to 6 months with migalastat was seen in diarrhea symptoms compared with placebo (P = 0.03). Based on the minimally clinically important difference for diarrhea, an improvement from baseline of ≥0.4 units was seen: 69 % of patients on migalastat had a clinically relevant change vs. 11 % of patients on placebo (P = 0.012). A statistically significant improvement in reflux was seen at 6 months compared with baseline (P = 0.047). During the open-label extension (months 6–24), a statistically significant improvement from baseline was also observed for indigestion (P < 0.05), and there was a trend toward improvement in constipation. Discussion: Treatment with migalastat led to statistically significant, clinically relevant improvements in GI symptoms assessed by GSRS in patients with Fabry disease with amenable GLA mutations. Conflict of Interest declared.

P-542

A novel delivery platform for intracellular and extralysosomal targeting of enzyme-replacement therapeutics Armstrong D D 1 1

Valerion Therapeutics, Concord, United States

Background: Valerion has developed a humanized monoclonal antibody, VAL-1205, capable of serving as a cell-penetrating delivery vehicle. A FAb fragment of the VAL-1205 antibody has been fused with human acid alphaglucosidase to generate an enzyme-replacement candidate, (VAL-1221; FAbrhGAA), capable of being internalized into both lysosomes and into extralysosomal areas via antibody-mediated uptake. Methods: VAL-1221 biodistribution and efficacy has been examined in multiple glycogen storage disease models with both lysosomal and extralysosomal glycogen accumulation. VAL-1221 safety has been demonstrated in mice, rats, and non-human primates. Results: VAL-1221 demonstrated significant greater tissue delivery to skeletal muscle following systemic injection when compared to rhGAA-alone with a percent injected dose to skeletal muscle of greater than 5.0 %. VAL-1221 demonstrated efficacy in multiple mouse models of glycogen storage disease; including, enhanced GAA activity in tissue lysates and improved clearance of

P-543 Comparison of α-galactosidase A activity in white blood cells of patients (pts) with Fabry disease after 2 weeks of exposure to migalastat, agalsidase beta, or agalsidase alfa Johnson F K 1, Valenzano K J 1, Castelli J 1 1

Amicus Therapeutics, Cranbury, United States

Background: Fabry disease is an X-linked disorder of lysosomal αgalactosidase A (α-Gal A) deficiency, leading to substrate accumulation and multiorgan disease. Migalastat HCl, an oral pharmacological chaperone, stabilizes specific mutant forms of α-Gal A, increasing trafficking to lysosomes. α-Gal A activity was assessed in circulating white blood cells (WBCs) due to ease of sampling and exposure to both migalastat and enzyme replacement therapy (ERT). Methods: WBC α-Gal A activity following migalastat was assessed at selected visits in male and female pts with Fabry disease with amenable mutations from randomized phase 3 studies (Study 011, double-blind, placebo-controlled trial; Study 012, open-label trial). WBC α-Gal A activity following single infusions of agalsidase beta or alfa was assessed in a separate open-label trial conducted in male pts with Fabry disease with any mutation (Study 013) at 0, 2, 4, 24, 168, and 336 hr postdose. Two-week exposure of WBC α-Gal A activity was predicted for migalastat using data modeling and simulations and estimated for ERT with noncompartmental analyses. Results: Seven QOD doses of 150 mg migalastat resulted in a simulated overall AUC (2969 hr[pmol/mg/hr]) for WBC α-Gal A activity that was comparable to a single dose of 1 mg/kg agalsidase beta (3091 hr[pmol/mg/hr]) and approximately 6-fold greater than agalsidase alfa (485 hr[pmol/mg/hr]). A single dose of agalsidase beta resulted in higher Cmax (106 pmol/mg/hr), with rapidly declining α-Gal A activity. The simulated AUC for migalastat represented an attenuated Cmax (39.9 pmol/hr/mg) with more consistent levels of activity as a result of QOD dosing over the same 14-day interval. Discussion: In this analysis of 2-week exposure data, α-Gal A activity exposure was comparable between migalastat and agalsidase beta, but was 6-fold greater for migalastat than for agalsidase alfa. Levels of α-Gal A activity were more consistent following 150 mg migalastat QOD compared with ERT. Conflict of Interest declared.

P-544

Adaptive functioning and parental stress in patients with lysosomal storage diseases treated with enzymatic replacement therapy Caviglia S 2, Bottari A 2, Tondo I 2, Deodato F 1, Taurisano R 1, Dionisi-Vici C 1 1 Div Metab Dis, Bambino Gesu Hospital, Rome, Italy, 2Clinical Psychology Unit,Bambino Gesu Ch, Rome, Italy

Background: Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic disorders that result from defects in lysosomal function. LSDs are

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 generally multysistemic diseases with reduced life expectancy. For some LSDs the enzymatic replacement therapy (ERT) is available and it has proven to improve many somatic symptoms and signs of some of these disorders. To our best knowledge, no study has explored the adaptive functioning and the parental stress in this unique population of patients undergoing ERT treatment Methods: The parents of 28 LSDs patients amongst MPS (N = 13; I, I H-S, I H, I S, II, IV-Morquio, VI) Gaucher (N = 6; types I and III) and Pompe (N = 9; infantile and late onset forms) were asked to complete the Parenting Stress Index, Short Form (PSI-SF) questionnaire and take the Vineland Adaptive Behavior Scales (VABS) Survey Form interview. Data collection happened when patients frequented the Hospital for their ERT treatment. Results: The total sample reached significantly lower scores in the daily living skills domain of the VABS compared to the scores of the other domains and the general adaptive quotient of the VABS. Significant differences were also found comparing adaptive functioning with different age groups. No significant result was found comparing parents who reached scores indicative of stress on the PSI-SF (>85 %) and patients with low scores ( A LIPA mutation). SA was hard to get. Treatment started at 1 mg SA/kg/wk and increased to 3–5 mg SA/kg/5 days due to clinical deterioration (twice needing ICU). Key features of physical examination and blood tests were compared from baseline pre-treatment to 5 months of treatment giving also the worst data after some month of treatment. Results: Weight was 4.5 kg (with enlarged organs and ascites; z = −1), and now 8.5 (z = 0). Liver size and spleen size (by ultrasound) of 10/10 cm at baseline (worst after 1 month: 11/13 cm) now 9/11 cm. At baseline (worst) and last results were for ALT/AST 33/214 (worst 569/165) now 97/89 U/l, albumin 30 (23) 39 g/l, total bilirubin 12 (60) now < 10 μmol/l; Hb 4.6 (4.3 after red blood cell transfusion) now 6.6 mmol/l; TG 13.3, now 1.5 mmol/l, LDL-Chol < 0.1 now 2.3 mmol/l, ferritin 14472, now 94 ug/l. Discussion: Although especially TG and ferritin clearly improved in the first month, his hepatosplenomegaly, ascites and intestinal function deteriorated in that period. Growth is difficult to evaluate due to ascites and organomegaly. Clinical condition improved in parallel with liver/spleen size reductions. In patients with infant LAL-D rapid diagnosis and availability of SA is necessary, starting at least at a dose >1 mg/kg/week. To this aim it is of utmost importance to break down the barriers to get the first dose of SA without delay. Treatment needs intensive clinical and biochemical follow-up with careful nutritional adaptations (including the use of a modular feed and also possibly including temporarily total parenteral nutrition), albumin infusions, and ventilatory support. Conflict of Interest declared.

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Efficacy of migalastat in a cohort of male patients with the classical form of Fabry disease in a phase 3 study Germain D P 1, Giugliani R 2, Bichet D G 3, Wilcox W 4, Hughes D 5, Amartino H M 6, Schiffmann R 7, Viereck C 8, Yao M 8, Skuban N 8, Castelli J 8, Barth J 8 Univ Versailles St Quentin en Yvelines, Montigny, France, 2Med Gen Serv, HCPA-UFRGS Porto Alegre, Porto Alegre, Brazil, 3Hopital du Sacre-Coeur, Univ of Montreal, Montreal, Canada, 4Dept of Human Genetics, Emory Univ, Atlanta, United States, 5Royal Free Hospital, Univ College London, London, United Kingdom, 6Hospital Universitario Austral, Buenos Aires, Argentina, 7 Baylor Research Institute, Dallas, United States, 8Amicus Therapeutics, Cranbury, United States 1

Background: Fabry disease is an X-linked lysosomal storage disorder caused by GLA mutations, resulting in deficient lysosomal α-galactosidase A (α-Gal A) activity. Males with “classical” Fabry disease generally have an earlier onset of disease and more severe symptoms. Migalastat is an oral pharmacological chaperone that stabilizes mutant α-Gal A to facilitate normal trafficking to lysosomes. Subgroup analyses were undertaken to explore the benefit of migalastat in this cohort of male patients from a 24-month phase 3 study (ERTnaïve; FACETS). Methods: Male patients with classical phenotype were defined as having multiorgan system involvement (based on medical history and baseline assessments) and peripheral blood mononuclear α-Gal A activity < 3 % (n = 14; mean ± SD 42.4 ± 14 years). Changes from baseline (CFB) were calculated for estimated glomerular filtration rate (eGFRCKD-EPI), left ventricular mass index (LVMi), plasma lyso-Gb3, and diarrhea symptoms based on the Gastrointestinal Symptoms Rating Scale (GSRS-D; scored on a 7-point Likert-type scale [1 = absence of burden to 7 = severe discomfort]). Results: For male patients with classical phenotype, annualized CFB (mean [95 % CI]; mL/min/1.73 m2) to month 24 for eGFRCKD-EPI (baseline: 87.8 ± 33.6) was −0.3 (−2.8, 2.3). The CFB to month 24 for LVMi (baseline: 114 ± 27.3 g/m2; baseline left ventricular hypertrophy was present in 7/14 patients) was −16.7 (−31.1, −2.4). For plasma lyso-Gb3 , CFB (baseline: 99.8 ± 35.3 nmol/L) to month 12 was −36.8 (−69.9, −3.7). For GSRS-D (baseline: 2.5 ± 1.66), the CFB to month 24 was −0.9 (−2.1, 0.4). Discussion: These results demonstrate the clinical benefit of migalastat in male patients with amenable mutations and the classical phenotype of Fabry disease in terms of renal function, LVMi, plasma lyso-Gb3, and diarrhea symptoms. These results are consistent with those seen in the broader study population. Conflict of Interest declared.

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The validation of pharmacogenetics in the identification of patients with Fabry disease for treatment with migalastat Benjamin E R 1, Della Valle C 1, Wu X 1, Katz E 1, Valenzano K J 1, Bichet D G 2, Germain D P 3, Giugliani R 4, Hughes D 5, Schiffmann R 6, Wilcox W R 7, Yu J 1, Kirk J 1, Barth J 1, Castelli J 1 1 Amicus Therapeutics, Cranbury, United States, 2Hopital du Sacre-Coeur, Univ of Montreal, Montreal, Canada, 3Univ Versailles St Quentin en Yvelines, Montigny, France, 4Med Gen Serv, HCPA-UFRGS Porto Alegre, Porto Alegre, Brazil, 5Royal Free Hospital, Univ College London, London, United Kingdom, 6Baylor Research Institute, Dallas, United States, 7Dept of Human Genetics, Emory Univ, Atlanta, United States

Background: Fabry disease is an X-linked lysosomal storage disorder caused by CLN8p. mutations, resulting in deficient lysosomal α-galactosidase A (α-Gal A) activity and progressive cellular accumulation of globotriaosylceramide (GL3). Migalastat is a pharmacological chaperone that stabilizes specific mutant forms of α-Gal A (termed amenable), leading to increased physical stability, lysosomal trafficking, and intracellular activity of the enzyme.

S220 Methods: Each of 600 Fabry disease-causing mutations were expressed in HEK-293 (HEK) cells, and increases in α-Gal A activity in response to migalastat were measured using a Good Laboratory Practice (GLP)-validated in vitro assay. In total, 268 migalastat-amenable mutant forms of α-Gal A were identified, defined by a relative increase of ≥1.20-fold and an absolute increase of ≥3.0 % wild-type α-Gal A activity in the presence of 10 μM migalastat. Clinical validation was assessed using pharmacodynamic responses in phase 2/3 studies (73 unique amenable and nonamenable mutations in 160 male and female patients with Fabry disease). Results: Comparison of mutant α-Gal A responses in the GLP HEK assay and those of white blood cell α-Gal A to migalastat from male patients in phase 2 and 3 studies exhibited high sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively; ≥0.875). GLP HEK assay results were predictive of decreases in kidney GL-3 in males and plasma globotriaosylsphingosine in both males and females (sensitivity: 1.0 and 0.93, respectively; specificity: 1.0 and 0.69; PPV: 1.0 and 0.84; NPV: 1.0 and 0.85). The clinical study subset of amenable mutations (n = 51) were representative of all 268 amenable mutations identified by the GLP HEK assay. Discussion: The GLP HEK assay is now a clinically validated method of identifying male and female patients with Fabry disease who are candidates for treatment with migalastat. Conflict of Interest declared.

P-548

Clinical history of a cohort of Gaucher type 1 patients treated with ERT from childhood to adulthood. Moro A 1, Cecchinel S 1, Macor D 1, Ciana G 1, Da Riol M R 1, Dardis A 1, Bembi B 1 1

Reg Coord Cent of Rare Dis, Univ Hosp, Udine, Italy

Background: Gaucher disease (GD) is a lysosomal storage disorder characterized by a multiple organ involvement. Enzyme replacement therapy (ERT) has been available since 1991. The study was aimed to describe the clinical outcomes and costs burden of ERT in a pediatric cohort of GD1 patients treated with from childhood to adulthood. Methods: A retrospective cohort study of 15 pediatric GD1 patients receiving ERT for at least 10 years (y). Subjective symptoms, organomegaly, bone imaging (BMD z-score) and laboratory parameters (Hb, platelets, transaminases, IgG, lipidic pattern) were evaluated. A questionnaire was administered to 8 patients to evaluate yearly economical costs and travel burden to receive ERT. Results: The most prevalent GD1 genotype was N370S/L444P. The mean age at diagnosis was 4,66 y (range 1–13), while mean age at ERT beginning (T0) was 9,46 y (range 3–15). Three patients were splenectomized. At T0, ERT median dose was 42 U/Kg/month (range 25–60), while it was 50 U/kg/month (range 24–60) after 10 y of ERT (T10). At T10, 10 patients showed normal laboratory parameters, spleen volume < 5 x normal value (n.v) and liver volume < 1.25 x n.v. BMD z-score was normal in 14 patients. Pain symptomatology (not bone related) was present in 10 patients at T10. The mean travel distance to receive ERT was 230.9 km, while the mean cost was 540.8 euro. Discussion: The study confirmed the good clinical and laboratory response in a cohort of GD1 patients receiving a quite low (500 mutant forms of α-Gal A were expressed in HEK-293 cells, and α-Gal A activity was measured in the presence/absence of 10 μM migalastat; 268 amenable mutant forms were identified. The proportions of patients with Fabry disease-related involvement of ≥2 organ systems and the mutation phenotypes were determined (classical phenotype identified by baseline α-Gal A < 3 % of normal). Results: In FACETS, all male patients and 88 % of female patients with amenable mutations had disease involvement in ≥2 organ systems, >90 % of patients had elevated plasma globotriaosylsphingosine, and 60 % of all patients and 87 % of male patients had mutations associated with a literature-defined classical phenotype. In ATTRACT, 81 % of patients had involvement of ≥2 organ systems and 36 % had mutations associated with a classical phenotype. To date, 68 % of the known 268 migalastat-amenable mutations identified in the medical literature are associated with a classical phenotype. Discussion: A majority of patients with amenable mutations in the phase 3 migalastat trials had involvement of ≥2 organ systems and a genotype associated with a literature-defined classical phenotype. Conflict of Interest declared.

22. Glycosylation disorders/CDG, protein modification disorders

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Evaluation of the global coagulation balance among PMM2-congenital disorder of glycosylation patients, using the thrombin generation assay Pascreau T 1 2, Cathala F 1, Lasne D 1, Seta N 3, Guemann A S 4, De Lonlay P 4 , Borgel D 1 2 1 APHP, Lab Hematology, Necker hospital, Paris, France, 2INSERM UMR S1176, Univ Paris Sud, Le Kremlin Bicetre, France, 3APHP, Biochemistry, Bichat hospital, Paris, France, 4APHP, Metabolic dis, Necker hospital, Paris, France

Background: Patients who suffer from phosphomannomutase 2 deficiency (PMM2-CDG) may develop acute vascular events such as stroke-like episodes. Clotting factors IX and XI, and coagulation inhibitors, such as antithrombin and protein C, are often decreased, and the coexistence of combined coagulation factor impairments creates an unpredictable imbalance between hemorrhagic and thrombotic risk. To evaluate the coagulation balance in PMM2-CDG patients, we used a global test, the thrombin generation assay. By adding soluble thrombomodulin (sTM), both procoagulant and inhibitor factors are explored. Methods: 10 PMM2-CDG patients with a median age of 11 years (range, 3– 16 years) were enrolled in the study. 4 of them have already presented strokelike episodes. They were compared to 10 controls matched in age and sex. Thrombin generation was assessed using the calibrated automated thrombogram method with and without sTM.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Results: Thrombin generation, measured without sTM, was increased in PMM2-CDG patients compared to controls. Both endogenous thrombin potential (ETP) and the highest thrombin concentration reached (Peak) were increased (1322 [1092–2251] vs. 970 [915–1108] nM.min; p = 0.003 and 270 [221–288] vs. 210 [167–244] nM; p = 0.0433 respectively). However, no significant difference in thrombinogram parameters (ETP and Peak) was observed between PMM2-CDG patients who suffered from stroke-like episodes and those free of these events, even though a tendency to a more procoagulant state was observed. In the presence of sTM, ETP was significantly higher only when focused on patients that experienced stroke-like events compared to the others (1270 [1098–1771] vs. 329 [258–916] nM.min; p = 0.0381). Discussion: Despite a decrease in both procoagulant and anticoagulant factors, PMM2-CDG patients have a global hypercoagulant profile. This procoagulant balance is even more pronounced in patients that experienced stroke-like events because of an altered sensitivity to the protein C system.

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Liver involvement in congenital disorders of glycosylation (CDG): literature review Marques-da-Silva D 1 2 6, Monticelli M 1 3, Dos Reis Ferreira V 2 6, Ferro T 1 2 6 , Janeiro P 4, Videira P A 1 2 6, Jaeken J 5 6, Cassiman D 5 6 1 Universidade Nova de Lisboa, Caparica, Portugal, 2Portuguese Association for CDG, Lisboa, Portugal, 3Universita degli Studi Napoli FedericoII, Napoli, Italy, 4Centro Hospitalar Lisboa Norte HSM, Lisboa, Portugal, 5CDG and Allies PPAIN, Lisboa, Portugal, 6Center for Metabolic Disease, Leuven, Belgium

Background: Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases caused by defects in glycosylation. Nearly 100 CDG are known so far. Patients present a great phenotypic diversity ranging from poly- to mono-organ/system involvement with very mild to extremely severe expression. This literature review summarizes the liver involvement reported in human CDG. Methods: The present literature review is based on 192 papers identified via a search of the Medline database, using PubMed as a search engine. Results: Liver involvement was present in about 22 % of CDG. Two groups could be distinguished: 4 CDG with major or isolated liver disease, and 16 CDG with minor liver dysfunction. The CDG with major liver involvement comprise MPICDG, PIGM-CDG, CCDC115-CDG and TMEM199-CDG. Among the CDG with minor liver involvement are 7 N-glycosylation diseases (including PMM2CDG), 1 O-glycosylation disorder (B4GALTL-CDG), 1 GPI anchor synthesis disorder (PIGA-CDG), and 7 combined glycosylation defects. The major signs and symptoms reported are hepatomegaly, hepatic cirrhosis, fibrosis, steatosis, increased serum transaminases, coagulopathies and Budd-Chiari syndrome. Discussion: A minority of CDG show manifestations of liver involvement. In only four of these, liver disease is predominant or even isolated. While CDG, at the cause of the underlying liver disease, can often not be treated yet, the liver-related problems e.g. ascites, liver failure, variceal bleeding often can be treated or prevented, with the help of experienced Hepatologists. This makes it worthwhile to screen for underlying liver disease in CDG, as it is worthwhile to screen for CDG in patients with cryptogenic liver disease. This study is a result of a collaborative study between patient advocacy groups, families and professionals(CDG Professionals and Patient Associations International Network; CDG & Allies – PPAIN). P-552

CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation Jansen J C 1, Cirak S 3, Van Scherpenzeel M 1, Foulquier F 2, Marquardt T 4, Lefeber D J 1

1

Radboud University Medical Center, Nijmegen, Netherlands, 2University of Lille, Lille, France, 3Uniklinik Koeln, Koeln, Germany, 4Universitatsklinikum Muenster, Muenster, Germany Background: Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell biology in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Methods and results: Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation in CCDC115 (c.92 T > C; p.Leu31Ser), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G > T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated serum bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, restored after complementation with wild-type CCDC115. PSIBLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. Discussion: Our data, including the clinical spectrum which is distinct from that seen in defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.

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Exome sequencing of patients with positive screening of congenital disorders of glycosylation (CDG) type I revealed mitochondrial diseases due to POLG and Twinkle mutations Vuillaumier-Barrot S 1 2 6, Dupre T 1 2 6, Bruneel A 1, De Lonlay P 3 4, Servais L 5, Moore S 2 6, Seta N 1 3 1 Biochimie, Hop Bichat-Claude Bernard, PARIS, France, 2INSERM U1149 CRB3, PARIS, France, 3Universite Paris Descartes, PARIS, France, 4Hop Necker Enfants Malades, Pediatrics, PARIS, France, 5Inst de Myologie, Hop Pitie salpetriere, PARIS, France, 6Univ Denis Diderot, Paris 7, PARIS, France

Background: CDG-I is caused by defects in the biosynthesis of the lipid-linked oligosaccharide required for protein N-glycosylation in the endoplasmic reticulum. This group contains more than 30 different molecular subtypes. After exclusion of PMM2-CDG and MPI-CDG (normal leucocyte enzymatic activities), CDG-I patients (serum glycoprotein hypoglycosylation) are usually submitted to next generation sequencing. METHODS: Eighteen patients with CDG-I without PMM2-CDG and MPICDG, were submitted to whole exome sequencing (granted by “Fondation Maladies Rares” and “Connaître les syndromes cérébelleux” association). Identified mutations were confirmed by Sanger sequencing. Results: Among 18 exomes performed, we identified 9 patients with mutations in known CDG-related genes: MPDU1, ALG11, ALG3, DHDDS, RFT1, DPM2, SRR4 and two STT3A (with only one heterozygous mutation found, cDNA study underway); one patient with a likely new CDG (functional study underway) and 3 non-CDG patients: one adult patient presented with ataxia and severe hypo-albuminemia with a homozygous deletion in APTX, one patient with mutated POLG and another one with mutated Twinkle/C10orf2. Five patients remained unexplained (28 %). In the APTX deficient patient, results suggest that lack of aprataxin could affect mitochondrial functions and consequently glycosylation, although it could also be a false positive related to his alcohol consumption. Both mitochondrial patients had a partial abnormal

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Facile CDG diagnostics via mass spectrometry and clinical exome sequencing

(33 %) with identified mutations in one of the 16 known α-DGpathy genes. In a LGMD patient a heterozygous ins-del mutation was identified in SLC35A2 which is usually associated with another type of glycosylation (CDG). This gene encoding for a UDP-galactose transporter could be involved in glycosylation of α-DG.. The efficacy of mutation identification was comparable between NGS and Sanger sequencing. The only limit of our NGS panel is the low coverage of the region of the frequent FKRP mutation: c.826C > A, p.Leu276Ile. Discussion: This new technology, assessing in one single step all the genes linked to α-DGpathies, leads to a gain of several months in the establishment of molecular diagnosis and thus benefits the families waiting for prenatal diagnosis. In addition, the design of our panel allows screening in parallel for other glycosylation genes potentially involved in α-DGpathies.

Van Scherpenzeel M 1, AbuBakar A 1, Huijben K 1, Zijlstra F 1, Ashikov A 1, Lefeber D J 1

P-556

glycosylation profile, and both presented severe progressive disease with fatal outcome rather than fixed disease, as in true CDG patients. Discussion: Our results enlarge the group of genes associated with abnormal glycosylation of serum glycoproteins. CDG-I and progressive rather than fixed disease should suggest a mitochondrial disease and lead to POLG or Twinkle sequencing.

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1

Radboud University Medical Center, Nijmegen, Netherlands

Background: Subtype identification in the Congenital Disorders of Glycosylation (CDG) has been a challenge due to the large clinical heterogeneity and the poorly understood biochemical mechanisms. Introduction of clinical exome sequencing (CES) in CDG diagnostics has revolutionized gene identification, while CES in itself is yielding glycosylation defects with normal transferrin glycosylation in a growing number of cases. We are combining glycomics profiling with clinical phenotyping and CES to increase the percentage of solved cases. Methods and results: High resolution nanoLC-chip-QTOF mass spectrometry of intact serum transferrin shows highly characteristic, diagnostic glycoprofiles for several CDG-II subtypes, such as MAN1B1-CDG, SLC35A2-CDG, PGM1-CDG, etc. Total serum N-glycan profiling using Porous Graphitized Carbon chip-QTOF mass spectrometry shows highly characteristic profiles for several CDG-I subtypes, such as ALG1-CDG. Combination of these mass spectrometry approaches with clinical exome sequencing and clinical phenotyping allows facile identification of the majority of known CDG-I and CDGII subtypes in a diagnostic setting. An increasing number of genetic defects in sugar metabolism shows a normal transferrin profile, e.g. PGM3-CDG, GFPT1-CDG, GMPPB-CDG and GNE-CDG. To improve functional confirmation of these gene defects, we have established a highly sensitive and specific mass spectrometry assay of nucleotide sugars in patient cells, which provides actual intracellular levels and enzyme activities as a functional readout. Discussion: Application of diverse glycomics profiling methods in combination with clinical exome sequencing and clinical phenotyping has significantly increased the time-to-diagnosis in both CDG-I and CDG-II in our Center.

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Transferrin and total serum glycoprofiling for diagnosis and therapy monitoring in phosphoglucomutase 1 deficiency AbuBakar A 1, Voermans N C 1, Morava E 2, Van Scherpenzeel M 1, Lefeber D J 1 1 Radboud University Medical Center, Nijmegen, Netherlands, 2Tulane University Medical School, New Orleans, United States

Background: Phosphoglucomutase 1 (PGM1) deficiency has been shown to be a Congenital Disorder of Glycosylation (CDG). Recently, Nanochip-C8QTOF mass spectrometry (MS) of intact transferrin revealed features of CDGI (lack of complete N-glycans) and CDG-II (defect in N-glycan processing) as diagnostic for PGM1-CDG, while galactose supplementation improved glycosylation. Methods and results: We examined 18 PGM1-CDG patients by transferrinspecific and global serum N-glycoprofiling, some of them on galactose therapy. Nanochip-C8-QTOF MS and Nanochip-PGC-QTOF MS were used to profile the transferrin N-glycans and total serum N-glycans, respectively, and this profiling was compared with clinical assessments. We determined a specific PGM1-CDG glycobiomarker based on a combination of selected glycan signatures of transferrin N-glycans. Total serum N-glycan profiling showed an increased fucosylation and N-glycans lacking galactose; however, the profile was not diagnostic for PGM1-CDG. For treatment monitoring, we proposed three main biochemical indexes from transferrin N-glycan profiling: a normal glycosylation index, a lack of complete glycans index and a lack of galactose index. Data upon galactose treatment show that the lack of glycans is restored more slowly than the lack of galactose. Total serum N-glycan profiling showed improvement of the fucosylation status in PGM1-CDG patients. Discussion: Transferrin glycoprofiling showed specific glycobiomarkers for the diagnosis of mild and severely affected PGM1-CDG patients and can be used to monitor the effectiveness of galactose treatment in PGM1-CDG.

NGS: a new strategy for the molecular diagnosis of α-dystroglycanopathies Bouchet-Seraphin C 1 2, Chelbi M 2, Reocreux M 2, Gazal S 2, VuillaumierBarrot S 1 2, Boileau C 2, Seta N 1

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1 Biochimie, Hop Bichat-Claude Bernard, PARIS, France, 2Genetics, Hop Bichat-Claude Bernard, Paris, France

A population based study on congenital defects of protein N-glycosylation experience in clinical and genetic diagnosis

Background: α-Dystroglycanopathies are characterized by a broad clinical spectrum (from type II lissencephaly-LISII, to Congenital Muscular Dystrophy-CMD and Limb Girdle Muscular Dystrophy-LGMD) with poor phenotype–genotype correlation. Up to now mutations in 16 genes link to the glycosylation pathway of α-dystroglycan have been identified. αDGpathy is a good candidate for NGS strategy based on a glycosylation gene panel. Methods: Library preparation for NGS was accomplished using the HaloplexPCR target enrichment system with a Miseq reagent kit (209 kb – 45 glycosylation genes). Results: DNA from 23 new cases was tested, leading to the identification of mutations in 8 different genes. Among the 23 cases, a molecular diagnosis was established in 8 LISII fetuses (72 %) and 4 patients with CMD or LGMD

Perez-Cerda C 1, Giros M L 2, Serrano M 4, Ecay M J 1, Gort L 2, Perez Duenas B 4, Medrano C 1, Garcia-Alix A 3, Artuch R 5, Briones P 2, Perez B 1 1 CEDEM-UAM,CBM-SO,IDIPAZ,CIBERER, Madrid, Spain, 2Hospital Clinic-IBC,IDIBAPS,CIBERER, Barcelona, Spain, 3Div Neonatol, IRPHSJD, Univ Barcelona, Barcelona, Spain, 4Neuropediatr Dep, IRP-HSJD, CIBERER, Barcelona, Spain, 5Clin Biochem Dep, IRP-HSJD,CIBERER, Barcelona, Spain

BACKGROUND: Congenital disorders of glycosylation (CDG) covers a group of genetic diseases involving defects in the synthesis of glycans and their binding to proteins and lipids. They are characterized by great clinical and genetic heterogeneity. We describe the clinical, biochemical and genetic

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 features of patients with congenital defects of protein N-glycosylation, identified in Spain over the last 15 years. Case report: Patients were selected among those presenting with multisystem disease of unknown aetiology. Along with tests to detect the glycoprotein isoforms of transferrin and lipoprotein apoC3, dolichols were analysed in serum, phosphomannomutase and phosphomannoisomerase activities were measured in fibroblasts, and conventional (Sanger) or massive DNA sequencing was performed to identify genes and mutations. Results: 97 patients were diagnosed with 18 different congenital defects of glycosylation. Some 75 % of these had PMM2-CDG and presented with a very heterogeneous mutational spectrum. 37 different mutations of 126 alleles were detected (29 missense mutation, 3 nonsense mutation, 4 affecting mRNA processing, and one large deletion), with p.Arg141His the most common alteration. Four mutations were novel, p.Gln33Ter; p.Tyr64Thrfs*11; p.Val158Ala and p.Thr237Lys. The remaining 17 patients showed mutations in one of the following genes: MPI, PGM1, GFPT1, SRD5A3, DOLK, DPGAT1, ALG1, ALG6, RFT1, SSR4, B4GALT1, DPM1, COG6, COG7, COG8, ATP6V0A2 and CCDC115. Discussion: Based on this national population study of CDG, an algorithm that may guide and optimize CDG diagnosis and reduce the timeframe from clinical suspicion to genetic confirmation is proposed. The different CDG identified in Spain are in accordance with previously published reports from other countries, thus contributing to the knowledge of CDG world-wide. A clear predominance of PMM2 deficiency was detected, and four novel PMM2 mutations described.

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Galactose supplementation in SLC35A2-CDG: results after 24 weeks of treatment in an Italian patient Barone R 1 2 3, Striano P 4, Sturiale L 3, Garozzo D 3, Messina A 3, Jaeken J 5, Morava E 5, Fiumara A 2 1 Child Neuropsychiatry, Univ Child Hosp, Catania, Italy, 2Div Metab Dis, Univ Child Hosp, Catania, Italy, 3 IPCB - CNR, Catania, Italy, 4 Neuromuscular Dis, Gaslini Hospital, Genoa, Italy, 5Div Metab Dis, Univ Child Hosp, Leuven, Belgium

Background: SLC35A2-CDG (MIM314375) is an X-linked disorder due to UDP-galactose transporter mutations, and characterized by severe developmental disability, epilepsy, cerebral and/or cerebellar malformation and variable multisystem involvement. Biological evidence and preliminary results in one previous patient support a therapeutic approach by galactose supplementation. Case report: A female SLC35A2-CDG patient aged 49 months underwent galactose dietary control and oral galactose supplementation from 0.5 g/kg per day for 4 weeks to 1 g/kg per day for 20 weeks. She showed dysmorphism, early epileptic encephalopathy with burst suppression EEG pattern, severe developmental disability, Dandy-Walker malformation with posterior arachnoid pouch, and impaired vision with hypopigmentary retinal spots. Routine laboratory analyses, urinary galactose, EEG and liver ultrasound, serum Transferrin (Tf) isoelectrofocusing (IEF) and total plasma N-glycome by MALDI analyses were monitored during galactose treatment. Results: After 4 weeks of galactose supplementation (0.5 g/kg per day), serum Tf IEF improved and agalactosylated N-glycans from total plasma glycoproteins were significantly reduced. Increasing galactose supplementation to 1 g/kg per day did not result in further changes of protein glycosylation in the next 20 weeks. Liver tests and ultrasound remained normal throughout treatment. Seizure frequency and duration decreased over time. No side-effects were recorded. Discussion: Galactose supplementation was promptly effective in reversing glycosylation defects of serum glycoproteins in this patient with SLC35A2-CDG. Further studies are warranted to optimize clinical protocols.

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Metabolic insights into the pathomechanism of orofacial malformation: prevalence and clinical variability of cleft palate and other congenital malformations among PGM1-CDG patients Wong S Y 1, Rymen D 2, Beamer L 3, Perez S J 1, Perez S J 1, Kozicz T 1, Morava E 1 4 1

Hayward Genetics Center, Tulane Univ SOM, New Orleans, United States, Ctr for Metab Dis, University Hospitals, Leuven, Belgium, 3Biochem and Chem Depts, Univ of Missouri, Columbia, United States, 4Dept of Pediatrics, Tulane Univ SOM, New Orleans, United States 2

Background: Cleft palate is a common congenital malformation with an incidence of 1–2 in 1000 live births. Aberrations in well-studied signaling pathways have been shown to lead to pathogenesis of the palate. Deficiency in phosphoglucomutase 1 (PGM1) not only leads to aberrant carbohydrate metabolism and protein N-glycosylation, but is also unexpectedly associated with congenital anomalies. Methods: We characterized a cohort of 28 previously and newly identified patients with an inherited deficiency in phosphoglucomutase 1 (PGM1CDG) and studied the association between PGM1 enzyme activity and the severity of congenital malformations. In three unpublished patients, we conducted glycomics studies and RNA expression analysis in vitro. Results: Strikingly, 70 % of the cohort presented with cleft palate, with or without bifid uvula, Pierre Robin sequence, and other congenital malformations, such as VSD, mitral prolapse, missing vertebrae, and anal atresia. We did not find a correlation between PGM1 residual enzyme activity and the severity of congenital malformations. In vitro studies demonstrated a clear association between PGM1 deficiency and protein hypoglycosylation. In a panel of genes involved in glycosylation, we discovered aberrant RNA expression of B3GALTL, which is involved in O-glycosylation and implicated in the orofacial defects of Peters plus syndrome. Discussion: Our preliminary data suggest a potential novel role of PGM1 in the gene regulatory network of orofacial development, which to date has not been considered. Given that embryonic development is a metabolic demanding process, aberrant carbohydrate metabolism due to PGM1 deficiency may cause a secondary disruption in normal signaling. For example, congenital malformations have long been associated with chronic hyperglycemia, such as in the case of gestational diabetes mellitus; however, the mechanism has not yet been identified.

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Clinical description and long-term outcome in PMM2-Congenital Disorder of Glycosylation: a series of 96 French patients De Lonlay P 4, Roda C 1 4, Monin M L 6, Dupre T 1 2 5, Vuillaumier-Barrot S 1 25 , De Barace C 8, Francannet C 7, Heron D 6, Seta N 1 3, French Metabolic Disease Health Network N 4 1 Biochimie, Hop Bichat-Claude Bernard, PARIS, France, 2INSERM U1149 CRB3, PARIS, France, 3Universite Paris Descartes, PARIS, France, 4Hop Necker Enfants Malades, Pediatrics, PARIS, France, 5Univ Denis Diderot, Paris 7, PARIS, France, 6Genetics, Hop Salpetriere, Paris, France, 7Genetics, CHU Clermont, Clermont-Ferrand, France, 8Pediatrics, Saint-Brieuc, France

Background: The congenital disorders of glycosylation (CDG) comprise a rapidly growing group of inherited multisystem disorders commonly associated with severe psychomotor and mental disability, and defective glycosylation of glycoproteins and glycolipids. PMM2-CDG is the most frequently diagnosed protein N-glycosylation disorder. In order to better delineate the long-term outcome of patients with PMM2-CDG, we analysed the clinical and biological parameters in a large cohort of patients followed up in France. Methods: 96 patients (86 families, 41 males; mean age: 12.3 years [1 m-50.3 y]) with PMM2-CDG were included in this study. Clinical, biological and

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outcome data were retrospectively collected using a standardized questionnaire. Results: Diagnosis of PMM2-CDG was made mainly in front of neurological symptoms (hypotonia, mental disability, cerebellar ataxia). 15 % died at a mean age of 3.8 years (failure to thrive, pericarditis, infection, ..). Neurological symptoms (mental disability and cerebellar ataxia) were noted in all but one. 38 patients had a postnatal multivisceral presentation including at least one visceral feature (cardiomyopathy, pericarditis, chronic diarrhoea, ascites, hepatic failure, portal hypertension, nephrotic syndrome, tubulopathy, nutritional support). The neurological score was not significantly different between the patients with or without visceral involvement, except hypotonia and epilepsy. Ovarian dysplasia was very frequent. No genotype-phenotype correlation could be found, due to the wide variety of identified mutations. The biological presentations mainly included hepatic cytolysis, hypothyroidism, haemostatic anomalies (decrease of factor XI, antithrombin III and protein C). Since stroke-like episodes are frequent, prevention of thromboembolic and bleeding accidents is crucial. Discussion: An extensive description of PMM2-CDG patients should allow a better targeting of patients to screen.

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Diminished convergent extension and disturbed TGF-ß/BMP and FGF signaling in Xenopus Pmm2-CDG morphants Himmelreich N 1, Dimitrov B 1, Kaufmann L T 2, Bartram C R 2, Hoffmann G F 1, Thiel C 1 1 Center for Child and Adolescent Medicine, Heidelberg, Germany, 2Institute of Human Genetics, Heidelberg, Germany

Background: Monogenetic defects affecting the complex process of glycoconjugate biosynthesis in humans lead to Congenital Disorders of Glycosylation (CDG), presenting mostly as multi-organ disease with clinical characteristics such as mental and psychomotoric disability, seizures, hypotonia, cerebellar hypoplasia and ataxia, hepatomegaly, cardiomyopathy and dysmorphism [1]. Since the synergy between signal transduction and protein glycosylation for organ dysfunction is still insufficiently analyzed, we generated a frog model for the most common N-glycosylation defect, PMM2-CDG. The induced hypoglycosylation provoked an altered morphogenetic phenotype in Xenopus Pmm2 embryos due to an impaired non-canonical Wnt signaling [2]. Methods and results: Here we show that the lack of phosphomannomutase 2 (Pmm2) is associated with significantly disturbed tissue separation movements like the convergent extension. Additionally, in-situ hybridization experiments in combination with realtime analyses further revealed that the TGF-ß/BMP and FGF pathways are rely on glycosylation as well. Discussion and conclusion: Our data implicate that the organ dysfunction found in CDG patients is already induced by malfunction of glycosylation dependent signal cascades during the early embryogenesis which are essential for e.g. tissue and organ development [3; 4]. 1 Cylwik B, Naklicki M, Chrostek L et al. (2013) Congenital disorders of glycosylation. Part I. Defects in protein N-glycosylation. Acta Biochim Pol 60:151–161 2 Himmelreich N, Kaufmann LT, Steinbeisser H, Körner C, Thiel C (2015) Lack of phosphomannomutase 2 affects Xenopus laevis morphogenesis and the noncanonical Wnt5a/Ror2 signalling. J Inherit Metab Dis. 2015; 38:1137-46 3 Pires-da Silva A, Sommer RJ (2003) The evolution of signalling pathways in animal development. Nat Rev Genet 4:39–49 4 Heisenberg CP, Bellaiche Y (2013) Forces in tissue morphogenesis and patterning. Cell 153:948–962

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A new case of SLC35A2-CDG with relatively mild phenotype and our experience with D-galactose treatment Ounap K 1 2, Vals M A Reimand T 1 2 4

2 3

, Pajusalu S

2 4

, Lefeber D J 5, Morava E

6 7

,

1

Dept Pediatrics, University of Tartu, Tartu, Estonia, 2Dept Genetics, Tartu Univ Hospital, Tartu, Estonia, 3Child Clinic, Tartu Univ Hospital, Tartu, Estonia, 4Inst Biomed Transl Med, Univ Tartu, Tartu, Estonia, 5Radboud University Medical Center, Nijmegen, Netherlands, 6Univ Med School Leuven, Leuven, Belgium, 7Hayward Gen Ctr, Tulane Univ Med Sch, Tulane, United States Background: Congenital disorders of glycosylation (CDG) are a group of hereditary metabolic diseases characterized by abnormal glycosylation of proteins and lipids. Here we report on a new case with a defect of the UDPgalactose transporter, encoded by the SLC35A2, in the Golgi apparatus and our experience with treatment. Case report: He was born at term. During infancy delayed developmental milestones were noticed. He was investigated at the age of 17 m because of craniosynostosis (premature closure of sutura sagittalis superior). He showed developmental disability, short stature (−3 SD), facial dysmorphism and muscular hypotonia. EEG was normal. Brain MRI showed symmetric hyperintense signals in the basal ganglia which raised a suspicion of Leigh syndrome. Results: Transferrin isoelectric focusing for CDG screening showed a type 2 pattern. Isoelectric focusing of plasma apolipoprotein CIII was normal. Exome sequencing revealed a novel hemizygous de novo mutation c.670C > T (p.Leu224Phe) in the SLC35A2 gene. Transferrin QTOF mass-spectrometry analysis in two independent serum samples showed a prominent loss of one sialic acid (21 %, normal < 5–6 %) and several minor glycan peaks showing a lack of galactose and sialic acid as seen in SLC35A2-CDG. The loss of one sialic acid could not be explained by secondary causes. Treatment with Dgalactose was started at 2.5 y (1.5 mg/kg/day, 6 m ago). We have noticed normalization of coagulation factors and of transferrin glycosylation, and disappearance of the subhepatic fluid seen on ultrasound. He is growing well (3 cm during last 10 m), and is more active and bright. Craniosynostosis has not progressed during the evaluation period. Discussion and conclusion: We describe the use of dietary galactose supplementation in a patient with SLC35A2-CDG and we noticed some effect. Nevertheless, for definite conclusions, longer evaluation periods and larger groups of patients are needed.

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Screening of FDA approved drugs in fibroblasts derived from PMM2CDG patients Andreotti G 1, Cimmaruta C 1 2, Citro V 2, Liguori L 2, Minopoli N 2, Cubellis MV2 1 Institute of Biomolecular Chemistry-CNR, Pozzuoli-Naples, Italy, 2Dept Biology, Univ Federico II Naples, Naples, Italy

Background: PMM2-congenital disorder of glycosylation (CDG-1a or Jaeken syndrome) is an autosomal recessive disease without a cure. All patients have residual activity because complete loss of activity is not compatible with life. Patients could benefit from the enhancement of PMM2 expression or from the stabilization of the endogenous protein (1,2). Methods: Fibroblasts harbouring the mutations V231M/R141H, G15S/D223N, V129M/R141H, R141H/F119L and F119L/F119L were used. We mined the literature and the Gene Expression Omnibus (GEO) looking for FDA approved drugs associated with PMM2 and/ or with protein stability. Results: Lactacystin and bortezomib, which are proteasome inhibitors, had no effect. Insulin had a small effect on the amount of PMM2, but not on Icam-1, a biomarker of PMM2 activity in the cells. Glyburide and metformin were also tested, but had no effect. Fluoxetine was tested on healthy fibroblasts and fibroblasts harbouring the mutation V231M/R141H. Treatment with fluoxetine resulted in a higher PMM2 concentration and total activity. Discussion and conclusion: We believe that drug repositioning should be considered as the first option in the development of pharmacological therapies for rare diseases, because, if it is successful, the progress from test-tube to bed-side is greatly accelerated (3). 1) Andreotti G, Pedone E, Giordano A, Cubellis MV. 2013. Biochemical phenotype of a common disease-causing mutation and a possible therapeutic

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 approach for the phosphomannomutase 2-associated disorder of glycosylation. Mol Genet Genomic Med 1:32–44. 2) Andreotti G, Monti MC, Citro V, Cubellis MV. 2015. Heterodimerization of Two Pathological Mutants E nhances the Activity of Human Phosphomannomutase2. PLoS One 10:e0139882. 3) Hay Mele B, Citro V, Andreotti G, Cubellis MV. 2015. Drug repositioning can accelerate discovery of pharmacological chaperones. Orphanet J Rare Dis 10:55.

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Assessment of a CDT kit for screening of Congenital Disorders of Glycosylation and its comparison with transferrin isoform analysis by HPLC Dave M B 1, Dherai A J 1 2, Udani V P 3, Hegde A U 4, Desai N 3, Ashavaid T F 1 2 1 Res Lab, P D Hinduja Hosp and MRC, Mumbai, India, 2Biochem sec, P D Hinduja Hosp and MRC, Mumbai, India, 3Ped Neurol Dept, PD Hinduja Hosp and MRC, Mumbai, India, 4Ped Neurol Dept, Jaslok Hosp, Mumbai, India

charge, i.e. sialylation, or molecular mass changes, routinely evidenced in our lab by capillary electrophoresis (CE) or Western blot (WB), respectively. Case report: Among the 1,500 samples yearly screened for CDG using CE in our hospital laboratory, about 10 patients per year present a typical CDG 1 pattern. Lately, two of them were of special interest: one was a mentally disabled Lebanese newborn girl presenting cerebellar hypoplasia; her molecular diagnosis is in progress. The other one was a 1-year old boy presenting with epilepsy, cerebral atrophy, protein-losing enteropathy, hypoalbuminemia and abnormal coagulation. He was finally diagnosed with ALG8-CDG (CDG 1 h). Results: In these 2 patients, CE analysis of Trf revealed patterns showing abnormal peaks compatible with protein polymorphism and greatly complicating CDG screening. In order to eliminate an underlying glycosylation abnormality, a WB for different serum glycoproteins was performed and showed a typical CDG 1 pattern for transferrin, α1-antitrypsin, α1- acid glycoprotein and haptoglobin. The serum two dimensional electrophoresis patterns of various Nglycoproteins also showed abnormal spots corresponding to hyposialylation associated with a loss of molecular mass as typically seen in CDG-I. Discussion: Trf polymorphism is benign but can complicate the interpretation of the isoform patterns and thus CDG screening! One single technic is insufficient to consider a CDG screening positive.

P-566 Background: Healthcare advancements in India are backed by several laboratories of global standards equipped with techniques like HPLC, Capillary Zone Electrophoresis (CZE) and others. However, the expertise to detect rare genetic disorders is scarce thus limiting their diagnosis in spite of a high prevalence. Congenital disorders of glycosylation (CDG) are such group of rare disorders, with a wide clinical spectrum. Availability of a test in routine labs would help to look for CDG in many patients. In view of this, we evaluated a commercially available CDT kit and CZE for screening N-glycosylation defects. Methods: Isoform patterns of 80 serum samples including 30 healthy individuals and 50 patients received by the laboratory for CDG screening on HPLC were analyzed using the CDT kit. Isoform fractions for each sample were compared by both methods. In addition commercial as well as pooled control samples were analyzed for accuracy and precision. Results: Transferrin isoforms of healthy individuals were consistent with reported ranges. Bland-Altman plots comparing each isoform with CZE and HPLC were within the mean +/− 1.96 SD with no systemic bias. Two out of 50 patients showed a CDG type 1 by both methods. A CV of < 2 % for retention time and < 20 % for AUC for each isoform was obtained for commercial as well as pooled sera. Discussion: The CDT kit gives rapid and reproducible results and its availability in routine labs will offer CDG screening to clinicians who would otherwise miss the diagnosis. The ease of the ready to use kit with commercial controls assures interlaboratory quality standards. However, it is essential that the laboratory workers have a basic understanding of secondary glycosylation defects, transferrin variants and types of glycosylation defects.

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Beware of abnormal capillary electrophoretic patterns of serum transferrin: congenital disorder of glycosylation (CDG) type I can be associated with a protein variant Bruneel A 1 7, Dupre T 1 2 4, Chaabouni T 1, Dupont A 6, Mansour H 5, Seta N 1 3 1 Biochimie, Hop Bichat-Claude Bernard, PARIS, France, 2INSERM U1149 CRB3, PARIS, France, 3Universite Paris Descartes, PARIS, France, 4Univ Denis Diderot, Paris 7, PARIS, France, 5Pediatrics, St Georges Hospital, Beyrut, Lebanon, 6Pediatrics, CHU-Lenval, Nice, France, 7INSERM 1193, Chatenay-Malabry, France

Background: CDG-I is caused by defects in the biosynthesis of the lipid-linked oligosaccharide required for protein N-glycosylation in the endoplasmic reticulum. This group contains more than 30 different molecular subtypes. CDG screening is based on testing the glycosylation of serum glycoproteins, such as transferrin (Trf). Abnormal glycosylation can be revealed by loss of electric

New insights into glycosylation and Na+/Ca2+ exchangers in human cells Amorosi C A 2, Bistue Millon M B 3, Papazoglu M 3, Siravegna M 2, ElsoBerberian G 2, Asteggiano C G 1 1 CONICET-CEMECO-UNC-UCC, Cordoba, Argentina, 2CONICET-INIMECUNC, Cordoba, Argentina, 3CONICET-CEMECO-UNC, Cordoba, Argentina

Background: Ca2+ homeostasis is mainly regulated by the Na+/Ca2+ exchangers. Recent studies demonstrated that humans platelets express NCX1 (SLC8) and NCKX1 (SLC24) proteins. However little is known about the glycosylation and Ca2+ uptake. A tight ([Ca2+]i) is necessary in platelets to prevent inappropriate thrombus formation or bleeding which are severe clinical manifestations in patients with altered N-glycosylation due to Congenital Disorders of Glycosylation (CDG). CDG are genetic diseases due to defects in the synthesis of glycoconjugate glycans. The clinical features are often neurological impairments and thrombotic-hemorrhagic events. Methods: In this study, we wanted to look into various glycosylation states and Na+/Ca2+ exchange activity. We assayed the immunopurified NCX1 and NCKX1 proteins by Western blot and the 45− Ca2+ uptake in microsomal membrane fractions from human controls and PMM2-CDG platelets. Proteins were detected with anti-SLC8 and anti-SLC24 respectively and lectin staining (ConA and WGA). Additionally, enzymatic N- and Odeglycosylation strategies (PNGase F and O-glycosidases) were done. Results: We showed that NCX1 has N-linked glycans and that NCKX1 contains mainly O-linked sialo-oligosaccharides. We observed a greatly diminished NCX1 protein expression and 45− Ca2+ uptake in PMM2-CDG patients platelets, but no differences in NCKX1 activity. The same results were observed in transfected NCX1 cDNA HEK293 tunicamycin-treated cells. Discussion: Additional studies will be necessary to further elucidate the role and structure of glycans in Na+/Ca2+ exchangers and the altered platelet aggregation events in CDG patients. Supported by CONICET/FONCYT/UCC/Sanford-Burnham Research Institute. www.cdgargentina.com.ar.

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Evaluating galactose treatment in vivo and in vitro in TMEM165-CDG Witters P 1, Wong S Y 2, Cassiman D 1, Matthijs G 1, Foulquier F 3, Morava E 1 2 1

University Hospitals Leuven, Department, Leuven, Belgium, 2Tulane University Medical School, New Orleans, United States, 3Université des Sciences et Technologies, Lille, France

S226 Background: Oral galactose therapy has been shown to be clinically efficient in two types of congenital disorders of glycosylation, PGM1-CDG and SLC35A2-CDG, both affecting Golgi function and galactosylation. We follow two unrelated individuals with TMEM165-CDG and a combined N-linked and O-linked glycosylation defect. Our patients have developmental disability, skeletal dysplasia, growth hormone deficiency, liver disease and coagulation abnormalities. Methods: We evaluated the possible efficiency of galactose therapy in TMEM165 deficient cells and patient fibroblasts by glycomics, ICAM1 immunohistochemistry and Western blot analysis. Our patients also underwent a dietary protocol of incremental increase of oral galactose treatment with increase of the dosage from 0.5 g/kg/day to 1.5 g/kg/day every 6 weeks to a maximum dose of 50 g/day followed by clinical and laboratory investigations. Results: Glycan analysis, immunohistochemistry and protein expression of ICAM1 in TMEM165 defective cells confirmed a severe glycosylation defect. Galactose supplementation of the cell culture led to significant improvement of N-linked glycosylation in 5 days. Galactose concentrations between 0.75 mEq and 2 mEq were the most effective. Higher dose galactose concentrations (5– 10 mEq) inhibited glycosylation. Oral galactose supplementation was initiated in two patients. Laboratory evaluation showed improving endocrine and coagulation parameters during the trial. Galactose was well tolerated, without side effects. Discussion: TMEM165-CDG apeears to be a novel treatable CDG. Based on the shared feature of hypogalactosylation all CDG-II types could be screened in vitro for galactose treatment. The effect of galactose on O-linked glycosylation in vitro and in vivo remains to be demonstrated during long-term follow up.

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Expanding phenotype variability in RFT1-CDG: absence of deafness in two patients with novel mutations Bandeira A 1, Fortuna A M 5, Quelhas D 3, Jaeken J 4, Matthijs G 2, Martins E 1 1 Metab Dis Unit, Centro Hosp Porto, Porto, Portugal, 2Center Human Gen, UZ Leuven, Leuven, Belgium, 3Bioch Gen Unit, CGM, Centro Hosp Porto, Porto, Portugal, 4Metabol Center - UZLeuven, Leuven, Belgium, 5Med Gen Unit, CGM, Centro Hosp Porto, Porto, Portugal

Background: Congenital disorders of glycosylation (CDG) are genetic diseases due to defects in the synthesis of glycans and in the attachment of glycans to lipids and proteins. RFT1-CDG is a defect in the assembly of Nglycans. Rft1 protein is essential for translocation of the cytosolically oriented intermediate DolPP-GlcNAc2Man5 to the luminal side of the endoplasmic reticulum. The authors present two mild to moderately affected RFT1-CDG patients with an unusual clinical picture due to absence of deafness, a striking feature of this CDG subtype. Case report: The first patient, now a 33 years old male, presented at diagnosis mild mental retardation, with marked attention deficit, macrocephaly and slight dysmorphic features. No hearing impairment was found. The second patient, now an 18 years old female, presented at diagnosis non progressive moderated mental retardation. Brain MRI and hearing evaluation were normal. Results: In both patients, apart from an abnormal type 1 pattern on the transferrin isoelectric focusing, pointing to a CDG-I defect, no other relevant results were obtained in the metabolic study. Results of the CDG capture gene panel made on these patients revealed that both were compound heterozygous for alterations in the RFT1 gene. The first patient had the pArg25Trp (c.73C > T) and p.Cys70Arg (c.208 T > C) alterations and the second patients p.Gly276Asp (c.827G > A) and p.Cys70Arg (c.208 T > C). These alterations have not been reported until now, and results obtained by in silico analysis point out that all these three alterations are most likely pathogenic. Discussion: With these novel mutations here by reported, the authors widen the mutational spectrum of the RFT1 gene. As these two moderately affected RFT1-CDG patients are the first ones to be reported without deafness and have in common the p.Cys70Arg (c.208 T > C) mutation, this could indicate an association of this mutation and milder phenotypes without hearing impairment.

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Exuberant myopathic phenotype in a DPAGT1-CDG patient Quelhas D 3, Santos M 5, Jaeken J 4, Matthijs G 2, Lacerda L 3, Martins E 1 1 Metab Dis Unit, Centro Hosp Porto, Porto, Portugal, 2Center Human Gen, UZ Leuven, Leuven, Belgium, 3Bioch Gen Unit, CGM, Centro Hosp Porto, Porto, Portugal, 4Metabol Center - UZLeuven, Leuven, Belgium, 5Neuroped Unit, Centro Hosp Porto, Porto, Portugal

Background: Congenital disorders of glycosylation (CDG) are caused by enzymatic defects of the formation or processing of lipid-linked oligosaccharides and glycoproteins. DPAGT1-CDG is caused by a defect of the human DPAGT1 (UDP-GlcNAc: Dolichol Phosphate N-Acetylglucosamine-1Phosphotransferase), catalyzing the first step of N-linked glycosylation. The DPAGT1-CDG patient presented here has striking severe myopathic phenotype as main clinical phenotypic feature. Case report: B was born after a normal pregnancy and birth. He walked at 12 months . He had a torcicolis since birth. At 13 m he was hospitalized due to fever, diarrhea and vomits. He had regression, walking only at 3 years and language at 6 years. He was first observed at 5 years – he was retarded, limitation in ocular movements, pyramidal signs and a myopathic syndrome with a limb girdle distribution, mainly of lower limbs. At 17 years he had tonic clonic seizures. He had inverted nipples, abnormal fat distribution. At 20 years old he is unable to walk due to the myopathic syndrome. Results: Muscle biopsy, Autophagic vacuoles with cytoplasmic material, electro dense materials and myeloid bodies and normal mitochondria. An abnormal type 1 pattern on the serum transferrin isoelectric focusing, pointing to a CDG-I defect. Results of the CDG capture gene panel made on this patient revealed a compound heterozygoty for mutations in the DPAGT1 gene with p.Ser133Alafs*64(c.380_395dup) and p.Ala39Val (c.116C > T) mutations. The duplication of 16 bp of the p.Ser133Alafs*64 mutation creates a frame shift wich lead to a premature stop codon. Discussion: With these novel mutations here by reported, the authors widen the mutational spectrum of the DPAGT1 gene. Myopathy is mostly associated with O-glycosylation disorders, but recent reports suggest it in N-glycosylation defects and this patient reinforces the need to consider a N-glycosylation defect in a myopathic patient.

23. Neurotransmitter and creatine related disorders

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Challenges in diagnosis and treatment of dopa-responsive dystonia due to tyrosine hydroxylase deficiency in a single patient in Indonesia Hafifah C N 1, Sjarif D R 1 1

Div Nutr Metab Dis Cipto Mangun Hosp, Jakarta, Indonesia

Background: Dopa-responsive dystonia is occasionally caused by mutation in the TH gene resulting in tyrosine hydroxylase deficiency. Diagnosis of TH deficiency is difficult to establish due to limited laboratory facilities, while treatment with L-dopa is not yet available in Indonesia. This case study aims to demonstrate the first case of TH deficiency in Indonesia. Case report An 8 year-old boy with microcephaly, severe wasting, and spasticity was refered to our hospital with involuntary movements for 2 weeks prior to admission. He was the first child of two siblings from nonconsanguineous parents and had an unremarkable birth history. Growth and development were relatively normal until he was 3 years old when he began to lose postural stability and deteriorated gradually to severe psychomotor retardation with severe malnutrition. Brain MRI showed cerebral atrophy. Results: Based on his laboratory data showing low levels of HVA, dopamine, and BH4 with normal level of neopterin and 5-HIAA in CSF and normal levels of plasma tyrosine, tryptophan, and phenylalanine, he was diagnosed as tyrosine hydroxylase deficiency and severe malnutrition. Subsequently, L-dopa

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 250 mg/carbidopa 25 mg was given through SAS scheme with a starting levodopa dose of 4 mg/kg/day given in 3 divided doses. L-dopa/carbidopa dose was increased gradually to 14 mg/kg/day in 8 divided dose until he had minimal dystonia and no dyskinesia. Benzodiazepine as muscle relaxant was given 0.75 mg/kg 4 times/day. Following enteral nutrition management with 1.5 kcal/cc formula, the patient progressed to good nutritional status within 2 months. Inconsistent with TH deficiency, the patient had seizures; these had previously been treated with phenytoin, but due to possible interaction with levodopa/carbidopa, the dose was lowered. Discussion: Despite lack of diagnostic facilities, management based on clinical observation, and use of the SAS scheme for orphan drugs, dystonia in our patient could be controlled.

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biochemical change, but no neurological phenotype. We therefore use zebrafish as model organism for analyzing qdpr deficiency. Methods: We first created a Morpholino (MO) knockdown using both splice and translation blocking MOs to establish a qdpr deficient model. Expression patterns were determined by in situ hybridization and making use of transgenic lines to unravel the gene profile in the developing brain. Furthermore qPCR and biochemical measurements were taken to confirm the results. Results: Our studies with zebrafish indicate qdpr deficiency as cause for neurological changes in the embryonic brain development. Downregulation of the regarding homolog, causes a malformation of the diencephalic structure, as well as a lack of prethalamic and habenula neurons. Overall, the zebrafish model will help to unravel the pathophysiology of qdpr deficiency and pave the way for potential therapeutic targets. Discussion: Unlike the knockout mouse, the zebrafish model can be used to analyze a neurological embryonic phenotype specific for qdpr deficiency, induced by MO knockdowns. Both the specific phenotype and controls using qPCR and further validation test confirm the early role of qdpr.

Dopamine and serotonin turnover in neuronal cell models of mitochondrial complex I deficiency and Gaucher Disease. P-573 De La Fuente Barrigon C 2, Garcia Gomez M 2, Burke D G 1 2, Eaton S 1 2, Heales S J R 1 2 1

Great Ormond Street Hospital, London, United Kingdom, 2UCL Institute of Child Health, London, United Kingdom

Secondary brain creatine deficiency and neurological impairment in BDL rats, an in vivo model of chronic cholestatic liver disease Rackayova V 1, Loup M 2, Henry H 2, McLin VA 3, Cudalbu C 1, Braissant O 2

Background: Loss of either mitochondrial function (complex I) or Gaucher disease (glucocerebrosidase, GBA1) has been associated with Parkinson’s disease (PD). Whilst a number of pathogenic mechanisms have been proposed, there has been relatively little interest in dopamine metabolism, the neurotransmitter deficient in PD. Methods: A neuronal cell line (SHSY5Y) cell line was used. Complex I deficiency was created by the use of rotenone. GBA1 deficiency was created by treatment with conduritol beta epoxide (CBE). To stimulate dopamine metabolism, control and inhibited cells were incubated with L-dopa (100 μM). Dopamine turnover was assessed by quantification (HPLC and electrochemical detection) of HVA and DOPAC in the extracellular medium. For comparison, the serotonin metabolite, 5-HIAA was also quantified. Results: L-dopa treatment resulted in an increase, over time, in the extracellular concentration of HVA and DOPAC. 5-HIAA was not affected by the L-dopa treatment. For the rotenone and CBE treated cells identical results were obtained; significant 6 fold increase in DOPAC concentration accompanied by a 12 fold decrease in HVA. 5-HIAA concentration was increased 2 fold. Discussion: DOPAC and 5-HIAA are formed by the action of monoamine oxidase (MAO). HVA generation additionally requires catechol methyl transferase (COMT) activity. Consequently our data may suggest that in complex I or GBA deficiency there is increased catabolism of dopamine and serotonin via MAO. However, metabolism to HVA may be decreased. Further work is now required to evaluate the consequences of these changes with regards to identifying mechanisms to explain the parkinsonism associated with complex 1 and GBA1 deficiency. Supported by the EU - Marie Curie Training Felllowships (TINTIN).

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Brain development in qdpr (dihydrobiopterin reductase)–deficient Zebrafish

1 Lab Funct Metab Imaging, EPFL, Lausanne, Switzerland, 2Biomedicine, Lausanne Univ Hosp, Lausanne, Switzerland, 3Cent Liver Dis Child, Geneva Univ Hosp, Geneva, Switzerland

Background: Chronic cholestatic liver disease is associated with poorly characterized neurocognitive impairment. To evaluate its brain consequences, we used bile duct ligation (BDL) in rat pups and adults, an in vivo recognized model to study liver disease-induced hepatic encephalopathy (HE) and IEM affecting bile acids. Methods: Wistar male rats (21 days pups & adults) were BDL and followed longitudinally by in vivo 1H-MRS (9.4 T MRI) from t0 (surgery) and every 2 weeks (w2/4/6/8). Open field tests were done at w4/6/8 to evaluate motor activity and HE degree. Hippocampal 1H-MRS spectra (11.2 mm3) were acquired using the SPECIAL sequence (TE = 2.8 ms); metabolite concentrations were calculated by LCModel allowing in vivo measure of 18 CNS metabolites. Blood was sampled at w0/2/4/6/8 and animals were sacrificed at w8 for brain tissue analysis. Results: Pups showed stronger response to BDL than adults. As compared to sham-operated animals, BDL rats developed hyperbilirubinemia, hyperammonemia and bile acid (TαMCA/TβMCA/TCA/TCDCA) accumulation in blood, and showed decreased performance in behavioural tests. Hippocampal 1 H-MRS showed strong increase in Gln at w4 already, reaching 311 % (pups) and 236 % (adults) of t0 at w8, together with decreased other osmolytes (mIns, tCho). Interestingly, Creatine/Phosphocreatine (Cr/PCr) significantly decreased in BDL CNS (w8: pups 81/91 % / adults 89/94 % / sham 100/100 %). BDL brain metabolic changes were accompanied by astrocytic swelling, aquaporin 4 dysregulation at blood–brain barrier and increased apoptotic brain cells. Discussion: We show for the 1st time the in vivo longitudinal impairment of CNS metabolites after BDL. Chronic cholestatic liver disease may lead to a significant decrease of brain Cr and PCr, which could hamper CNS development and function and lead to cell death. More work is needed to better understand the mechanisms leading to brain dysfunction cholestatic liver disease of infants and adults.

Breuer M 1, Opladen T 1, Carl M 2, Sauer S W 1 1 Inborn Error Metab, Univ Child Hosp, Heidelberg, Germany, 2CBTM, Univ, Mannheim, Germany

Background: Tetrahydrobiopterin (BH4) is necessary in the synthesis of tyrosine, dopamine and serotonin. A lack of BH4 causes a severe depletion of the respective neurotransmitters and therewith neurological defects, mental retardation and hyptonia. In the special case of Dihydropteridine Reductase (qdpr) deficiency, BH4 is not properly recycled from quinonoid BH2 and additionally causes symptoms such as brain atrophy. The knockout mouse has shown a

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IPSCs from a patient suffering from tyrosine hydroxylase deficiency Jung-Klawitter S 1, Sebe A 2, Shen N 1, Hoffmann G F 1, Blau N 1, Opladen T 1 1 Univ Child Hosp, Metabolic Center, Heidelberg, Germany, 2Div Med Biotech, Paul-Ehrlich Institute, Langen, Germany

S228 Background: Human induced pluripotent stem cells (hiPSCs) represent an invaluable tool for regenerative therapy, drug screening and toxicity testing. Moreover, they can serve as model system to identify pathomechanisms in rare human diseases where patient material is often not easily available. Congenital defects in neurotransmitter biosynthesis lead to severe central deficiency of dopamine, serotonin, epinephrine and norepinephrine leading to progressive symptoms including psychometric retardation, muscular hypotonia, extrapyramidal movement disorders, epilepsy and oculogyric crisis starting in early childhood. Here, we report the generation of hiPSCs from a patient suffering from tyrosine hydroxylase deficiency as a model to study the underlying pathomechanisms of the disease. Methods: Fibroblasts from a patient suffering from tyrosine hydroxylase deficiency have been reprogrammed to iPSCs using episomal plasmids and further characterized for pluripotency marker gene expression and normal karyotype as well as transgene silencing and differentiation potential. Deficient iPSCs have been differentiated to neurons and characterized biochemically. Results: Tyrosine hydroxylase deficient iPSCs express all classical pluripotency marker genes (Oct4, Sox2, Nanog, SSEA-4, Lin-28), show transgene silencing, a normal karyotype and are able to differentiate into cells of all three germ layers. Cells can be differentiated to neurons expressing classical marker genes like Tuj-1. Tyrosine hydroxylase activity is barely detectable in the deficient cells. Discussion: Tyrosine hydroxylase deficiency is no roadblock for the generation of patient-specific iPSCs. They can be differentiated into cells of all three germ layers. Corresponding to the underlying deficiency, the iPSCs and differentiated neurons show the expected changed patterns of metabolites thereby providing a patient specific model for the identification of underlying pathomechanisms associated with the disease.

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2 siblings with a new genotype of GAMT deficiency and response to sodium benzoate therapy.

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Safety and efficacy of Rotigotine in 7 patients with monoaminergic neurotransmitter deficiency. Mastrangelo M 1, Giannini M T 1, Carducci C L 2, Carducci C A 2, Leuzzi V 1 Div Pediatr Neurol, Sapienza Univ, Rome, Italy, 2Dep Exp Med, Sapienza Univ, Rome, Italy 1

Background: Rotigotine is a dopamine agonist indicated for the treatment of Parkinson’s disease (PD) and restless leg syndrome (RLS). It is formulated as a once-daily transdermal patch which provides a slow and constant supply of the drug over the course of 24 h. We investigated the efficacy, tolerability and safety of transdermal rotigotine in patients with inherited defects of biogenic amine metabolism. Patients and methods: Our series included 5 males and 2 females (age range 5– 33 years): four patients with aromatic L-amino acid decarboxylase (AADC) deficiency, two patients with tyrosine hydroxylase (TH) deficiency, and one patient with dopamine transporter (DAT) deficiency. Rotigotine dosages ranged from 2 mg/24 h to 8 mg/24 h. Results: An improvement in asthenia and gross motor functions were observed in 3 patients with AADC deficiency. One wheelchaired patient with AADC deficiency improved head control. One patient with TH deficiency experienced a transient motor improvement. None of the rotigotine—treated patients evidenced improvements in cognitive profile, fine motor performances and autonomic symptoms. Side effects of rotigotine included paroxysmal dyskinesia (5/7 patients), worsening bradykinesia and on-off phenomena (2/7patients), confusional state (2/7patients), vomiting and hypotension (1/7 patients), increased sexual impulses (1/7patients). Significant reduction of prolactinaemia was rapidly observed in 3 patients while in 2 patients prolactin blood values remained as low as under other previously used dopamine agonists. Discussion: Rotigotine induced a significant improvement in asthenia and in gross-motor functions in patients with AADC deficiency and no benefits in TH and in DAT deficiency.

Fiori L 1, Leuzzi V 3, Carducci C L 4, Carducci C A 4, Uggetti C 2, Podesta’ A F 1 P-577 1

UO Ped -Pat Neo,AO San Carlo Borromeo, Milano, Italy, 2UO Sci Rad - AO S Carlo Borromeo, Milano, Italy, 3Dipt Ped e Neurop Inf Univ La Sapienza, Roma, Italy, 4Dipt Med Sper Univ La Sapeinza, Roma, Italy Background: Guanidinoacetate (GAA) methyltransferase (GAMT) defect causes the most severe phenotype among the disorders of the creatine metabolism. Treatment include creatine supplementation and GAA reducing strategies (ornithine supplementation, arginine restricted diet, sodium benzoate [SB]). We describe two new related patients with an atypical clinical course. Case report: Both were born from healthy consanguineous parents. A 17 -yearold male presented at the age of 6 months with progressive developmental delay, febrile seizures evolving in drug-unresponsive epilepsy, and, from the age of 21 months, generalized dystonic movements. Increased blood GAA (9.9 μM; r.v. 1.2–4.3), brain creatine depletion on 1HMRS and GAMT gene alteration (homozygous p.Ala54Pr; c.160G > C mutation) confirmed the diagnosis. Creatine monohydrate, low arginine diet and ornithine chlorhydrate resulted in remission of seizures and stable improvement of dystonia for the next 6 years. At the age of 8 neurological deterioration and increased blood GAA (18 μM) prompted the treatment with SB. The first degree proband cousin, a 11-year-old female, presented with dystonic limb movements at the age of 13 months. Under creatine and ornithine supplementation blood GAA decreased, creatine increased in plasma and brain, dystonic movements vanishing and she experienced a normal intellectual development until the age of 8 years when school performances declined and blood GAA increased up to 18 μM. SB was added to therapy. Results: SB treatment resulted in a stable 50 % decrease of GAA with remarkable improvement of the neurological status. Discussion: Our patients showed a biphasic course with a rapid positive response to treatment followed by a late clinical and biochemical worsening, which was reversed by SB therapy. The genotype we detected has not been reported so far. We suggest it may have influenced the specific pattern of response to the treatment and the relatively good prognosis.

Analyses of cerebrospinal fluid neopterin: inborn errors of metabolism versus neuroinflammatory diseases Molero-Luis M 1 3, Ormazabal A 1, Sierra C 1, Cuadras D 1, Garcia-Cazorla A 23 , Jordan Y 4, Perez-Duenas B 2, Artuch Iriberri R 1 3 1

Biochemical Lab, Hosp Sant Joan de Deu, Esplugues de Llobregat, Spain, Neurological dep, Hosp Sant Joan Deu, Esplugues de Llobregat, Spain, 3 CIBER-ER, Instituto Salud Carlos III, Madrid, Spain, 4Ped Intens Care Unit, Hosp Sant Joan Deu, Esplugues de Llobregat, Spain 2

Background: Cerebrospinal fluid (CSF) pterins are useful for analyzing tetrahydrobiopterin metabolism errors. However, a high level of CSF neopterin is also a marker of central nervous system inflammatory-immune mediated processes. Currently, it is still difficult to do the differential diagnosis among the etiologies leading to neuroinflammatory diseases. We aimed to define a statistical approach to define different groups of neuroinflammatory diseases using diverse CSF biomarkers. Methods: We analyzed CSF neopterin, protein and leukocytes in a cohort of 706 neuropediatric patients classified in 4 groups: viral infections (A), bacterial infections (B), central autoimmune processes (C) and other neuropediatric disorders (D). We did lineal combinations of the 3 CSF biomarkers to assess the best discriminative biomarker values for the studied cohort (canonical population test). Results: We obtained 2 canonical equations: Can1 = −0.78logNeopterin - 0.27logProtein - 0.66logLeukocytes Can2 = −0.98logNeopterin + 0.003logProtein + 0.68logLeukocytes Since Can1 have negative coefficients in each biomarker, low biomarker values will result in a high Can1 value. Group D-patients presented the lowest biomarkers values resulting in a high Can1 value. The other groups presented higher biomarker values and Can1 value were lower.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 In Can2 low neopterin with high leukocytes values will result in a high Can2 value. Patients from group A and B had lower neopterin and higher leukocytes values than those of group C permitting differentiate groups A-B from C. Discussion: Both canonical equations are able to differentiate the groups of patients. High CSF neopterin concentration with high leukocyte values is suggestive of an infectious event whereas high CSF neopterin concentration with lower leukocytes values orientate to autoimmune diseases. Those neurological disorders without implication of inflammatory-immune mediated processes had low CSF proteins, leukocytes and neopterin values.

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Two New Cases with Hereditary Dopamine Transporter Deficiency Syndrome Tokatli A 1, Yildiz Y 1, Pektas E 1, Haliloglu G 2 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 2Div Ped Neuro, Hacettepe Univ Child Hosp, Ankara, Turkey

Background: Dopamine transporter deficiency syndrome (DTDS) is a recently discovered neurotransmitter disorder caused by a defect in neuronal uptake of dopamine. Less than twenty patients have been reported so far. Here, we present two new patients with DTDS, harboring novel homozygous SLC6A3 gene mutations. Case report: Patient A is an 8-month-old male with neonatal-onset hypotonia, who developed orolingual dyskinetic movements and oculogyric crises after the age of 4 months, with a progressive evolution to episodes of status dystonicus. Patient B is a 4-year-old male who also had hypotonia since birth, whose severe limb contractions and oculogyric crises started at the age of 3 months, leading to a misdiagnosis of epileptic encephalopathy. Both patients had consanguineous parents and similar CSF neurotransmitter profiles with markedly elevated levels of homovanillic acid and increased ratio of homovanillic acid to 5-hydroxyindoleacetic acid, which is suggestive of DTDS. Diagnostic delay is 4 months, and 3 years 9 months, respectively. Results: Clinical and CSF neurotransmitter findings led to the diagnosis of DTDS in these two patients. Novel homozygous mutations in SLC6A3 gene were detected in both patients: c.418 + 3800_653 + 3058del in Patient A and c.1156G > A (p.Gly386Arg) in Patient B. Therapeutic trials with levodopa up to 5 mg/kg/day combined with benserazide during a follow-up period of 1 and 2 months, respectively, were unsuccessful. Dopamine receptor agonist pramipexole also failed to provide significant benefit. Discussion: Management of hyperkinetic movement disorder, status dystonicus, and severe feeding difficulties are challenging at the bedside. Detailed phenotyping of individual patients along with treatment response should provide insight into this rare disease and into the complex system of dopamine homeostasis.

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Substantial psychiatric symptoms and reduced quality of life in welltreated patients with GTP-cyhydrolase deficienct dopa-responsive dystonia. Kuiper A 1, Smit M 1, Timmers E R 1, Bartels A L 1, Tijssen M A J 1, De Koning T J 1 2 1 Univ Med Centr, Dep Neurol, Groningen, Netherlands, 2Univ Med Centr Dep Pediatr Metab Dis, Groningen, Netherlands

Background: The aim of this study was to systematically investigate the prevalence of psychiatric disorders and health-related quality of life (HRQoL) in patients with GTP-cyhydrolase deficient dopa-responsive dystonia (DRD). Methods: We assessed psychiatric co-morbidity and HR-QoL in 7 adult patients with a confirmed CGH1 mutation and 7 healthy controls matched for age and sex. All patients received levodopa treatment. Psychiatric assessment included the Mini International Neuropsychiatric Interview - PLUS (MINI-

PLUS) and quantitative validated questionnaires: Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI) and Yale Brown Obsessive Compulsive Scale (Y-BOCS). HR-QoL was determined with the RAND-36 item Health Survey. Results: In DRD patients the MINI-PLUS revealed a high life-time prevalence of psychiatric disorders of 57.1 %, vs 42.9 % in the control group. Particularly more anxiety disorders were reported (42.9 % vs 14.3 %) On the BDI, DRD patients scored on average almost 2 points higher (6.9 vs 5.0) but this did not reach significance. In total 2/7 DRD patients had an aberrant BDI score > 9. In both the DRD and control group one patient had a BAI score > 9; there were no differences in current anxiety scores between groups. No obsessivecompulsive disorders were seen. Concerning HRQoL, DRD patients scored significantly lower on the general health domain compared to the control group (53.6 vs 71.1, p = 0,033). Discussion: In our DRD cohort psychiatric co-morbidity was highly prevalent despite dopaminergic treatment with good dystonia control. Our findings support the accumulating evidence of an important nonmotor phenotype in DRD. This highlights the need for systematic research into psychiatric disorders and the underlying neurobiology. Adequate treatment of psychiatric symptoms could significantly contribute to better quality of life in DRD patients.

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Expanded phenotype in creatine transporter deficiency: identification of two novel mutations correlated with mild clinical presentation Mancardi M M 6, Battini R 1, Schiaffino M C 7, Alessandri M G 5, Gherzi M 6, Viglione V 5, Battaglia F M 6, Carducci C L 3, Moro F 5, Carducci C 3, Morana G 8, Tosetti M 5, Cioni G 2 5, Leuzzi V 4 1

IRCCS FSM and Ped Neurol Unit UCSC, Pisa and Rome, Italy, 2University of Pisa, Pisa, Italy, 3Dpt Experim Med, Sapienza Univ, Rome, Italy, 4Dpt Ped Child NeuroPsy Sapienza Univ, Rome, Italy, 5Dpt Develop Neurosci IRCCS FSM, Pisa, Italy, 6Dpt Head Neck Neuro IRCCS G Gaslini Univ, Genova, Italy, 7Dpt Pediatric IRCCS G Gaslini, Genova, Italy, 8Unit Neurorad IRCCS G Gaslini, Genova, Italy Background: Creatine transporter deficit (CT1) is an inherited metabolic disease that causes intellectual disability usually moderate to severe, epilepsy, language and behavioural disorders. Findings of different therapeutic approaches did not show an agreement in this condition and the disease remains without therapy up to now. Case report: We report on two new Italian families affected by CT1 defect with peculiar mild features, whose diagnosis was possible because of a reduced Creatine (Cr) peak at magnetic resonance spectroscopy (MRS) and of altered Cr/Crn ratio in urine performed during routine investigations for developmental delay. Results: First family: two brothers, 9 and 32 years; the younger has a borderline intellectual functioning, attention and working memory disorder and social skills impairment; the older has no apparent intellectual disability but an history of speech delay. MRS was diagnostic for the younger brother, confirmed by genetic testing (inherited mutation p.Pro268Arg c.803C > G, not described to date in the literature). We performed a MRS also in the older brother because of the reported language delay and found out a pathologic peak, molecular analysis confirmed the mutation. Second family: a boy of 5.6 years with clinical picture characterized by hyperactivity and attention deficit (ADHD) with oppositiveprovocative behaviour and speech disorder associated to social communication skills impairment. MRS revealed a reduction of Cr peak at MRS and a Cr/Crn ratio in urine showed a value of 2.24 (n.v. < 1). A new de novo intronicdeletion (c.1255-14_1255-24del) was found by CT1 sequence analysis causing a premature protein truncation.). Discussion: We describe two new CT1-deficit Italian families with mild phenotype mostly characterized by language delay, attention deficit and hyperactivity. One patient is an adult with pathologic MRS but no history of intellectual disability or behavioural disorder.

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Case report 2: A 4-year old girl was referred to our hospital for seizures and psychomotor retardation. Prenatal/natal history was unremarkable. She is the third child of nonconsanguineous Turkish parents. She was bedridden. Metabolic investigations revealed heavy-persistent pyroglutamic aciduria. EEG was compatible with burst suppression. A novel compound heterozygous mutation p.S518L (c.1553C > T)/p.G825 (c.2473G > A) was detected in OPLAH gene. Her 2 asymptomatic brothers had also the same compound heterozygous mutation, mild pyroglutamic aciduria, and completely normal neurological examination. Results: The mostly discussed feature of 5-Oxoprolinase deficiency was the neurological involvement. Discussion: We report 4 patients with 2 novel compound heterozygous mutations, 2 with severe psychomotor retardation, seizures, and heavy-persistent pyroglutamic aciduria, and 2 without any neurological involvement and mild pyroglutamic aciduria.

Glomerular filtration rate (GFR) strongly influences guanidinoacetic acid (GAA) in plasma and urine Salvador C L 1, Rowe A D 1, Bjerre A 2, Tondel C 3, Brun A 4, Brackman D 3, Morkrid L 1 1 Nat Unit Lab diagn IEM, Oslo Univ Hosp, Oslo, Norway, 2Div Paed and Adolesc Med, Oslo Univ Hosp, Oslo, Norway, 3Dep Paed, Haukeland Univ Hosp, Bergen, Norway, 4Lab Clin Bioch, Haukeland Univ Hosp, Bergen, Norway

Background: Age strongly influences plasma and urine concentrations of creatinine (crn), creatine (CRE) and guanidinoacetic acid (GAA) and subsequently the urine-ratios U-CRE/crn and U-GAA/crn which are used in screening for creatine deficiency syndromes (CDS). How renal function affects these variables in children with different stages of kidney function is unknown. Methods: We examined 96 children (median age 9.2, range 0.25–17.5 years and 55 males/41 females) with different stages of chronic kidney disease. GFR was (median 65.9, range 6.3–153 mL/min/1.73 m2) measured by iohexol clearance using seven venous blood samples after iohexol injection, and fasting GAA and CRE were analyzed in plasma collected before iohexol injection. Urinary GAA and CRE were measured in first morning void urine and given as ratio to creatinine. After appropriate transformation of the markers, a linear regression analysis examined the influence of age, gender and measured GFR. Results: The level of GFR significantly affects plasma GAA (p = 2 × 10−4) and U-GAA/crn (p = 5 × 10−11) leading to decreased values in renal impairment. In contrast, GFR does not correlate significantly with the level of CRE and only to a minor degree U-CRE/crn ratio (p = 0.54 and 0.01, respectively). Age strongly influences the level of GAA, CRE and U-CRE/crn (p < 0.001), but not U-GAA/crn in this population. U-GAA/crn also differs with respect to gender with lower values in males. Discussion: GFR strongly influences the levels of GAA and U-GAA/crn with lower values as renal function declines. This may be attributed to decreased production of GAA in the renal tubular cells. Evaluation of kidney function is essential when interpreting screening results for CDS.

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5-Oxoprolinase deficiency: report of three siblings and a case with two new compound heterozygous mutations and the clinical diversity even in the same family Onenli Mungan N 1, Seker Yilmaz B 1, Bulut F D 1, Oktem M 3, Kor D 1, Ceylaner G 2, Herguner O 4 1 Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Intergen Genetic Lab, Ankara, Turkey, 3 Duzen Lab, Ankara, Turkey, 4 Div Ped Neu, Univ Cukurova, Adana, Turkey

Background: 5-Oxoprolinase deficiency is an extremely rare autosomal recessive disorder of gamma-glutamyl cycle. The clinical picture is not well defined due to the limited numbers of patients and some authors asked whether it is a real metabolic disorder. 5-Oxoprolinuria is the diagnostic clue for this disorder. Nephrolithiasis, enterocolitis, and psychomotor retardation are the reported symptoms. Case report 1: A 9-month old girl admitted to clinic for seizures and mental-motor retardation. She is the only child of consanguineous Turkish parents, born at term without a complication. Except psychomotor retardation, axial hypotonia, and peripheral spasticity physical examination was normal. Metabolic investigations revealed heavypersistent pyroglutamic aciduria. We did not detect metabolic acidosis, hemolytic anemia, hyperammonemia or lactic acidosis. All the other causes of pyroglutamic aciduria were excluded. A novel compound heterozygous mutation p.P868L (c.2603C > T)/p.G825 (c.2473G > A) was detected in OPLAH gene.

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Abnormal CSF phenylalanine level in patients presenting with disorders of tetrahydrobiopterin (BH4) metabolism with hyperphenylalaninemia Celato A 2, Mastrangelo M 4, Burlina A P 1, Polo G 2, Carducci C 3, Carducci C 3, Leuzzi V 4, Burlina A B 2 1 Neurological Unit, St Bassiano Hosp, Bassano del Grappa, Italy, 2Div Inherited Metab Dis, Univ Hosp, Padova, Italy, 3Exp Med Dep, Sapienza Univ, Roma, Italy, 4Child Neurol and Psyc, Dep Ped, Sapienza, Roma, Italy

Background: Atypical hyperphenylalaninemia represents an heterogeneous group of disorders affecting the biosynthesis or the regeneration of tetrahydrobiopterin (BH4), an essential cofactor for phenylalanine (Phe), tyrosine, and tryptophan hydroxylases. This treatable disorder causes a combined deficiency of dopamine and serotonin precursors together with high Phe levels. This disorder encompasses several forms of primary autosomal recessive monoamine neurotransmitter encephalopathies including: autosomal recessive guanosine triphosphate cyclohydrolase (AR-GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and dihydropteridine reductase (DHPR) deficiencies. Case report: 16 patients, 10 males and 6 females (4 DHPR, 10 PTPS and 2 AR-GTPCH deficiency affected patients), ranging from 7 months to 38 years old, were included and were monitored clinically andbiochemically. Molecular genetic analysis (performed in 13 of 16 patients) showed 3 novel pathogenic mutations. Plasma and CSF Phe levels were compared and CSF biogenic amines were assessed, prior and after pharmacological treatment. Results: 5/16 patients showed an alteration of plasma/CSF Phe ratio ( A];[1506 + 4A > G]. Since the 7th month he has thrived with no neurological or hematological complications. With oral OH-Cbl therapy (1000–3000 μg weekly) P-tHcy was 7–20 μmol/l, U-MMA < 15 to 51 mg/g creat), holoTCII 96 to >256pmol/l. Discussion: In contrast to other combined defects of intracellular cbl metabolism, this case of cblJ deficiency presented with low serum vitamin B12 as reported in another cblJ patient. These cases of cblJ defect emphasize the need to monitor tHcy and MMA metabolism in patients with apparent normalization of cobalamin levels upon vitamin B12 treatment, to distinguish nutritional and genetic causes. Supported by MZ VZ RVO VFN64165.

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Molecular characterization of the cblC disease reveals new pathways in pathogenesis Hannibal L 1, Behringer S 1, Klenzendorf M 1, Jacobsen D W 2, Spiekerkoetter U 1, Blom H J 1 1 Lab Metab Dis, Univ Child Hosp, Freiburg, Germany, 2LRI, Cleveland Clinic, Cleveland, United States

Background: Nutritional and functional deficiencies of vitamin B 12 (cobalamin) lead to severe neurological and hematological dysfunction, and if left untreated, to death. Intracellular processing of dietary cobalamins is catalysed by the CblC enzyme, which precedes the delivery of cobalamin to cytosolic methionine synthase and mitochondrial methylmalonyl-CoA

S232 mutase. Absence of or presence of a mutated cblC leads to combined hyperhomocysteinemia and methylmalonic acidemia (MMACHC, OMIM 609831) the most common inborn error of cobalamin metabolism. In spite of over 500 cases identified to date, little is known about the gene regulation and protein changes that accompany cblC disease onset and progression. Methods: The production of homocysteine (Hcy), methylmalonic acid (MMA), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) was examined in cultured fibroblasts from healthy neonates and patients with early onset cblC disease. Protein expression levels were determined by global 2D-DIGE proteomics, and post-translational modifications were identified in parallel cultures grown in SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) medium. Results: Cells from cblC patients with neonatal onset demonstrated changes in protein expression levels involving the neurological, musculo-skeletal and detoxification systems. While a global downregulation of protein expression was identified in cblC mutant cells, a subset of proteins exhibited higher expression levels compared to controls. Several genes encoding proteins up and down-regulated in the cblC proteome are subject to epigenetic regulation. Discussion: Data from metabolite, protein expression levels and gene regulation analysis concur with the clinical manifestations of the cblC disease and the insufficient response to treatment with hydroxocobalamin. Our study identifies new pathways in pathogenesis and suggests an abnormal methylation status in the cblC disorder.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Pyridoxal-5’-phosphate (PLP), the active form of vitamin B6 (VB6), plays an essential role in brain metabolism and development. PLP is an essential cofactor in more than 140 metabolic reactions making the metabolic consequences of VB6 deficiency difficult to predict. Four inborn errors of metabolism are known to affect VB6 concentrations in the brain: pyridoxine-dependent epilepsy (PDE), hyperprolinemia type II, pyridox(am) ine 5’-phosphate oxidase (PNPO) deficiency and hypophosphatasia. Although VB6 deficient patients clinically present with severe neonatal seizures, little is known about the biochemical alterations. Methods: We investigated the metabolic consequences of VB6 deficiency on a neuroblastoma (Neuro-2A) cell line using targeted (UPLC-MS/MS) and untargeted (DI-HRMS) mass-spectrometry methodologies. To evaluate serine de novo biosynthesis, we performed a metabolic flux analysis where 13C3serine and 13 C 2 -glycine formation was measured in cells incubated with13C6-glucose, in the absence and presence of PL. Results: The untargeted data unveiled serine as one of the most significantly altered metabolites. Furthermore, the metabolic flux analysis revealed that serine de novo formation was significantly impaired upon VB6-deprivation (P < 0.01) while glycine formation was decreased to a lesser extent. In addition, the intracellular 5-methyltetrahydrofolate (5-mTHF) levels were significantly decreased in VB6-deprived cells. Discussion: The severe impact of vitamin B6 deficiency on intracellular serine, glycine and 5-mTHF concentrations clearly shows the importance of VB6 on serine de novo biosynthesis in a neuronal cell model. Low concentration of serine and 5-mTHF may play a role in the pathogenesis of genetic VB6 deficiencies. Furthermore, our findings may explain the response of some patients with PDE to folinic acid supplementation.

Disruption of the metabolome in a zebrafish model of PNPO deficiency P-590 Albersen M 1, Savelberg S M C 1, Bosma M 1, Gerrits J 1, Willemsen M 1, De Sain-van der Velden M G M 1, Prinsen H C M 1, Van der Ham M 1, Pras-Raves M L 1, Bakkers J 2, Tessadori F G 1 2, Jans J J M 1, Van Haaften G 1, Verhoeven-Duif N M 1

Long-term visual and electrophysiological follow-up in early onset cblC patients

1 Dept Genet,Univ Med Center Utrecht, Utrecht, Netherlands, 2Cardiac Dev and Genet,Hubrecht Institute, Utrecht, Netherlands

Garcia Segarra N 1 2, Rigaudiere F 3, Benoist J F 4, Delouvrier E 5, Pichard S 2, Ogier De Baulny H 2, Schiff M 2

Background: Patients with pyridox(am) ine phosphate oxidase (PNPO) deficiency suffer from severe neonatal seizures. Current treatment with vitamin B6 is successful in reversing the epilepsy while it often fails to prevent intellectual disability. To optimize treatment, we studied the biochemical phenotype of PNPO deficiency in a zebrafish model. Methods: CRISPR/Cas9 technology was used to generate PNPO knockout zebrafish. Wildtype, heterozygous and homozygous knockout zebrafish larvae were homogenized at 5 days post fertilization. B6 vitamers, amino acids and neurotransmitters were quantified with UPLC-MS/MS. Untargeted analyses of the metabolome were performed using a Q-Exactive Orbitrap-MS and a bioinformatics pipeline. Results: B6 vitamers, amino acids and neurotransmitters were successfully quantified in wildtype zebrafish larvae. In homozygous PNPO knockout larvae, accumulation of the B6 vitamers pyridoxamine and pyridoxamine phosphate was found besides decreased concentrations of pyridoxal and pyridoxal phosphate (PLP). Concentrations of several amino acids and neurotransmitters were increased. The metabolome showed changes in multiple pathways. Discussion: The zebrafish is a suitable model to study PNPO deficiency at the biochemical level. PNPO deficiency not only results in a disbalance between B6 vitamers and impaired functioning of PLP-dependent enzymes involved in amino acid and neurotransmitter metabolism, but also in broader disruption of the metabolome.

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Vitamin B6 is essential for serine de novo biosynthesis Ruben R J 1, Pras-Raves M L 1, Gerrits J 1, Bosma M 1, Prinsen H C M 1, Jans J J M 1, Verhoeven-Duif N M 1 1

Dep Med Gen, Univ Med Center Utrecht, Utrecht, Netherlands

1 Center Mol Dis, Univ Hosp, Lausanne, Switzerland, 2Ref Cent Met Dis, Robert Debre Univ Hosp, Paris, France, 3Clin Physiology, Lariboisiere Univ Hosp, Paris, France, 4Biochemistry, Robert Debre Univ Hosp, Paris, France, 5 Dep Ophthalm, Robert Debre Univ Hosp, Paris, France

Background: CblC defect is the most frequent inborn error of intracellular cobalamin (vitamin B12) metabolism caused by mutations in the MMACHC gene. Maculopathy and progressive retinal dysfunction are observed in most patients with early onset cblC defect. Methods: In order to longitudinally characterize the retinal dysfunction, fundus, electroretinogram (ERG) and visual evoked potentials (VEPs) were recorded yearly in 6 vigil patients (3 males and 3 females without sedation) with early onset cblC defect (confirmed at the molecular level and appropriately treated with intramuscular hydroxocobalamin and betaine), during 5 to 10 years. Results: At the first exam (mean age: 7 months), 5/6 had nystagmus with normal fundus: 3/6 with normal ERGs and normal VEPs and 2/6 with electronegative scotopic ERGs and normal VEPs. 1/6 had no nystagmus with maculopathy, normal retinal periphery, normal ERG and abnormal VEP. Two to four years later, the fundus of 5/6 patients with initial normal fundi showed maculopathy. Preceding appearance of maculopathy, 4/5 had electronegative scotopic-photopic ERGs and abnormal VEPs, 1/5 had normal ERG with abnormal VEP. After their fifth year of age, all 6/6 patients showed maculopathy, 2/6 with normal ERGs and abnormal VEPs, 4/6 with abnormal scotopic and photopic reduced ERGs and abnormal VEPs. Discussion: These data show extensive characterization of the progression of eye disease over time in cblC defect. In 4/6 patients, our findings suggest that the retinal dysfunction preceding the onset of maculopathy, is located in the inner retina (electronegative ERG i.e. inner retinal dystrophy). To our knowledge, this level of early retinal dysfunction has been described for the first time. Over time, the retinal dysfunction evolved towards the outer retina (conerod or rod-cone dystrophy) as already described.

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Role of intramuscular Levofolinate in treatment of Hereditary folate malabsorption. Manea E 1, Gissen P 1, Pope S 2, Heales S J R 3, Batzios S 1 1

Dep Metab Dis, Great Ormond St Hospital, London, United Kingdom, Neuro Metab Unit, National Hosp, London, United Kingdom, 3Dept Chem Path, Great Ormond St Hospital, London, United Kingdom

1397 to 28: normal range < 17 μmol/mol/creat) and plasma Hcy (Patient 1: 197, 1 to 19,1; patient 2: 174, 6 to 17, 5 patient 3: 184, 4 to 13,5: normal range < 15 μmol/l). At 6 months of age the 3 patients had almost normal neurological examination including eye fundus. In patients 1 and 2 nystagmus disappeared following higher dose of OHB12. Only in patient 3, nystagmus tended to persist. Discussion: Identical mutation may cause different clinical severity. OHB12 dose intensification strategy may also better control early onset cblC deficiency and clinical phenotype.

2

P-593 Background: Hereditary folate malabsorption (HFM) is a rare autosomal recessive disorder caused by impaired active folate transport across membranes and into the central nervous system due to loss-of-function mutations in proton-coupled folate transporter (PCFT). Significant neurological morbidity usually follows the initial non-specific clinical presentation and delayed initiation of treatment. Oral and parenteral folinic acid treatment is used with the aim to normalise the neurological features. We report a single center’s experience in achieving normal cerebrospinal fluid 5-Methyl tetrahydrofolate (5MTHF) levels following daily intramuscular administration of levofolinic acid in two patients. Case report: Both patients presented at 2 months of age with failure to thrive, pancytopenia with megaloblastic elements and non-specific immunodeficiency. Both serum and CSF folate were undetectable. Developmental arrest, movement disorder and seizures developed after their first birthday and neurological features dominated the clinical picture. The first patient inherited c.204-205delCC in exon 1 resulting in a frameshift, N68Kfs and a mutant allele in exon 2 (c.1004 C > A) resulting in a loss-of-function point mutation, A335D. The second patient’s PCFT gene mutation analysis revealed a homozygous mutation, c.198C > A, p.Cys66*, causing a premature stop codon. Although oral folinic acid was initially prescribed this was switched to intramuscular folinic acid at a dose of 0.5–2.5 mg/kg/day. Results: Haematological normalization was associated with an initial modest neurological improvement while CSF folate levels remained suboptimal. Levofolinic acid, at a dose of 1–3 mg/kg/day, doubled the CSF folate levels in 1 year with subsequent normalization. Seizures have stopped in one patient. Discussion: The experience of our center confirms previous literature reports and reaffirms biological efficacy of individualized levofolinate dosage to achieve normal age dependent trough CSF 5-MTHF levels.

Bone mineral density and vitamin D status in inborn errors of metabolism Olgac A 1, Tumer L L 1, Inci A 1, Karaoglu B 1, Okur I 1, Ezgu F S 1 1

Div Ped Metab Dis,Gazi Univ Hosp, Ankara, Turkey

Early onset of cobalamin C deficiency in 3 neonates: clinical and biochemical features following parenteral hydroxycobalamin dose intensification strategy

Background: Vitamin D is actively involved in multiple metabolic pathways including calcium metabolism and bone health. We evaluted the vitamin D status of patients with various subtypes of inborn errors of metabolism (IEM) and its relationship with bone mineral density (BMD), anthropometric parametres and restricted diets. Methods: 262 patients were evaluated in total (84 amino acid metabolism disorders, 43 mucopolysaccharidoses (MPS), 29 sphingolipidoses, 22 biotinidase deficiency, 24 organic acidurias, 17 galactose/fructose metabolism disorders, 17 glycogen storage diseases, 13 energy metabolism defects, 8 cerebral organic acid disorders, 5 osteogenesis imperfectas). Results: 37.5 % of all patients showed vitamin D deficiency being most commonly seen in the MPS (61,5 %), osteogenesis imperfecta (61 %) and sphingolipidoses (56 %) disorders. Rate of osteoporosis and osteopenia among all patients was 22 % and 15.5 % respectively. Osteoporosis was most common among MPS patients (60 %). No significant relationship between vitamin D status and BMD scores was detected. Regardless of disease type, vitamin D defcient patients had significantly higher incidences of failure to thrive. Vitamin D levels of patients that were on protein restricted diets were not different to patients without special diets. Discussion: Vitamin D levels may be reduced in IEMs due to several factors. Especially in patients with skeletal system involvement, immobilisation may further contribute to the deficiency. Although supplementation is recommended for any patient with known vitamin D deficiency, special attention should be given to IEMs with skeletal system involvement and patients with failure to thrive, and supplementation should be initiated at the time of diagnosis to support bone health. Also, it should be kept in mind that normal levels of vitamin D may not be indicative of normal BMD and analysis of BMD should be performed for any patient with suspected osteopenia/osteoporosis.

Scalais E 1, Osterheld E 1, Amrom D 1, Geron C 1, Pierron C 1, Bindl L 1, Schlesser V 1, Regal L 2, Martens G 2, De Meirleir L 2

P-594

P-592

1 Centre Hospitalier de Luxembourg, Luxembourg, Luxembourg, 2Ped Neurology,AZ-VUB, Vrije Universiteit, Brussels, Belgium

Background: Cobalamin C (cblC) deficiency, a combined form of methylmalonic aciduria (MMA) and hyper- homocystinemia (Hcy) is characterized in the early onset, by failure to thrive, hypotonia, or with acute metabolic crisis. Parenteral hydroxycobalamin (OHB12) is the main treatment and dose escalation strategy may improve metabolic control. Methods: In three patients with early onset of cblC, we described the clinical and biochemical evolution following OHB12 dose intensification strategy. Results: Patients 1 and 2 were twins and before 1 month of age had failure to thrive, hypotonia, nystagmus and neutropenia. Patient 3 had also failure to thrive before 1 month of age. Subsequently, he developed acute metabolic crisis with hemolytic uremic syndrome, dermatitis enteropathica, neutropenia and nystagmus. For the 3 patients, molecular analysis disclosed homozygous mutations (c.271 dup A) in MMACHC. For patients 1 and 2, parenteral OHB12 was progressively increased from 1 to 5 mg per day and for patient 3 from 1 to 25 mg per day. In the three patients, we observed a reduction of both urinary MMA (patient 1: 2220 to 17; patient 2: 1425 to 14; patient 3:

Evaluation of genetic and biochemical profiles of patients with biotinidase deficiency Kose E 1, Onay H 2, Unal O 4, Gunduz M 4, Bulbul S 3, Arslan N 1 1 Div Metab Dis, Dokuz Eylul Univ Hosp, Izmir, Turkey, 2Div Medical Genetics, Ege Univ Hosp, Izmir, Turkey, 3Div Metab Dis, Kirikkale Univ Hosp, Kirikkale, Turkey, 4Ankara Children’s Hem Oncology Div Metab, Ankara, Turkey Background: Biotinidase deficiency (BTD) is an autosomal recessive inborn error of biotin metabolism and has been screened for nationwide in Turkey since 2007. The aim of this study was to evaluate the mutations and biotinidase activities of BTD patients. Methods: The study group consisted of 48 children (26; 54.2 % male) with BTD from three different centers in Turkey. Results: Among all, 45 (93.8 %) were found via the neonatal screening programme (SP) and the other 3 were diagnosed before the SP was started. The leading symptoms in these 3 patients were hearing loss and neurological

S234 detoriation. At present, the mean age of the group is 32.2 ± 39.8 months (median: 14, min: 2 - max:212 months). Consanguinity existed in 14 patients’ parents (29.2 %) and a family history of BTD was present in 7 (14.6 %) children. The mean serum biotinidase activity was 1.6 ± 1.1 nmol/min/mL (N: 4.4–12.2). Enzyme activities were profound (0.42 ± 0.25 nmol/min/mL) in 12 (25.0 %) patients and in 36 (75.0 %) the deficiency was partial (2.06 ± 0.98 nmol/min/mL). Mutation analyses revealed 8 (17.0 %) homozygous (D444H) and 21 (44.7 %) compound heterozygous (one allele D444H and a different mutant allele) mutations. 18 patients (38.3 %) had homozygous or heterozygous mutations other than D444H. In the heterozygous group two novel mutations (p.W140X and p.R209C) in one allele and D444H in the other allele were detected in two patients. Serum biotinidase activities of these patients were 0.7 and 3.08 nmol/min/mL, respectively. There was no significant difference between the four groups regarding biotinidase enzyme activity levels. All patients underwent biotin treatment (5–20 mg/day). DISCUSSION: Partial BTD is higher than expected in Turkey. We describe two novel mutations in two of our patients. More case series are required for evaluating the effect of these two mutations on enzyme levels and clinical spectrum of the BTD patients.

P-595

Abnormal folate metabolism is associated with metabolic syndrome components in spontaneous hypertensive rats (SHR) Kozich V 1, Pravenec M 2, Krijt J 1, Kazdova L 3, Sokolova J 1 1 Charles University- First Faculty of Med, Prague, Czech Republic, 2Institute of Physiology, Czech Acad Sci, Prague, Czech Republic, 3Inst Clin Exper, Czech Acad Sci, Prague, Czech Republic

Background: Metabolic syndrome (MS) is a multifactorial common risk factor for cardiovascular disease. A number of studies have indicated that abnormal metabolism of sulfur amino acids and folates is associated with MS components. Methods and results: We analyzed the role of folate deficiency in the pathogenesis of the metabolic syndrome in the spontaneously hypertensive rat (SHR) model. The SHRs fed a folate-deficient diet showed significantly reduced serum folate (104 ± 5 vs. 11 ± 1 nmol/L) and urinary folate excretion (4.3 ± 0.6 vs. 1.2 ± 0.1 nmol/16 h), ectopic fat accumulation, impaired glucose tolerance and increased systolic blood pressure. Next, by using BXH/HXB recombinant inbred strains derived from the spontaneously hypertensive rat (SHR) and Brown Norway progenitors we detected a deletion variant in the folate receptor 1 (Folr1) promoter resulting in decreased expression of Folr1 mRNA in the kidney. Analysis of the SHR.BN-chr.1 congenic strain confirmed that the Folr1 promoter variant in SHR cosegregates with reduced renal expression of the mRNA and renal folate reabsorption, decreased serum folate levels, ectopic fat accumulation, reduced muscle insulin sensitivity, and increased blood pressure. Transgenic rescue experiments performed by expressing a Folr1 transgene in the SHR ameliorated most of the metabolic disturbances. Discussion: These data support hypothesis that genetic and nutritional folate deficiency is associated with development of the metabolic syndrome in SHR model and constitutes a previously neglected mechanism that may contribute to increased risk for cardiovascular disease. Supported by grant GA CR 14-09283S, institutional support by PRVOUK P24 and RVO-VFN 64165/2012.

P-596

Postnatal hepatocyte transplantation in a child with Molybdenum cofactor deficiency type B Schwahn B C 1, Bansal S 2, Fitzpatrick E 2, Lemonde H 3, Champion M 3, Turner C 4, Fairbanks L 5, Mitry R 2, Lehec S 2, Filippe C 2, Chong K 6, White F 1, Okokon E V E 7, Dhawan A 2

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 1

Willink Biochem Genet Un, St Mary’s Hosp, Manchester, United Kingdom, Paed Hepatol, King’s College Hosp, London, United Kingdom, 3Paed Metab Unit, Evelina Hosp, London, United Kingdom, 4WellChild Laboratory, Evelina Hospital, London, United Kingdom, 5Purine Res Lab, Viapath, St Thomas Hosp, London, United Kingdom, 6 Dept of Radiology, Great Ormond St Hosp, London, United Kingdom, 7Clin Biochem, Viapath, King’s Coll Hosp, London, United Kingdom 2

Background: Neonates affected with Molybdenum cofactor deficiency (MoCD) typically present with early postnatal severe neurological symptoms due to excitotoxic effects of accumulating metabolites. Whereas MoCD type A can be treated with cPMP substitution, there is no known effective treatment for MoCD type B. Case report: A prenatally diagnosed child with MoCD type B was given a total of 1 billion donor hepatocytes in 6 intraportal doses between days 1 and 18 of life, combined with pharmacological treatment and dietary reduction of sulphur intake. Relevant biomarkers were monitored daily and brain imaging was performed at 1, 10 and 44 days after birth. Results: The hepatocyte transplantation was tolerated well. The patient remained intubated and ventilated until day 5. He had short clonic seizures at 4 h of age and was started on phenobarbitone. Levetiracetam was added from day 3 for remitting seizures which disappeared from day 18 but for intermittent myoclonic jerks. The patient was hypoactive with poor truncal tone and peripheral hypertonicity and non-nutritional suck only. Markers of sulphite and xanthine oxidation capacity indicated a partial correction of the metabolic defect within the first 5 days, which has persisted for over 2 months. Brain imaging at 44 days however showed extensive cystic degeneration and atrophy of the cerebral white and deep gray matter. Discussion: Postnatal hepatocyte transplantation and frequent metabolic monitoring was achieved through effective collaboration between multiple teams from different sites. The transplant led to a persistent improvement of seizure control, irritability and biochemical profile in comparison with a previous, pharmacologically and dietetically treated sibling. Treatment effects were seen within 5 days but were not sufficient to prevent neurological injury. Given the biochemical response antenatal intraperitoneal hepatocyte transplantation may be an option to improve the outcome of this otherwise fatal condition.

P-597

Preponderance of c.394C > T mutation in MMACHC gene in Indian patients with combined methylmalonic aciduria and homocystinuria due to cobalamin C deficiency Bijarnia-Mahay S 1 7, Gupta D 1 5, Puri R D 1, Kohli S 1, Saxena R 1, Shigematsu Y 3, Yamaguchi S 4, Sakamoto O 2, Deb R 5, Udani V 6, Verma I C 1 1 Inst Med Genet Genomics, SGR Hosp, New Delhi, India, 2Tohuku Univ Sch Med, Aobaku, Sendai, Japan, 3Sch Med Science, Univ of Fukui, Fukui, Japan, 4 Shimane Univ Sch Med, Shimane, Japan, 5Amity Sch Biotech, Amity Univ, NOIDA, UP, India, 6Dept Ped Neurol, PD Hinduja Hosp, Mumbai, India, 7 GRIPMER, Sir Ganga Ram Hosp, New Delhi, India

Background: Methylmalonic aciduria with homocystinuria due to cobalamin C deficiency (MMACHC) is the most common inborn error of cobalamin metabolism, caused by mutations in MMACHC gene. Recessive mutations in MMACHC causes a deficiency of the cofactors for methionine synthase (methylcobalamin) and methylmalonylCoA mutase (adenosylcobalamin) enzymes resulting in elevated methylmalonic acid and homocysteine with ensuing symptoms. Methods: Twenty biochemically proven cases of MMACHC deficiency from 16 families were enrolled. Sequencing of MMACHC gene, either full or targeted exons was performed in 18 cases. Results: All cases presented beyond neonatal period up to 12 years of age. Common clinical presentations were: developmental delay / neuroregression (70 %), seizures (50 %) and behavioral abnormalities. One-thirds of cases presented in acute crisis. All had hyperhomocysteinemia. Molecular analysis in 17 children detected homozygous mutation; c.394C > T (p.Arg132Ter) in exon 3 of MMACHC gene. One child showed compound heterozygous

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 mutations, c.394C > T and c.331C > T (p.Arg132Ter and p.Arg111Ter) in exon 3. Two prenatal diagnoses were carried out in one family. Discussion: This study shows the preponderance of c.394C > T mutation in MMACHC gene in Indian population, reportedly associated with late-onset phenotype. This simplifies the molecular analysis in cases and enables prenatal diagnosis, at least in the Indian sub-continent.

P-598

The challenge of treatments in three patients affected by pyridoxine dependent epilepsy (PDE) Celato A 1, Fasan I 1, Zanonato E 1, Cazzorla C 1, Toldo I 2, Sartori S 2, Polo G 1 , Burlina A B 1 1 Div Inherited Metab Dis, Univ Hosp, Padova, Italy, 2Div Ped Neurol, Dep Ped, Univ Hosp, Padova, Italy

Background: Pyridoxine dependent epilepsy(PDE) is a metabolic epileptic encephalopathy responsive to pyridoxine. Vitamin supplementation is effective in controlling seizures while adjunctive medications are required to prevent developmental retardation Case report: Three males were studied (one Italian and two North Africans) presenting early onset drug-resistant seizure. Pipecolic acid (PA) and αaminoadipic semialdehyde (α-AASA) were measured in cerebrospinal fluid (CSF), plasma and urine; pyridoxal phosphate (PLP) and amino-acids in plasma and CSF; biogenic amines in CSF. Pyridoxine therapy was started, after diagnosis, in 1 patient (30 mg/kg/day) while 2 patients were initially treated with PLP (30 mg/kg/day) and switched afterwards to pyridoxine or to a mixed regimen of the vitamins. Furthermore, 1 patient started a lysine-restricted diet, 1 L-arginine supplementation (400 mg/kg/day) and 1 a mixed treatment with diet together with L-arginine (150 mg/kg/day) Results: All patients showed high PA and α-AASA levels which turned to near normal values on pyridoxine; PLP was markedly low while lysine levels, though always normal, further decreased after diet and/or L-arginine. Biogenic amines displayed a secondary aromatic amino acid decarboxylase defect. Molecular genetics confirmed ALDH7A1 pathogenetic mutations with a patient homozygous for a previously unreported missense mutation (c.1256C > T, p.Ser419Leu) Discussion: PDE is characterized by 1) frequent incidence in formerly malaric areas 2) the absence of typical dysmorphic features though, in our patients, minor facial characteristics (carp-like mouth, flat nose, mongoloid eye shape) were present 3) variable electroencephalographic patterns but our cases shown generalized/multifocal discharges and delta brush activity 4) taking advantage of pyridoxine in controlling seizure and nearly normalizing PA and α-AASA 5) lowering lysine both through diet and/or L-arginine with clinical/ developmental effects yet to be proved.

25. Miscellaneous / New Disease Group

P-599

Recessive Pathogenic Variants in the MICU1 Gene: Expanding the Phenotypic and Genotypic Spectrum Ben-Omran T 1 1

Hamad Medical Corporation, Doha, Qatar

Background: Recently MICU1 gene, which encodes a subunit of the mitochondrial uniporter and regulates the opening of the dimeric calcium channel, MCU, has been deemed responsible for a novel mitochondrial disorder. Methods: Using WES, we identified 9 affected individuals from 9 additional unrelated families, who were either homozygous or compound heterozygous for pathogenic variants, most of which being novel in MICU1 gene

Results: Six Middle Eastern families (Qatari origin) were confirmed to be homozygous for a novel nonsense variant, p.Q185*. Another unrelated patient of Middle Eastern descent was compound heterozygous for p.Q185* and an intragenic duplication of exons 9 and 10 in MICU1 gene. One unrelated patient of European ancestry was found to be homozygous for an intragenic deletion encompassing exons 2 through 9 of MICU1 gene and another unrelated European individual was compound heterozygous for a novel nonsense variant, p.R119*, and a rare missense variant, p.R129P Discussion: Six patients presented with developmental delay and hypotonia. Three other patients with developmental regression. Most of the patients developed weakness and abnormal movements including ataxia, chorea or other stereotypic movement phenotype. All had elevated serum creatine phosphokinase and mild elevation of hepatic transaminases with hepatomegaly. Liver biopsy from one patient showed chronic active hepatitis. Additional features including ptosis were rarely observed, however ophthalmoplegia was not present in our patients. In summary, with this detailed phenotypic and genotypic characterization of 9 additional individuals in this study, we further expand the spectrum of MICU1-related mitochondrial disorder and highlight myopathy, movement disorders and the newly described liver involvement as major clinical features. Furthermore, targeted mutation analysis of MICU1 gene in Middle Eastern patients is warranted.

P-600

MBTPS2 mutations in X-linked osteogenesis imperfecta Lindert U 1, Cabral W A 2, Ausavarat S 3 4, Tongkobpetch S 3 4, Ludin K 5, Barnes A M 2, Yeetong P 3 4, Weis M 6, Krabichler B 7, Srichomthong C 3 4, Makareva E 9, Janecke A R 7 10, Leikin S 9, Roethlisberger B 5, Rohrbach M 1, Kennerknecht I 8, Eyre D R 6, Suphapeetiporn K 3 4, Giunta C 1, Marini J C 2, Shotelersuk V 3 4 Div Metab Dis, Univ Child Hosp, Zurich, Switzerland, 2Bone Extrac Matrix, NIH, Bethesda, United States, 3Dept Pediat, Chulalongkorn University, Bangkok, Thailand, 4Exc Cent Med Genet, Chulalongkorn Hosp, Bangkok, Thailand, 5Dept Med Genet, Aarau Hospital, Aarau, Switzerland, 6Dept Orth Sports Med, Univ Washington, Seattle, United States, 7Div Human Genet, Univ Innsbruck, Innsbruck, Austria, 8Inst Human Genet, Westfaelische Univ, Muenster, Germany, 9Physical Biochemistry, NIH, Bethesda, United States, 10Department Pediatrics, Univ Innsbruck, Innsbruck, Austria 1

Background: Osteogenesis imperfecta (OI) is a connective tissue disorder characterized by low bone mass and reduced bone mineral strength, leading to increased bone fragility and deformity. Up to now 16 different genes, which encode proteins involved in collagen synthesis and posttranslational modification, in bone mineralisation and osteoblast development, have been reported in autosomal dominant or recessive OI. We present the characterization of the first X-linked recessive form of OI which is caused by defects in MBTPS2. In a process called regulated intramembrane proteolysis (RIP), MBTPS2 a site-2 metalloprotease proteolytically cleaves diverse substrates thus releasing their N-terminal parts that act as transcription factors. Case report: In a small German family and in a large Thai pedigree with a moderate-severe form of OI linkage analysis and sequencing of the entire chromosome X-exome by next generation sequencing (NGS) was performed. Results: NGS of the X chromosome identified two hemizygous changes in MBTPS2: a p.L505F in the German family, and a p.N459S in the Thai family. In 4 affected individuals tested so far, the urinary pyridinoline ratios LP/HP were elevated (LP/HP 0.40 ± 0.03) compared to controls (LP/HP 0.19 ± 0.02). Mass spectrometry on a patient bone sample confirmed abnormal collagen crosslinking by showing a reduced hydroxylation of the K87 residues, which are critical for collagen crosslinking, in both collagen I α-chains. Discussion: MBTPS2 missense mutations have been identified in IFAP/KFSD spectrum diseases, which are regarded as disorders of cholesterol and/or lipid metabolism. The LP/HP ratio measured in an IFAP patient was normal. Thus, OI and IFAP/KFSD are not only different in the clinical presentation but likely also in the pathomechanism. Our results provide evidence that RIP plays a fundamental role in normal bone development.

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P-601

Al Menabawy N M 1, Ismail R I 1, Mohamed M A 2, Mehany D 2, ElSaeedy S 1 , Abdel Sattar I 1, Amar R 1, Selim L A 1, Seliem Z S 1

Discussion: PYCR2-related syndrome represents a clinically recognizable condition in which PYCR2 mutations lead to protein dysfunction, not detectable on routine biochemical assessments. Mutations predict a poor outcome, probably as a result of impaired mitochondrial function. Although this condition has been classified as a hypomyelinating leukodystrophy, we found that the imaging features could be variable, ranging from subtle white matter T2 hyperintensities to progressive brain atrophy. The variant c.796G > A (p.Arg266*) was the most common mutation suggesting common founder mutations in our population.

pediatric dep, Cairo University, Cairo, Egypt, 2clinical pathology dep, Cairo University, Cairo, Egypt

P-603

Clinical and biochemical spectrum of metabolic cardiomyopathies in Egypatian children

1

Background: Pediatric cardiomyopathy has a number of different, both genetic and non-genetic, causes and poses a formidable diagnostic challenge to clinicians. Identification of the underlying causes of cardiomyopathy may lead to improved outcomes with disease specific treatments. Methods: The clinical, biochemical, radiological and neurophysiologic data of all pediatric patients with a confirmed inborn error of metabolism (IEM) as an underlying cause of cardiomyopathy were analyzed and their prevalence was calculated among a cohort of patients with suspected metabolic cardiomyopathy (229) gathered from cardiomyopathy and metabolic clinics at the Cairo University Children Hospital. Results: In the current study, 57/229 patients (24.4 % of total) with cardiomyopathy were diagnosed with an IEM by metabolic work-up. Their mean age at diagnosis was 2.6 years. Within patients with metabolic cardiomyopathy, 75.4 % had hypertrophic cardiomyopathy, 22.8 % had dilated cardiomyopathy, while only 1.8 % was diagnosed with mixed cardiomyopathy and none of them had restrictive cardiomyopathy. The most common IEM causing hypertrophic cardiomyopathy were glycogen storage diseases (48.8 %), followed by mucopolysaccharidoses (25.5 %), mitochondrial disorders (9.3 %), lipid storage diseases (4.6 %) and aminoacidopathies (4.6 %). In the dilated cardiomyopathy group, fatty acid oxidation defects were the most common (38.5 %), followed by mucopolysaccharidoses (30.7 %). Mitochondrial disorders were 15.3 % while organic acidoses (Barth syndrome) and neutral lipid storage disease (Chanarin-Dorfman syndrome) were each 7.6 %. Discussion: IEM are an important group of genetic disorders amongst children suffering from cardiomyopathies. Early diagnosis will allow early therapeutic intervention with potential reversal of the disease and prevention of morbidity and mortality.

P-602

Mutations in PYCR2 define a lethal syndrome of failure to thrive, microcephaly, and brain atrophy in 10 Egyptian families Zaki M S 1, Bhat G 2, Issa M Y 1, Dikoglu E 2, Selim L A 3, Gamal I 3, Abdel Hamid M S 4, Marin-Valencia I 2, Gleeson J G 2 1 Clin Genet Depart, National Res Cent, Cairo, Egypt, 2Lab Ped Brain Dis, HHMI, Rockefeller Un, NY, United States, 3Pediatrics Depart, Cairo Univ, Cairo, Egypt, 4Med Molec Genet Dep, National Res Centre, Cairo, Egypt

Background: Hypomyelinating leukodystrophy type 10 with microcephaly is a newly delineated autosomal recessive disorder mapped to chromosome 1q42.12 due to mutations in PYCR2 gene. This gene encodes an enzyme involved in proline synthesis in mitochondria. Methods: We describe detailed clinical, radiological and mutational spectrum of twelve patients from ten consanguineous Egyptian families with PYCR2 mutations identified either by whole exome or targeted sequencing. Selective mutations from patients were tested for effect on protein function. Results: The characteristic clinical presentation of patients with PYCR2 mutations includes failure to thrive, microcephaly, craniofacial dysmorphism, progressive psychomotor disability, hyperkinetic movements, axial hypotonia and appendicular hypertonia with variable spasticity. Patients did not survive beyond the first decade of life. Brain magnetic resonance imaging (MRI) showed global brain atrophy and white matter T2 hyperintensities. Routine serum metabolic profiles were unremarkable. Both nonsense and missense mutations were identified, which impaired protein multimerization.

Asfotase alfa treatment in a prematurely born baby with hypophosphatasia Niinikoski H 1, Nanto-Salonen K 1, Korhonen K 1 1

Dept of Paediatrics, Turku Univ Hosp, Turku, Finland

Background: ALPL-gene codes tissue non-spesific isoenzyme of alkaline phosphatase (TNAP). In hypophosphatasia (HPP), loss of function in ALPL gene leads to high levels of inorganic pyrophosphate (PPi) and pyridoxal phosphate. Accumulation of PPi inhibits bone matrix calcification. In HPP, alkaline phosphatase (AFOS) is characteristically very low while plasma vitamin B6 (vitameric form of pyridoxal phosphate) is high. Perinatal form of HPP has been almost universally lethal but recent discovery of alfotase alfa enzyme (=TNAP) has changed the prognosis of this ultra rare disease. Asfotase alfa was approved by EMA in 2015 for treatment of childhood HPP Case Report: A baby with skeletal dysplasia was born at 34 weeks of gestation with BW of 2.090 kgs. She was intubated immediately after birth due to breathing difficulty and tracheomalacia. Plasma AFOS was < 5 U/L (ref. 60– 270) and B6-vitamin >1012 nmol/L (ref. 35–110). She had initial hypercalcemia and hyperphosphatemia. Skeletal survey revealed bone demineralization and changes including narrow chest and ribs and a bony spur in fibula with an adjacent skin dimple, i.e. Bowlder sign. She had a homozygous mutation in ALPL gene. Results: Subcutaneous asfotase alfa therapy (2 mg/kg x3/week) was started in this child at 10th day of life and is continuing. During enzyme therapy AFOS has increased to 2000–6000 U/L and plasma Ca, Pi and PTH have remained within reference ranges. The baby remained intubated for 2.5 months in NAVA-mode (neurally adjusted ventilation assist) and was gradually weaned from high-flow nasal cannula over the few days after extubation. At 5 months she is at home without extra oxygen or breathing difficulties but needs gastrostomy feeding. Her cognitive development is normal and she has not developed craniosynostosis. Bone mineralization has shown substantial improvement. Discussion: Asfotase alfa seems effective in perinatal HPP.

P-604

Gene Therapy for Canavan disease in the Knockout Mouse using rAAVs at a 20-fold lower dose Matalon R 1, Ahmed S 2, Su Q 2, Trapasso J 1, Gao G 2 University of Texas Medical Branch, Galveston, United States, 2Univ of Massachusetts Medical School, Worcester, United States 1

Background: Canavan disease is a lethal CNS disorder which is inherited in an autosomal recessive pattern. The mechanism of the disease is a deficiency of aspartoacylase (AspA) which leads to severe white matter degeneration of the brain with early death. No treatment has been found as of yet to correct the lethality in Canavan disease patients Methods: In previous studies the serotype rAAV9 was used and reported to prevent brain degeneration when injected intravenously. In this report we have tested rAAV9 as well as rAAV8 and rAAV10. Intracerebroventricular

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 injections were used instead of intravenous as in previous studies and the dose was lowered 20 fold to 2 microliters. Results: The intracerebroventricular injections resulted in significant alleviation of the brain pathology and also resulted in significantly decreased NAcetylaspartic acid. Weight gain and life span all improved and were close to normal with the intracerebroventricular injection. General health was assessed by blood biochemistry and showed improvement with both rAAV8, 9 and 10. Visual acuity assessed by Electroretinography studies also showed normalization when vector was administered irrespective of route of delivery. All the rAAV treated mice exhibited extended growth and survival by up to 2 years which were statistically significant (p value < 0.0001). Discussion: Gene therapy with rAAV8, 9, and rh.10 on mice with Canavan disease shows promising results. The Canavan mice treated with gene therapy had significant weight gain and life expectancy which were near the average of non-Canavan mice. When comparing our results with previous studies, the increased life span and weight gain are comparable. Also, our intrecerebroventricular injections used much lower dosages of the vector compared to the IV injections which can induce aspartoacylase activity in tissue that normally do not have it, such as the liver.

P-605

Increase in the diagnostic rate by exome sequencing in patients with neurometabolic disorders Kilic E 2, Kilic M 1, Ozgul R K 3, Yucel-Yilmaz D 3, Kavak P 4, Yuceturk B 4, Demirci H 4, Sagiroglu M S 4 1 Div Metab Dis, Sami Ulus Child Hosp, Ankara, Turkey, 2Div Pediatr Genetic, Hem-Onco Child Hosp, Ankara, Turkey, 3Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 4Bilgem, TUBITAK, Kocaeli, Turkey

Background: The aim of the study is to identify disease causing genes and mutations in undiagnosed neurometabolic patients. Methods: Twenty three patients from fifteen families, who had no diagnosis in spite of detailed biochemical, radiological and genetic studies were taken to study. Results: Exome sequencing resulted in identification of disease-causing mutations in 12 of 15 families. Consanguinity rate was 9/15 in this cohort. The diagnosis of patients and mutated genes were respectively; altered channel function, epilepsy (CACNA1H gene), mitochondrial-membran protein associated neurodegeneration (C19orf12 gene), 14q11-q22 deletion syndrome (CHR14: 20182017–20404268 duplication), 17p13.1 deletion syndrome (CHR17: 9943008–10416015 duplication), mitochondrial DNA depletion syndrome, encephalopathic form (SUCLA2 gene), spinocerebellar ataxia (ARSACS-Charlevoix-Saguenay) (SACS gene), bilaterally frontotemporal polymicrogyria (GPR56 gene), Dravet syndrome (SCN1A gene), congenital myopathy (RYR1 gene), ‘rhabdomyolyses’ (RYR3 gene, ACTN3 gene), combined oxidative phosphorylation deficiency 5 (MRPS22 gene). One family had a novel homozygous mutation in the MRPS24 gene which has not previously been identified and further studies are needed to prove the diagnosis. No disease associated gene was found in the remaining three families. Discussion: Next generation DNA sequencing methods are very useful diagnostic tools in undiagnosed patients with neurometabolic disorders.

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A floppy infant with slit ventricles on brain imaging - absence of SLC4A10 may cause the first human cerebrospinal fluid synthesis disorder Wortmann S B 1, Mayr J A 1, Spenger J 1, Mayr D 1, Sperl W 1, Koch J 1, Alhaddad B 2, Meitinger T 2, Prokisch H 2, Huebner C 3, Wolf N 4, Haack T B 2 1 Dep of Ped, SALK and PMU, Salzburg, Austria, 2Inst of Hum Gen, Techn Univ Munich, Munich, Germany, 3Inst of Hum Gen, Univ of Jena, Jena, Germany, 4Dep of Ped Neurol, VUMC, Amsterdam, Netherlands

BACKGROUND: SLC4A10 is a sodium/bicarbonate cotransporter in exchange for intracellular chloride, expressed in principal and inhibitory neurons as well as in choroid plexus epithelial cells of the brain. Slc4a10 knockout mice have collapsed brain ventricles and display an increased seizure threshold. Heterozygous deletions encompassing SLC4A10 in man have been associated with epilepsy and autism. CASE REPORT:This male is the first child of non-consanguineous Austrian parents. Child movements during pregnancy were experienced as normal, there was no polyhydramnios or breech presentation. From birth on he showed severely decreased muscle tone (“floppy infant”) and failure to thrive. Neonatal as well as follow up brain ultrasound at the age of 5.5 months showed collapsed (“slit”) brain ventricles. Currently, aged 10 months, he has an elongated face with bilateral ptosis, tented mouth and high-arched palate. He has high hyperopia (+8,5/+7 dpt), hearing tests were unremarkable. His passive muscle tone is very low (no head control, no sitting), but he is able to move his limbs against gravity. He sleeps longer and more frequent than peers, but when awake, he is alert, smiles, babbles and makes good contact. Parents report no seizures. A recent MRI at the age of 10 months shows small ventricles, especially slit anterior horns of the lateral ventricles. RESULTS: Exome sequencing revealed a homozygous nonsense variant in SLC4A10, c.2269C > T, p.(Arg757*) predicting a prematurely truncated protein and loss-of-function. There were no pathogenic variants in genes, or copy number variations affecting chromosomal regions, known to be related to neuromuscular disorders. DISCUSSION: In line with the existing mouse model, we assume that the biallelic mutations in SLC4A10 underly the paucity of cerebrospinal fluid in this individual. How this leads to muscular hypotonia needs further evaluation. Detection of further patients would be of enormous value.

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Rotavirus: The Implications on Children with Inherited Metabolic Disorders Mannion M A 1, Smith A 1, Hughes J 1, Knerr I 1, Monavari A 1 1

Temple Street University Hospital, Dublin, Ireland

Background: Rotavirus is the leading cause of diarrhoea worldwide in children < 5 years. Although mortality rates are low in Ireland, certain populations are more vulnerable to the associated morbidity and mortality of infection, particularly children with inherited metabolic disorders (IMDs). Rotavirus can result in acute decompensation, lactic acidosis, liver dysfunction and death in this population. The aim of this study was to evaluate children with IMDs who had confirmed rotavirus and compare the investigations, management and outcome with controls with rotavirus. We aim to highlight the importance of introducing the rotavirus vaccination in this vulnerable population. Methods: Patients with confirmed rotavirus was obtained from the microbiology laboratory over the last 5 years in Temple Street Children’s Hospital, Dublin. 14 patients with confirmed IMDs who were admitted with rotavirus were evaluated for details in terms of initial presentation, investigations and management. They were compared with 19 randomly selected age matched controls; children without IMDs who were also admitted with rotavirus infection. A retrospective chart review was conducted along with a review of the hospital laboratory system. Result: The median length of stay was 7 days in IMD patients versus 2 days in the controls. IV fluids were required on average for 4.57 days (SD 4.27) in IMD versus 1.05 days (SD 0.8) in controls. 2 IMD patients required a central line and 7 required total parental nutrition (TPN) compared to zero controls. Bloods were performed on average 8.53 times (SD 5.71) in IMD patients versus 2.5 times (SD 2.65) in the controls. Discussion: The rotavirus vaccination has been proven to be highly effective in reducing hospital admissions and mortality worldwide. As demonstrated in the IMD cohort, it leads to significant decompensation and morbidity in this population. The introduction of the rotavirus vaccine would offer additional protection to this vulnerable cohort.

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markers were early events, preceding the drop in expression of glycolytic enzymes. This suggests that glycolysis and the branching biosynthetic pathways respond to changing cellular biomass demand. Moreover, we observed that proliferation requires glycolysis but inhibition of glycolysis does not induce differentiation. Discussion: We provide the most comprehensive characterization of the metabolic configuration in proliferating and differentiated epithelial adult stem cells to date. Through this work, we reveal a crucial role for glycolysis in the production of cellular building blocks in proliferative epithelial stem cells to meet biomass demands. This might well represent the optimal metabolic configuration for any highly proliferative cell in physiology and malignancy.

High Incidence of Hereditary Liver Diseases as an Indication for Pediatric Liver Transplant in Saudi Arabia: comparison to the Hungarian Data. Barr M A 1 2, Burkholder J 1, Shagrani M A 1, Algoufi T 1, Dezsofi A 3, Szonyi L 1 King Faisal Spec Hosp, Riyadh, Saudi Arabia, 2Tanta Univ, Tanta, Egypt, Semmelweis Univ, Budapest, Hungary

1 3

Background: Liver transplantation is a therapeutic option for children with certain hereditary and metabolic liver diseases; some which cause liver injury, cirrhosis, and liver failure; and others lead to the accumulation of toxic metabolites with extra-hepatic effects. Aims and Methods: To compare hereditary liver disease as an indication for pediatric liver transplantation in two different centers; King Faisal Specialist Hospital and Research Center (KFSH&RC), Riyadh, Saudi Arabia, (2011– 2016) and I. St. Department of Pediatrics, Semmelweis University, Budapest, Hungary (1987–2016). Results: The following data were retrieved from KFSH&RC and Semmelweis University, respectively; total children who received liver transplantation were 212 vs 140 patients; for hereditary liver diseases (n = 103, 48.5 %) vs (n = 31, 22.1 %), biliary atresia (n = 61, 28.7 %) vs (n = 66, 47.1 %), and other liver diseases (n = 48, 22.6 %) vs (n = 43, 30.7 %). Of the patients with hereditary liver diseases, patients had: familial cholestasis (n = 53, 51.5 %) vs (n = 10, 32.3 %), Alagille syndrome (n = 10, 9.7 %) vs (n = 8, 25.8 %), and metabolic liver diseases (n = 40, 38.8 %) vs (n = 13, 41.9 %). Moreover, some children with hereditary liver disease had end-stage liver disease (ESLD) (n = 63, 61 %) vs (n = 18, 58 %), while others did not have ESLD (n = 40, 39 %) vs (n = 13, 41 %). Discussion: The number of liver transplanted children in KFSH&RC in the last 5 years has significantly exceeded that in the Hungarian data over the last 30 years. The main indication for pediatric liver transplantation in KFSH&RC was hereditary liver diseases, while biliary atresia was the main indication in the Hungarian data. The ratio of children transplanted for familial cholestasis in KFSH&RC is significantly higher than that in the Hungarian data, while the ratio of metabolic diseases is similar. The ratio of patients with hereditary ESLD was almost equal in both study groups, and accordingly, that of patients without ESLD.

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Glycolysis and the formation of building blocks in proliferating epithelial cells Fuchs S A 1, Schene I 1, Hasselt P M 1, Houtkooper R H L 3, Verhoeven-Duif N M 2, Nieuwenhuis E E S 1 1 Div Metab Dis, Wilh Child Hosp Utrecht, Utrecht, Netherlands, 2Lab Metab Dis, Wilh Child Hosp, Utrecht, Netherlands, 3Lab Metab Dis, Acad Med Center, Amsterdam, Netherlands

Background: The Warburg effect (aerobic glycolysis) is a well-known hallmark of proliferating cancer cells. Recently, this glycolytic phenotype was detected in some healthy proliferating cell types, but the function remains controversial. Despite their importance in cancer development and prospects for regenerative strategies for metabolic disorders, little is known about the metabolic phenotype in epithelial adult stem cells. Methods: We applied expression profiling and functional metabolic analyses to liver and intestinal organoids to unravel the metabolic configuration of proliferating and differentiating epithelial stem cells under controllable conditions. Results: We show that proliferating epithelial adult stem cells display high glycolytic transcription, activity and capacity but similar oxidative phosphorylation when compared to differentiated cells. We observed upregulation of the pathways branching from glycolysis during proliferation, fueling generation of building blocks needed for cell replication. During differentiation, the decrease in expression of stem cell markers, regulators of proliferation and cell cycle

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Diagnostic biochemical abnormalities masked by early testing or prospective treatment in newborns at risk of multiple acyl-coA dehydrogenase deficiency and maple syrup urine disease Aitkenhead H 1, Lam A A J 1, Prunty H 1, Cleary M A 2, Davison J 2 1

Chem Path,Gt Ormond St Hosp for Children, London, United Kingdom, Metab Med,Gt Ormond St Hosp for Children, London, United Kingdom

2

Background: Newborn siblings of patients with known metabolic diagnoses have a 25 % risk of being affected, and rapid confirmation of whether the newborn is affected is required for on-going treatment and to reduce the psychological burden on the family. Prospective treatment is usually initiated until results are available, however this may alter or mask biochemical findings, complicating diagnostic results. Case Reports: We describe two cases of newborns who were treated prospectively as their siblings had metabolic diseases. Case 1: sibling of a patient who died suddenly with suspected multiple acyl CoA dehydrogenase deficiency (MADD) but without a confirmed diagnosis. The newborn was treated with a low fat, high energy feeding plan plus riboflavin. Diagnostic samples for acylcarnitine analysis were collected on day 1, 3, 5 and day 18 (off treatment). Case 2: sibling of a patient with MSUD. The newborn was treated with a branched chain amino acid (BCAA) free feed. Blood spots were collected for BCAA analysis on day 1, 4, 5 and 6 during the first week of life. Results: Case 1: The initial acylcarnitine profile was unremarkable however by day 18 the acylcarnitine profile was consistent with MADD. Case 2: On day 1 the blood spot allo-isoleucine concentration was less than the detection limit and blood spot valine, isoleucine and leucine concentrations were within reference intervals. By day 4 and day 11, allo-isoleucine concentration was raised at 11 and 82 micromol/L respectively. Discussion: These cases highlight the need to interpret the results from samples collected shortly after birth with extreme caution especially if the patient is treated prospectively. Further samples should be collected and analysed quickly so that the diagnosis can be made or excluded. Good communication between the clinical team and the laboratory is vital so that decisions about treatments and results interpretations are fully informed.

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Developing next-generation pharmacological chaperones by fragment screening and crystallography. Mackinnon S R 2, Kopec J 2, Von Delft F 2 3, Brennan P E 1 2, Yue W W 2 1 Target Discov Inst, Univ of Oxford, Oxford, United Kingdom, 2Struct Genom Consortium, Univ of Oxford, Oxford, United Kingdom, 3Diamond Light Source, UK, Oxford, United Kingdom

Background: Pharmacological chaperones (PCs) hold promise as small molecule therapies for inborn errors of metabolism (IEM), potentially

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 crossing the blood–brain barrier to selectively stabilize mutant proteins to aid folding and activity. However, PCs developed to date are mainly substrate/cofactor mimetics that bind the active site and inhibit enzyme activity. To address this limitation, we have combined highthroughput crystallography and fragment screening, to survey target proteins for allosteric/cryptic binding pockets and to identify chemical starting points for non-inhibitory PCs. Methods: We selected 10 proteins from various IEM classes (e.g. organic aciduria, urea cycle, lysosomal storage disorders), based on tractability in protein biochemistry, prevalence of missense mutations, and unmet need. Using previously developed methodology, each target was subjected to the screening of up to 2000 fragment compounds (MW < 300 Da) where each compound was soaked into pre-formed protein crystals for x-ray structure analysis. Results: For our 10 IEM proteins, we have streamlined the entire geneto-structure process, implementing throughput parallel expression and purification platforms, robot-assisted crystallography and fragment soaking. To date, we have generated recombinant proteins for 5 of the 10 targets, reproducibly crystallized 3 of them, confirmed diffraction quality for 2, and completed an initial screening campaign for 1 target. Alongside our medicinal chemistry collaborators, fragment hits will be developed into potent PCs using structure based drug design and biophysical testing. Discussion: The screening approach developed here can be systematically applied to diverse protein targets linked to IEM, because of its independence from enzyme function or disease rarity. Therefore we anticipate that the new molecular entities and novel binding modes can provide the step change to transform small molecule therapies in the IEM field.

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Follow-up of a patient with cobalamin F deficiency El Habbas M 1, Sechter C 2, Joncquel M 3, Fontaine M 3, Morin G 5, Dobbelaere D 1, Benoist J F 4, Mention K 1 1 Div Metab Dis, Lille Univ Hosp, Lille, France, 2Div End Dis, Besancon, France, 3Bio lab, Lille Univ Hosp, Lille, France, 4Robert Debre Hosp, Paris, France, 5Amiens Hos Cent, Amiens, France

Background Cobalamin F (cbl F) deficiency is a rare disorder of intracellular cobalamin metabolism caused by mutation in LMBRD1 gene associating methylmalonic acidemia and homocystinuria. We distinguish the early and the late onset presentation of cbl F disorder. Our case illustrates the two clinical presentations of the disease. Case Report We report a case of an 8-year-old boy who presented in neonatal period with hypotonia, feeding difficulties and bone marrow aplasia with no dysmorphic features. Metabolic work up revealed methionine depletion, accumulation of methylmalonic acid (950 mmol/ mol of creat) and low carnitine. Genetic studies for transcobalamin II and cbl C were normal. He was treated with vitamine B12 and carnitine for 1 year with improvement of hematologic abnormalities and decrease of methylmalonic acid level to 30 then the patient lost to follow-up and the treatment was stopped. The parents resought medical advice for their 8-year-old-child for his behavior disorders. He exhibited a mild developmental and speech delay, limited cognitive skills, reccurent infections, presence of skin pigmentation and glossitis. Results Methylmalonic acid was 38.5 mmol/mol creat, the total plasma homocysteine levels was 72 μmol/l and no hematologic abnormalities. The diagnosis of cbl F deficiency was confirmed by genetic studies. Intramuscular vitamin B12, oral vitamin B6 and folic acid were instituted. Follow-up for 1 year revealed an improvement i n t h e p a t i e n t ’s b e h a v i o r w i t h c o r r e c t i o n o f b i o c h e m i c a l abnormalities. Discussion The interest of this case lies in its rarity, less than 20 patients were described so far in the literature. Left untreated, it could lead to neurodevelopmental and speech delay, recurrent infections, glossitis and skin pigmentation with eventual normal hematologic markers.

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Minimal prevalence and incidence of inherited metabolic disorders in Austria Karall D 1, Herbst S 1, Scholl-Buergi S 1, Brunner-Krainz M 2, Emhofer J 4, Huemer M 5, Kircher S G 6, Koch J 7, Konstantopoulou V 3, Lagler F 7, Loescher W N 8, Maier E M 10, Mayr J A 7, Moeslinger D 3, SunderPlassmann G 9, Sperl W 7, Stulnig T M 9, Wortmann S B 7, Zschocke J 11 1 Pediatrics, Medical University, Innsbruck, Austria, 2Pediatrics, Medical University, Graz, Austria, 3Pediatrics, Medical University, Vienna, Austria, 4 Pediatrics, Regional Hospital, Steyr, Austria, 5Pediatrics, Regional Hospital, Bregenz, Austria, 6Pathobiochemistry and Genetics, Med Univ, Vienna, Austria, 7Pediatrics, Medical University, Salzburg, Austria, 8Neurology, Medical University, Innsbruck, Austria, 9 Internal Medicine, Medical University, Vienna, Austria, 10von Hauner Children’s Hospital, LMU, Munich, Germany, 11Div Human Genetics, Medical University, Innsbruck, Austria

Background: One of the requirements to establish a national service for the care of patients with inherited metabolic disorders (IMD) is to know how frequent they are. Methods: Within the network of the Austrian metabolic group, we retrospectively retrieved data of patients with IMD according to the SSIEM ICD11 catalogue. Results: Altogether, 2287 minimal data sets of patients with confirmed IMD (biochemically, enzymatically and/or molecularly) were reported. Of these, 364 were diagnosed before 1994, 1310 between 1994 and 2014; no information for 613. The mean prevalence from 1994 to 2014 for all disorders was calculated to be 1/1286; the overall incidence for all disorders was calculated to be 1/3705 or 27/100000. The five most common condition are: Phenylketonuria (415/2287 patients), galactosemia (149/2287), Fabry disease (117/2287), X-linked adrenoleukodystrophy/-myeloneuropathy (104/2287), biotinidase deficiency (84/2287). The three most common metabolic disorder groups are: amino and organic acid disorders (701/2287 patients), lysosomal storage disorders (478/2287) and disorders of energy metabolism (342/ 2287). 12 % of patients were reported twice, only very few by more than two centers. Of all, 782 were diagnosed through newborn screening, 553 through selective screening and 130 through family screening; there are no data from 822 patients. To date, 641 are well under treatment, 309 are symptomatic and 179 have died (no outcome data for 1158 patients); 990 are female and 1266 are male, gender is unknown for 31. Discussion: The mean minimal prevalence of IMD in Austria (from 1994 to 2014) is 1/1286, the minimal incidence 1/3705 or 27/ 100000. As the reporting was done through the members of the Austrian metabolic group to warrant freedom of commercial and health insurance conflicts of interest, we are aware that there is a probably substantial number of patients unknown to us, e.g. those taken care by primarily family doctors and/or adult physicians only.

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Artefactual increase in urine thymine concentration affecting GC-MS but not HPLC analysis Mozley E 1, Fairbanks L 1, Carling R 1 1

Biochem Sci, Viapath, St Thomas’ Hosp, London, United Kingdom

Background: Increased thymine excretion in urine occurs in two pyrimidine metabolism disorders; dihydropyrimidine dehydrogenase (DPD) deficiency and dihydropyriminidase (DHP) deficiency. In these disorders an increase in the excretion of uracil is also usually observed.

S240 This laboratory uses GC-MS for urine organic acid analysis, which can be used to identify both thymine and uracil. Increases in thymine and uracil are re-analysed and quantified by HPLC. We have a number of examples where a gross increase in the excretion of thymine has been observed by GC-MS, but was measured within the reference range when using HPLC. Uracil was not increased in any case. There are no described pathological causes of an isolated increased thymine. Methods: Sample preparation for GC-MS analysis involves an extensive extraction process including acidification, solvent extraction and trimethylsilyl (TMS) derivatisation at 75 °C. Sample preparation for HPLC analysis is much simpler; the sample is diluted with water before direct injection onto the HPLC column. Results: We have a number of cases where a gross increase in thymine was observed when analysed by GC-MS, but within the reference range when measured by HPLC. In each case either the patient themselves, or the neonatal patient’s mother, was receiving a nucleoside analogue reverse-transcriptase inhibitor (NRTI) such as azidothymidine (AZT; zidovudine). The mechanism of action of these compounds is by insertion into replicating viral DNA in the place of thymine, therefore blocking transcription and preventing viral replication. Discussion: Due to the difference in thymine quantitation between the two methods, it is hypothesised that the analytical conditions used for GC-MS analysis cause hydrolysis of the drug compound present in the sample, causing release of thymine and a falsely increased concentration. Quantitation of uracil in these samples is important to exclude a disorder of pyrimidine synthesis.

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GMP isolation and biodistribution of human Amnion Epithelial Cell for Clinical Therapy. Raghuraman Srinivasan C 1, Kannisto K 1, Hammarstedt C 1, Zabulica M 1, Strom C S 1, Gramignoli R 1, Askelof U 2 1 Karolinska institute, Huddinge, Sweden, 2Karolinska university hospital, Huddinge, Sweden

Background: Human placenta is a non-controversial and readily available source of stem cells that can be used in regenerative medicine. We previously reported that transplanted human amnion epithelial cells (hAEC) differentiate into hepatocyte-like cells resulting in correction of mouse models of metabolic liver disease, such as MSUD and PKU. These successful preclinical results have motivated isolation and banking of hAEC at Karolinska Institutet. As part of preclinical safety studies, we investigated the bio-distribution of hAECs after different routes of administration. Methods: During the last year we have standardized reagents and procedures in accordance with current Good Manufacturing Practice (GMP). Release criteria have been designed, and biodistribution has been monitored after infusion via the spleen or the tail vein. Cryopreserved hAECs were labeled with the near IR lipophilic dye (DiR) and monitored by in vivo imaging system (IVIS) and the content of human DNA in different organs was quantified by qPCR. Results: After splenic infusion of hAECs (1 million cells/mouse) the DiR signal was detected in liver and spleen at 3 and 24 h post-transplant. The distribution was confirmed by analysis of human DNA content at 24 h posttransplant. Tail vein infusion resulted in detection of DiR mainly in the lungs, with some detection in the liver, also confirmed by human DNA content at 24 h post-transplant. Discussion: Preclinical studies suggest that amnion-derived epithelial cells might be a useful alternative to hepatocytes for the treatment of liver-based inborn errors of metabolism. Isolations according to GMP procedures and bio-distribution evaluation support the translation to clinical applications. As for clinical hepatocyte transplants, hAEC infusion via the spleen/portal vein resulted in cell engraftment in the liver parenchyma, with minimal leakage to lungs or other organs.

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Congenital myopathy associated with splice mutation in PAX7 gene Ahting U 1, Makowski C 2, Juenger H 2, Schoser B 3, Mayr J 4, Alhaddad B 1, Prokisch H 5, Rolinski B 6, Haack T 1, Freisinger P 7, Meitinger T 1 5 1 Human Genetics Technische Univ Munich, Munich, Germany, 2Pediatrics Technische Univ Munich, Munich, Germany, 3Friedrich Baur Insitut LUM, Munich, Germany, 4Pediatrics Paracelsus Med Univ, Salzburg, Austria, 5 Human Genetics Helmholtz-Zentrum, Munich, Germany, 6Laboratory Pathology Elblandliniken, Meissen, Germany, 7Pediatrics Kreisklinikum Reutlingen, Reutlingen, Germany

Background: Myopathies are common in pediatric neurology. They can have various aetiologies, e.g. metabolic or structural. It is estimated that one quarter of them remain unresolved. Here, we suggest a new cause of pediatric myopathy. Case Report: During pregnancy reduced foetal movements were noted. Delivery was normal. Newborn sepsis with increased plasma lactate resolved after antibiotic treatment. The patient then presented with signs of congenital myasthenia such as hypotonia and ptosis. Slight developmental delay was noted in the first few months. Mental and motor development normalized within the second year of life. Independent ambulation was achieved at a normal age. Pronounced ptosis with reclined head position remained a main sign of disease. The child is now 4 years-old. Results: Metabolic screening was negative. Myasthenia gravis was excluded. Muscle biopsy at 6 months of age revealed increased content of as well as ballooned mitochondria. However, no abnormalities in respiratory chain and pyruvate dehydrogenase activity were found. Whole exome analysis identified a homozygous splice site mutation in PAX7: c.86-1G > A. PAX7 is a transcriptional factor involved in development of early skeletal muscle fibers. Segregation analysis and prediction methods are consistent with a pathogenic role of mutated PAX7. Transcriptional and immunohistological studies showed reduced wt-PAX7 levels and altered expression of downstream factors. Discussion: We present a patient with myasthenic-like phenotype associated with loss of function mutation in a transcriptional factor for muscle development. To our knowledge, PAX7 has not yet been described as a disease-causing gene.

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HACE1 deficiency mimicking mitochondrial disorder Petkovic Ramadza D 1, Mayr J A 2, Haack T 3 4, Prokisch H 3 4, Zarkovic K 5, Fumic K 6, Baric I 1 7 1 Dep Pediatr, Univ Hosp Cen, Zagreb, Croatia, 2Dep Pediatr, Paracelsus Med Univ, Salzburg, Austria, 3Inst Hum Gen, Helmholtz Zentrum, Munchen, Germany, 4Inst Hum Gen, Technische Univ, Munchen, Germany, 5Dep Pathol, Univ Hosp Cen, Zagreb, Croatia, 6Dep Lab Diagn, Univ Hosp Cen, Zagreb, Croatia, 7School Med, Univ Zagreb, Zagreb, Croatia

Background: HACE1 is E3 ubiquitin ligase involved in Golgi membrane fusion and regulates the activity of a number of small GTPases. It is expressed in the brain and at lower levels in other tissues. HACE1 deficiency is an autosomal recessive disease reported by Hollstein et al. in 2015 in 8 patients from 2 families with developmental delay, hypotonia later progressing to lower limb spasticity, epilepsy, brain atrophy, ocular abnormalities (strabismus, myopia, retinal dystrophy), and intellectual disability. Patients were either homozygotes or compound heterozygotes for loss-of-function mutations of the HACE1 gene. Case Report: Our patient had generalised hypotonia and delayed milestones since early infancy. At the age of 2 years she had delayed

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 motor and speech development, neurogenic bladder requiring catheterisations, recurrent febrile seizures, and recurrent unexplained episodes of fever of central origin during summer. Brain MRI showed gray and white matter atrophy and hypoplastic corpus callosum. Imunological work-up was normal. Plasma alanine and proline were mildly elevated. In muscle tissue there were abnormally shaped mitochondrial cristae with myelin-like figures. Now, at the age of 3.5 years, the patient shows slow motor and intellectual improvement but is still unable to stand unsupported. Results: Enzyme activities of respiratory chain complexes in fresh frozen muscle were reduced, with clearly decreased cytochrome c oxidase. Whole exome sequencing revealed in HACE1 gene the likely pathogenic homozygous mutation c.1765G > A, p.(Glu589Lys) which was confirmed by Sanger sequencing. Parents were found to be heterozygotes for the mutation. Discussion: Recurrent fever and laboratory signs of mitochondriopathy are novel findings in HACE1 deficiency. It is unclear and still needs to be elucidated how HACE1 influences mitochondrial function. Considering the role of HACE1 protein in ubiquitination, recurrent fever might be due to altered immune and/or inflammatory response.

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Bi-allelic variants in PRUNE cause early manifestation of severe epileptic encephalopathy with muscular hypotonia Alhaddad B 1, Schossig A 2, Haack T 1 3, Kovacs-Nagy R 1, Sperl W 4, Prokisch H 1 3, Mayr J 4, Senderek J 6, Zschocke J 2, Distelmaier F 5, Koch J 4 1 Human Genetics Technische University, Munich, Germany, 2Human Genetics Medical University, Innsbruck, Austria, 3Human Genetics Helmholtz Zentrum, Munich, Germany, 4 Pediatrics Paracelsus Medical University, Salzburg, Austria, 5Pediatrics University Children Hospital, Duesseldorf, Germany, 6 Pediatric Neurol University of Munich, Munich, Germany

Background: PRUNE encodes a member of the aspartic acid-histidinehistidine (DHH) protein superfamily of phosphoesterases. This protein functions as both a nucleotide phosphodiesterase and an exopolyphosphatase and is highly expressed in the human fetal brain. In Drosophila, Prune has been suggested to stabilize the mitochondrial transcription factor A (TFAM) and promote mitochondrial DNA (mtDNA) replication through down-regulation of cAMP signaling in mitochondria. PRUNE mutations have been recently described in four unrelated cases with severe developmental delay, microcephaly, fronto-temporal cortical atrophy, and cerebellar atrophy. Case Report: We applied whole exome sequencing to investigate seven patients from four families presenting with infancy-onset therapyresistant epilepsy, severe developmental delay, and profound intellectual disability. All patients were hypotonic and some developed dysphagia, scoliosis, and spastic-dystonic quadriplegia. Plasma lactate in all patients was normal. Three of the patients died before the age of 12 years. Results: Whole exome sequencing identified the previously reported homozygous PRUNE missense variant (Asp106Asn) in affected individuals from three families. In the fourth family, an extended search for copy number variants (CNVs) detected a homozygous contiguous gene deletion encompassing exons 2–8 of PRUNE and exons 1–7 of the neighboring BNIPL open reading frame. Sanger sequencing and allele-specific PCR (for deletion) confirmed the variants in all patients with the parents being heterozygous carriers. Preliminary data from experiments in patient-derived fibroblasts showed no clear evidence of impaired mitochondrial function or changes in TFAM protein levels. Discussion: Our findings extend the phenotypic spectrum of the disorder and provide further evidence for a loss of function pathogenic mechanism in PRUNE resulting in this severe form of encephalopathy.

P-619

Mutations in RC3H1 in a boy with ethylmalonic aciduria and hemophagocytic lymphohistiocytosis Verloo P 1, Bogaert D 2, Haerynck F 3, Vanlander A 1, Menten B 4, Van Coster R 1, Dullaers M 2 1 Div Ped Neur, Univ Hosp, Ghent, Belgium, 2Clin Immunol Res Lab, Univ Hosp, Ghent, Belgium, 3Div Ped Pulm, Univ Hosp, Ghent, Belgium, 4Cent Med Genet, Univ Hosp, Ghent, Belgium

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare acute immune dysregulation syndrome characterized by fever and systemic inflammation. HLH was reported as a complication in several metabolic disorders. Case Report: An 11-year-old male patient became acutely ill. He was admitted to our hospital and was found to fulfill all criteria for HLH, including fever, thrombocytopenia (42.000/uL), anemia, liver failure with signs of necrosis on biopsy, increased ferritin concentration (35199 ng/mL), hypofibrinogenemia, hypertriglyceridemia, splenomegaly and signs of hemophagocytosis in bone marrow aspiration. He was followed for several years at another hospital because of mild intellectual disability, sensorineural hearing loss, chronic chronic diarrhea and petechia. Initial work-up in that center had shown ethylmalonic acid in the urine and the tentative diagnosis of ethylmalonic aciduria was made. At that moment, the underlying molecular defect was not clear. Before he presented with acute signs HLH he never had been acutely ill. The acute problems prompted us to do further etiological work-up. Results: Pathogenic mutations in ETHE1 and ACADS, two genes associated with ethylmalonic aciduria, could not be detected. OXPHOS activity was normal. Using whole exome sequencing a homozygous novel stop codon mutation was detected in the RC3H1 gene (c.2414G > A, p.688R/*) encoding Roquin, an E3 ubiquitin ligase that prevents autoimmunity and inflammation by repressing target mRNAs including ICOS, OX40 and TNF-α. The propositus had expanded but mainly naive B cells, increased expression of ICOS and OX40 in T-cells and extremely increased pro-inflammatory cytokines including TNF-α in serum, even under cyclosporine. Discussion: This is the first report of a pathogenic mutation in RC3H1 encoding Roquin in a subject with ethylmalonic aciduria and severe HLH. The RC3H1 mutation results in increased expression of ICOS, OX40 and pro-inflammatory cytokines which may predispose to HLH.

P-620

Diagnosis and discovery of treatable neurometabolic diseases via an integrated –omics approach Van Karnebeek C D 1, Salvarinova R 1, Horvath G 1, Stockler S 1, Vallance H 3 , Sinclair G 3, Wassermann W 2, Tarailo-Graovac M 1 2 1 Dept of Pediatr, Univ British Columbia, Vancouver, Canada, 2Dept Of Med Genet, Univ British Columbia, Vancouver, Canada, 3Dept of Pathology, Univ British Columbia, Vancouver, Canada Background: Whole exome sequencing (WES) has transformed rare diseasegene discovery and diagnosis. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorders (IDD). However, neurometabolic diseases (NMDs) are the exception; late in 2014, 89 were known to be responsive to causal therapy, i.e. targeting pathophysiology at molecular or cellular level. Methods: To uncover the genetic basis of potentially treatable NMDs, we combined deep clinical phenotyping with WES analysis and metabolomics via an unbiased semi-automated bio-informatics pipeline, in consecutively enrolled patients with IDD and an unexplained biochemical phenotype. Results: WES analysis was completed in 59 IDD patients (from 47 families); 8 patients were excluded due to other identified etiologies. The remaining 51 patients in 42 families were predominantly single cases born to nonconsanguineous Caucasian parents. Likely pathogenic variants were identified in probands of 38 families, in 43 different genes: 11 genes not previously linked to a human disease phenotype, 23 disease genes with novel patient

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phenotypes and 9 genes with expected phenotypes. In 5 families, complex phenotypes were explained by co-existing monogenic conditions. In 18 families the diagnosis impacted management beyond genetic counseling, including the discovery of 4 novel NMDs potentially amenable to causal therapy Discussion: Our diagnostic yield and discovery rate exceeded expectation, likely due to enrichment of our cohort for new NMDs and phenotypes, semi-automated bio-informatics pipeline and close collaboration between families, clinicians and scientists. In 43 %, WES diagnosis allowed for precision medicine, varying from prevention and tailored symptom management, to causal therapy.

P-621

Screening for treatable inborn errors of metabolism in 500+ intellectual developmental disorder patients Van Karnebeek C 1, Salvarinova R 1, Horvath G 1, Sinclair G 2, Ghani A 1, Vallance H 2, Stockler S 1 1 Dept of Pediatr, Univ British Columbia, Vancouver, Canada, 2Dept of Pathology, Univ British Columbia, Vancouver, Canada

Background: Intellectual developmental disorders (IDD) are characterized by significant impairment of cognitive functions, affecting 2.5 % of the population worldwide with significant morbidity and associated healthcare costs. Inborn errors of metabolism (IEM) currently constitute the only group of genetic defects amenable to causal therapy. Early diagnosis prevents or minimizes brain damage. Our literature review identified 89 such treatable IEM; although evidence is limited, therapies are often effective, safe, accessible. Methods: We translated this knowledge into the TIDE diagnostic protocol: The 1st tier comprises metabolic screening tests in blood/urine with potential to identify 62 % of treatable IDs. The second tier focuses on remaining disorders, requiring ‘single test per disease’ approach. A freely available App (www.treatable-id.org) supports the protocol. The protocol was implemented during 3 years in our 3 divisions in our tertiary care centre. Results: Treatable IEMs in >5 % of 518 IDD patients (n = 27), including creatine deficiencies, amino-acidopathies, serine deficiencies, metal disorders, vitamin responsive disorders, neurotransmitter diseases, organic acidurias etc. Analysis comparing these patients to those diagnosed in our hospital between 2000 and 2009 revealed that the TIDE protocol reduced ‘time to diagnosis’ by 6 months (range 1-50 months) as well as costs of unnecessary testing (>$1500per patient). Discussion: Our protocol for treatable forms of ID has proven effective in terms of increasing the diagnostic yield and reducing costs and diagnostic delay. Treatment effects vary from improvement of cognitive development, behavior, epilepsy, psychiatric disturbances or stabilization of disease. Overall better outcomes can be achieved via standard screening for treatable conditions in IDD patients.

P-622

Analysis of intensive care service (ICU) utilisation for metabolic patients over a 9-year period. Dalkeith T 1 2, Bhattacharya K Zurynski Y 1 2 3

1 2

, Biggin A

1 2

, Christodoulou J

1 2 4 5

,

1 Childrens Hospital at Westmead, Sydney, Australia, 2University of Sydney, Sydney, Australia, 3Australian Paediatric Surveillance Unit, Sydney, Australia, 4 Murdoch Childrens Research Institute, Melbourne, Australia, 5University of Melbourne, Melbourne, Australia

Background: Inborn errors of metabolism (IEM) place unquantified demands on health service utilisation. Our aim was to describe the burden of admissions for children with an IEM requiring ICU care during admission to our centre.

Methods: A cohort of children aged ≤18 yrs from the Genetic Metabolic Disorder Service (GMDS) representing 125 different genetic metabolic disorders were identified (July 2004-June 2013). Data detailing service utilisation and costs for inpatient admissions was obtained from our Management Support Analysis Unit. Results: 783 GMDS patients (428 M; 355 F) were identified; excluding day procedures, 354 patients accounted for 1682 admissions. ICU management was associated with 181 admissions (112 patients), with patients 0–2 yrs old accounting for 60 % of ICU admissions. Median ICU time was 98.5 hrs (4–1869 hrs), with a 7 -day median hospital admission length of stay (LOS), whereas a median admission LOS of 2 days was observed for those not requiring ICU care. 30 % of admissions were direct GMDS admissions and 27 % of admissions were primarily for surgery. The top 4 ICU admission disorder categories were: mitochondrial (24 %), lysosomal (16 %), amino acid (14 %), and urea cycle disorders (13 %). Neonates (≤40 days of age) accounted for 23 % of ICU stays, with a 12day median total LOS, compared to a median LOS of 7 days for older patients. 44 % of ICU admissions were directly attributable to metabolic decompensation. Twelve children (6.6 % of admissions) died during an admission requiring ICU care. Total cost for admissions associated with ICU care were 5,437,978€, with a median cost of 14,737€ compared to non-ICU admissions which were substantially less, with a median cost of 3,384€ Discussion: Metabolic patients have significant health care costs which are attributed primarily to the disorder or to associated complications or treatments. We continue to develop strategies to reduce hospital LOS, support optimum medical management of these children, while providing a family-centred, cost effective service.

P-623 The burden endured by caregivers of metabolic patients: mucopolysaccharidoses compared to intoxication disorders Nichelli F 1, Meregalli P 1, Gasperini S 1, Galimberti C 1, Schivalocchi E 1, Parini R 1 1

Dept Pediat MBBM Found S Gerardo Hosp, Monza, Italy

Background: Few data are available about the global burden among primary caregivers of metabolic disease patients (pts). More accurate information is needed to address a specific psychological support. Methods: An anonymized self–reported satisfaction and Quality of Life questionnaire (Majani and Callegari, SAT-P- Satisfaction Profile Erickson 2006) validated for adults of both sexes age 20– 65 was administered to 50 parents of 29 MPS pts and 32 parents of 21 pts with urea cycle disorders, organic acidemia and glycogenoses (overall defined OTHERS). SAT-P consists in five domains (1- psychological functioning, 2-physical well-being, 3-job, 4- sleep and free time, 5- social function), investigated through 32 items which are answered chosing a point from 0 to 100 over a line. The results are differently weighted depending on age and sex and transformed in a zeta-score where a result −1 to +1 corresponds to normal healthy individuals, −2 to −1 is considered a borderline result needing attention, below −2 is definitely pathological and needs psychological assistance. Results: In the group MPS 66 % of caregivers had normal results and 22 % had 2 or more borderline/pathological items compared to only 22 % with normal results and 32 % with 2 or more borderline/ pathological items in the group OTHERS. These results could not be attributed to a more recent diagnosis in the OTHERS nor to different extra support in the families of the two groups. Discussion: We hypothesize different psychological behaviors in the 2 groups due to disease charateristics. MPS parents can stabilize after having reached an adaptation to progressively slowly worsening disease. OTHERS’ parents have to cope with a permanent risk of sudden decompensation and need to manage anxiety everyday. Different psychological approaches are probably advisable for the 2 groups.

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P-626

A homozygous splice mutation in SLC25A42, encoding a mitochondrial transporter of coenzyme A and adenosine 3’,5’-diphosphate, causes metabolic crises and epileptic encephalopathy

Influence of altered ROS levels on expression and activity of sirtuins. Potthast A B 1, Fitter A 1, Das A M 1

Iuso A 1 2, Bader A 1 2, Meitinger T 1 2, Prokisch H 1 2, Strom T M 1 2, Weigel C 3, Haack T B 1 2 1

2

IHG, TUM, Munich, Germany, IHG, Helmholtz Zentrum Muenchen, Neuherberg, Germany, 3Dept of Paediatrics, Univ of Erlagen, Erlangen, Germany Background: The mitochondrial matrix hosts a variety of vital enzymatic functions supporting fatty acid and pyruvate oxidation, and the citric acid cycle. As it is surrounded by a double membrane, mitochondrial carriers are required for an efficient exchange of metabolites, nucleotides, and cofactors between the cytosol and mitochondrial matrix. Impairment of mitochondrial carriers have been linked to different clinical conditions, including disturbed cofactor metabolism. Only recently, it has been shown that SLC25A42 codes for an inner mitochondrial membrane transporter which imports coenzyme A (CoA) into mitochondria in counter-exchange for intramitochondrial (deoxy) adenine nucleotides and andenosine 3’,5’-diphosphate. Together with its thioester derivates (i.e. acetyl-CoA, malonyl-CoA, 2-hydroxy-3-methylglutaryl-CoA), CoA plays an essential role in various anabolic and catabolic pathways as well as regulation of cellular processes via allosteric interactions and gene expression. These include the biosynsthesis of fatty acids, ketone bodies, and cholesterol, amino acid metabolism, fatty acid oxidation, biosynthesis of the neurotrasmitter acetylcholine, and acetylation of histones and regulation of gene expression. Defects within two of the five enzymatic steps involved in CoA biosynthesis have linked to childhood-onset forms of neurodegeneration with brain iron accumulation (PANK2, COASY). Case Report: We have applied exome sequencing in a patient with a metabolic encephalopathy. Result and Conclusion: We have identified a homozygous canonical splice site variant in SLC25A42 rendering the encoded mitochondrial CoA transporter a new candidate for human disease.

1

Ped Metab Dis, MHH, Hannover, Germany

Background: In mammals 7 sirtuins with different subcellular localisations are known. Target proteins of the sirtuins are e.g. proteins in metabolic pathways. Radical oxygen species (ROS) are highly reactive molecules which can react with lipid membranes, nucleic acids and proteins, and thereby damage the biological integrity. ROS are produced as a side product of the mitochondrial respiratory chain. Methods: We analysed ROS levels via FACS-analysis during our experiments on regulation of sirtuins in mitochondriopathies (COX-deficiency). Additionally, we assayed SIRT1 and SIRT3 at transcript, protein, and activity levels. Results: COX-deficient fibroblasts showed decreased ROS levesl as well as decreased sirtuin levels. Further analyses with a ROS-scavenger (Tocopherol) and inducers (Paraquat, Diclofenac) led to altered sirtuin levels. ROSinduction resulted in increased sirtuin levels, while ROS depletion resulted in sirtuin inhibition. Discussion: In contrast to common concepts, we found reduced ROS-levels in COX-deficient cells. This reduction of the second messenger ROS resulted in decreased sirtuin levels in our experiments.

P-627

Sirtuin 4 is up-regulated in the HELLP-syndrome Sandvoss M 1, Potthast A B 1, Das A M 1 1

Ped Metab Dis, MHH, Hannover, Germany

P-625

Modulation of Sirtuins under Ketogenic Diet Potthast A B 1, Das A M 1 1

Ped Metab Dis, MHH, Hannover, Germany

Background: Sirtuins are NAD + −dependent protein deacetylases. In mammals 7 sirtuins with different subcellular localisations are known. Target proteins of the sirtuins are e.g. proteins in metabolic pathways. Ketogenic diet (KD) is a low carbohydrate, high fat diet, which is a therapeutic option in therapy -refractory epilepsy. Under KD the neuronal cells predominantly use ketone bodies as energy source. The molecular mechanism of the antiepileptic effect of KD is still unclear. Methods: We cultured HT22 neuronal cells with decanoic acid which is elevated in KD, for 7 days. The cells were analysed for SIRT1, SIRT2 and SIRT3 on transcript-, protein- and activity-level. The essential cofactor NAD+ and the NAD/NADH ratio were determined as well. Results: We found an inhibitory effect of decanoic acid on SIRT2 at protein and activity level while SIRT3 expression and protein levels were increased. SIRT1 protein level was slightly decreased while there was no effect on expression and protein level. The concentration of the essential cofactor NAD+ remained unchanged, while there was an increase in NADH concentration, so that there was a shift towards NADH in the NAD+/NADH equilibrium. Discussion: Changes in SIRT1 and SIRT3 levels can modulate the mitochondrial metabolism as well as general energy metabolism. A reduction of SIRT2 may hamper neuronal differentiation. In summary, our data suggest a modulation of sirtuins under ketogenic diet.

Background: The haemolysis, elevated liver enzymes and low platelet count (HELLP)-syndrome is frequently observed in mothers whose offspring suffer from long chain-fatty acid oxidation defects. We observed that fatty acid oxidation (FAO) is not only compromised when the fetus suffers from these inborn errors of metabolism but in human umbilical vein endothelial cells (HUVEC) from all pregnancies complicated by the HELLP syndrome. Sirtuins are NAD + -dependent deacetylases, directly linked to the metabolic status of the cell. SIRT4 is known to regulate FAO by repressing malonyl CoA decarboxylase. Furthermore, we measured SIRT1 and SIRT3 in HUVEC as these are important regulators of energy metabolism. Methods: Sirtuin levels were measured in HUVEC from pregnancies with a healthy fetus complicated by the HELLP syndrome and compared to uncomplicated pregnancies exposed to hypoxia (n: 7 controls, 7 HELLP; 0, 10, 60 or 120 min of 2 % O2). Transcript (qRT-PCR) and protein (Western Blot) levels of cytosolic SIRT1, mitochondrial SIRT3 and SIRT4 were determined. Enzymatic capacities of SIRT1 and SIRT3 were measured fluorometrically, NAD+-levels were assayed spectrophotometrically. Results: Protein levels of SIRT4 were significantly higher in HUVEC from HELLP-cells compared to controls after 60 and 120 min of hypoxia. NAD+levels increased in a time dependent manner. Discussion: Elevated SIRT4 levels result in compromised FAO which seems to be a common pathophysiological mechanism associated with the HELLP syndrome. SIRT4 increases mitochondrial coupling leading to a more efficient ATP synthesis with elevated ATP-levels. NAD+-synthesis seems to be stimulated under hypoxic conditions. Via increased substrate saturation this increases flux across sirtuins which further compromises FAO. This may give rise to toxic intermediates and elevated fatty acids which are known to result in fetal complications, namely preeclampsia, oxidative stress and inflammation.

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P-628

neonate mouse mortality in the efficacy study, higher doses of scPMP were required to increase liver SO activity in order to further reduce brain SSC levels. These findings may inform dose selection of scPMP.

Basal ganglia in neurometabolic disorders Paprocka J 1, Machnikowska-Sokolowska M 2, Rzepka B 1 1 Pediatr and Dev Age Neurol Dept, Katowice, Poland, 2Pediatr Radiol Dept, Katowice, Poland

Background: The basal nuclei (basal ganglia) are functional part of the extrapyramidal system. They are associated with involuntary movements coordination, cognitive functions, and emotional reactions. The main components of basal ganglia are: caudate nucleus, putamen, nucleus accumbens, globus pallidus, lenticular nucleus, and substantia nigra. The aim of the study is to define a correlation between different neurological disorders especially neurometabolic diseases in children and changes in basal ganglia and the thalamus. Methods: The study group consisted of 152 children, aged 2 months to 18 years (mean age 5.98 years), hospitalized in the Department of Pediatrics and Developmental Age Neurology in Katowice, Poland. The patients were divided into 8 groups: ischaemic encephalopathy (n = 43; 28.3 %), neurometabolic diseases/progressive encephalopathies (n = 28, 18.4 %), fakomatosis (n = 18; 11.8 %), cerebral palsy and intellectual disability (n = 12; 7.9 %), vascular diseases of the brain (n = 17; 11.2 %), neuroinfections (n = 12; 7.9 %), congenital defects of brain (n = 6; 3.9 %), and other disorders (n = 16; 10.5 %). Results: The most relevant percent of changes detected in the area of the basal nuclei was in group of ischaemic encephalopathies (n = 75/257; 29.2 %) and in the group of neurometabolic disorders/progressive encephalopathies (n = 47/257; 18.3 %). Discussion: From whole basal ganglia, changes in lenticular nucleus were the most frequent (n = 98; 38.1 %). In the group of neurometabolic disorders generalized involvement of basal ganglia were seen. Isolated changes were observed in 23.7 % of examined patients, the others have changes in multiple locations.

P-629

Synthetic cyclic pyranopterin monophosphate (scPMP) rescues the lethal phenotype of molybdenum cofactor (MoCo) deficient mice: relationship of scPMP doses, liver sulfite oxidase activity, body weight, and the detoxification of sulfite in vivo Liu-Chen S1 , Watsky E1 , Devore D1 , Kuklin N1 , Marozsan A1 , Wang Y1

01. Inborn Errors of Metabolism in Adults

A-001

Screening for Fabry disease using dried blood spots: an Australasian experience Stark S L 1, Dobbins J R 1, Fong B 1, Chin S 1, Fuller M 1, Fletcher J M 1 1

SA Pathology, Women’s Children’s Hosp, North Adelaide, Australia

Background: The National Referral Laboratory (NRL) has provided leukocyte and plasma testing for Fabry disease (FD) for the Australasian region since the late-1970s. FD is an X-linked recessive lysosomal storage disorder resulting from a deficiency in a-galactosidase A (a-gal). Symptoms of FD can include renal failure, stroke, neuropathic pain crises, and organ dysfunction, with significant variation in severity. Glycosphingolipid deposition, including lysoglobotriaosylsphingosine (lyso-Gb3), occurs throughout the body. Diagnosis of heterozygotes can be challenging, as many display a-gal activity within the normal range. The NRL began screening for Fabry disease using dried blood spots in 2013. Methods: Dried blood spot (DBS) a-gal activity was determined using a 4methylumbelliferyl-conjugated substrate. Results: Hemizygous males showed markedly reduced levels of a-gal activity (0.11 ± 0.02 nmol/h/mL, mean ± SEM, (NR = 2.0–6.9,)). Confirmed FD heterozygotes also displayed reduced levels of a-gal activity (1.17 ± 0.11 nmol/h/ mL, n = 51). 6/51 heterozygotes had a-gal activity in the normal range and notably, 2 of these women displayed elevated plasma lyso-Gb3. Current data stands at 100 % sensitivity/100 % specificity for males and 88 % sensitivity/ 99 % specificity for females. Discussion: Measurement of a-gal activity in DBS is a fast and inexpensive method for screening for FD. While a-gal-deficient males are clearly identified by the assay, caution must still be exercised when interpreting a-gal activity in females. More than one of enzyme activity, clinical presentation, lyso-Gb3 and GLA sequence variations should be evaluated when considering a diagnosis of FD.

1

Alexion Pharma Inc, New Haven, United States

Background: MoCo deficiency (MoCD) type A is caused by mutations in the MoCo synthase 1 (MOCS1) gene, which is responsible for synthesis of MoCo precursor cyclic pyranopterin monophosphate (cPMP). MoCD leads to loss of liver sulfite oxidase (SO) activity, causing accumulation of sulfite and Ssulfocysteine (SSC), irreversible brain damage, and infant mortality. Recombinant cPMP administration in Mocs1-/- mice and in patients with MoCD type A has been shown to produce favorable outcomes. In patients with MoCD, plasma SSC can be readily measured but brain SSC and liver SO are not accessible. We evaluated the dose relationship of scPMP to plasma concentrations of SSC, changes in brain SSC and liver SO activity, and sulfite metabolism in vivo in Mocs1-/- mice. Methods: In an efficacy study, a fixed dose of 2–4µg of scPMP was administered 3 times per week for 107 days. In a dose escalation study, Mocs1-/neonate mice were given scPMP at 0.1, 1.1, 2.2 and 4.4 mg/kg daily for 28 days to evaluate SSC in plasma and brain as well as SO activity in the liver. Results: In the efficacy study, scPMP extended survival for the majority (11/ 15) of Mocs1-/- mice beyond 107 days vs a placebo group (median survival of 9 days). However, in mice administered scPMP, body weight (BW) was significantly (p < 0.05) lower than age-matched heterozygote controls. In the dose escalation study, administration of scPMP from 0.1–2.2 mg/kg produced a dose-dependent reduction of SSC in plasma and brain. Administration of scPMP at 4.4 mg/kg enabled Mocs1-/- mice to achieve BW and liver SO activity comparable to that of wild type mice. Discussion: scPMP showed a dose-relationship to plasma and brain SSC, liver SO, and BW in Mocs1-/- mice. Although lower doses of scPMP prevented

A-002 The importance of MTHFR testing in woman with recurrent spontaneous abortions Serapinas D 1, Bartkeviciute A 2, Bartkeviciene D 2 1 Lith univ heal scien, Dept gen mol med, Kaunas, Lithuania, 2Viln Univ, Fac Med, Dep Ob Gyn, Vilnius, Lithuania

Background: Recurrent pregnancy loss (RPL) is a heterogeneous condition affecting up to 5 % of women of reproductive age. Pregnancy itself represents a protrombotic state, which combined with thrombophilic gene mutations may contribute to adverse pregnancy outcome. Methods: MTHFR C677T and A1298C mutation shave been tested in 30 consecutive women with recurrent spontaneous abortion of unknown etiology (other causes, such as anatomical or endocrine factors, antiphospholipid syndrome, or chromosomial anomalies were excluded by specific investigations). The methods used were - PCR-based assays. Results: At least a variant allele of the MTHFR mutations was seen in 23 patients (77 %), of which 12 patients (40 %) were either homozygotes for C677T or A1298C mutations, or compound heterozygotes. The mean homocystein level in patients with 2 mutations was 11.2 mcmol/L, in patients with one or none nutations—7,3 mcmol/L.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Discussion: A great proportion of women with recurrent spontaneous abortion harbour thrombophilic mutations. It is important to test these mutations in these cases, since the results can guide towards appropriate therapeutic measures, that lower homocystein level, during next pregnancies. A-003

Valproic acid-induced severe hyperammoniemia unmasked by a protein loading test Tran C 1, Royer Bertrand B 1, Rossetti A O 2, Bonafe L 1, Bonafe L 1 1 Center Mol Dis, Univ Hosp, Lausanne, Switzerland, 2Neurol Service, Univ Hosp, Lausanne, Switzerland

Background: Hyperammonemia has been reported to be associated with patients who receive valproic acid (VPA). The mechanism responsible for this side effect remains unsolved and might be related to polymorphism in the genes encoding components of urea cycle. We describe a 20 year old male who was treated with VPA due to refractory generalized epilepsy, presenting with mental slowing. His fasting ammonia level was slightly elevated (67 μmol/l; N < 50) under this treatment, and raised the question of the impact of protein intake on his ammonia level. Methods: Ammonia levels were measured during 2 conditions: 1. Pre/ postprandial (standard meal) and 2. Preprandial and 90 min after an oral protein load (total 1 g/kg body weight). The same measures were repeated after VPA withdrawal. Whole exome sequencing (WES) was performed to identify genetic variation causing progressive myoclonic epilepsy and mitochondrial disorders. It was also used to screen genes involved in fatty acid oxidation, urea cycle, and to detect polymorphism in the glutamine synthase and carbamoyl-phosphate synthetase 1 gene. Results: Preprandial and postprandial ammonia was 76 μmol/l and 203 μmol/l, respectively. Pre and post protein load ammonia was 81 and 217 μmol/l, respectively. Ammonia decreased to 76 μmol/l 4 h later, without any scavengers. The test was repeated 5 months after withdraw of VPA and revealed a level within normal pre and post protein load (21 and 39 μmol/l, respectively). WES did not reveal mutations or polymorphisms in any of the screened genes. Discussion: Clinicians should increase their awareness for potential valproate induced transient severe hyperammoniemia after protein intake, which can be missed based on fasting and preprandial value of ammonia. Further studies are needed to elucidate the role of protein intake on ammonia level during VPA therapy and its underlying causal mechanism. Mental slowing may represent a clue for testing ammonia levels in patients taking VPA.

A-004

An unexpected differential diagnosis for a severe deterioration in an adult with glycogen storage disease type 1 Bosanska L 1, Schroeder T 2, Hoff U 2, Koerner R 2, Kruse J M 2, Senf R 2, Enghard P 2, Vietzke M 2, Tiling N 1, Ploeckinger U 1 1 Centre Rare Metab Dis, Charite Univ, Berlin, Germany, 2Dep Nephrol and Intensive Care, Charite, Berlin, Germany

Background: Gastrointestinal symptoms or intercurrent infections may lead to a metabolic decompensation in patients with glycogen storage disease type 1. However, other causes should be considered in a prolonged / repeated decompensation. Case Report: We report about a 26-year-old patient admitted due to vomiting resulting in hypoglycemia and lactic acidosis. As the continuous PEG-tube feeding was not possible due to gastrointestinal complaints intravenous glucose was initiated. Microbiology testing as well as gastroscopy findings were without pathologies. A few days after the initial recovery the symptoms worsened and the patient had to be transferred to the intensive care unit (ICU). Despite normoglycemia the lactic acid blood levels (40–70 mg/dl) were above the target range and normalization was slow. A second deterioration 24 h after

ICU care led to a reduction in consciousness and acute respiratory failure, acute renal failure, hepatic dysfunction, bleeding complications and heart failure with severe lactate acidosis (lactic acid 340 mg/dl). Continuous hemodialysis was initiated. Results: Diagnostic testing unexpectedly showed a high blood concentrations of metformin and acetylsalicylic acid, none of the drugs had been prescribed. Intoxication by proxy was supposed to be the cause of the critical illness, patient contact was restricted and the police was contacted. With hemodialysis the drugs were removed, lactate acidosis normalized and a full recovery was observed. Discusssion: In conclusion we observed a severe lactate acidosis due to intoxication with metformin and acetylsalicylic acid in a patient with glycogenosis type 1 possibly due to Munchhausen syndrome by proxy.

02. Novel diagnostic/laboratory methods

A-005 GC /MS analysis of urinary mono and disaccharides following mixed-bed resin purification and acetylation in methyl sulfoxide /1-methylimidazole reagent Habbal M Z 1 1

pathology lab medicine americ university, beirut, Lebanon

Background: A variety of sugar derivatives are used to analyze these compounds by GC/MS which include silylation, reduction and acetylation, oximation and acetylation. All these methods are not without shortcomings and makes interpretation difficult. In their seminal paper, LI et al. (J .Agricultural and Food Chemistry 2013, 61, 4011400-18) described an optimized GC/MS method to quantify plant sugars based on derivatisation in methyl sulfoxide / 1methylimidazole system. Methods: Application of their method showed that various sugars found in human urine can be quantitatively acetylated at room temperature and short reaction time. The acetylated derivatives can be extracted with hexane rather than CH2CL2. In order to purify urine from interfering compounds, an amount of urine equivalent to 1 micromole of creatinine was added to a short column of mixed-bed resin, eluted with water which is lyophilized and the residue acetylated. Results: The retention time in minutes of the following was obtained: erythritol 8.370, Threitol 8.532, xylose 10.45, arabinose 10.631, ribitol 11.896, arabitol 12.060, xylitol,12.337, fucose 10.601, α-fructose 14.798, β-fructose 14.993, α-galactose 15.113, β-galactose 15.227, α-glucose 15.359, β-glucose 15.457, mannitol 15.768, sorbitol 17.133, sucrose 27.339, maltose 27.804, lactose 28.00. Discusssion: Mixed bed resin was the only method that gave clean urine extract for acetylation of carbohydrates. GCMS analysis proved to again be the method of choice to separate various urine carbohydrates succesfully, even the anomers.

A-006

Metabolome analysis by tandem mass spectrometry as a diagnostic tool for inborn errors of metabolism – preliminary studies. Polawski T 1, Kusmierska K 1, Sykut-Cegielska J 1, Oltarzewski M 1 1

Screen Dep, Inst of Mother and Child, Warsaw, Poland

Background: Targeted metabolomics techniques provide a promising followup of standard diagnostic procedures (amino acids profile/urine organic acid analysis). It involves a wide biomarkers spectrum and offers a simultaneous quantitative analysis of biologically active compounds without isotope analog. We have adapted tandem mass spectrometry (LC/MS/MS) to investigate

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plasma, urine and cerebral metabolome of the patients suspected for inborn errors of metabolism (IEM) to asses usefulness of quantitative metabolomics. Methods: We investigated healthy children (1 month–10 years old) and patients at the age 1 month–10 years with clinical symptoms of unknown etiology and drug resistant epilepsy. More than 50 metabolites were analysed by dansyl chloride derivatisation. Analytes derivatised with deuter labeled dansyl chloride were used as internal standard. The samples were seperated by HPLC using kinetex PFP column and detected by LC/MS/MS. Results: Reproducible results with linear standard curve was obtained for all analytes. Adapted method allows quantification of low abundant analytes even with concentration below 10 nmol/L. In patient group with symptoms of unknown etiology there were found abnormal metabolites concentration of: sulphocysteine, cystinie, pipecolic acid, sarcosine, corresponding to final diagnosis of molybdenium cofactor deficiency, cystinuria, Zellweger syndrome, glutaric aciduria type 2 respectively. Patient with drug resistant epilepsy had abnormal concentration of β-alanine and γ-aminobutyric acid (GABA)—suspected for GABA transaminase deficiency or hyper-beta-alaninemia. Discusssion: Targeted metabolome profiling can be useful as followup tool in IEM diagnostics by expanding profile for analytes not routinely analysed by standard amino acids and/or urine organic acids profile and by achieving low limit of quantification. Further studies are needed to evaluate more complex metabolome quantitative analysis.

1

Div Metab Dis, Univ Child Hosp, Utrecht, Netherlands, 2Hubr Inst, Utrecht, Netherlands, 3Div Gen, UMCU, Utrecht, Netherlands, 4Div Ped, UMCU, Utrecht, Netherlands, 5Ophthal, UMCU, Utrecht, Netherlands Background: The zebrafish (Danio rerio) is a powerful model system to study genetic defects underlying ophthalmological pathogenicity. As the lens mirrors systemic signs of inborn errors of metabolism, the zebrafish lens is expected to be a useful model to study early metabolic consequences of genetic mutations. No method to routinely image detailed cataractogenic structures of the zebrafish embryo lens has been described in the literature. Methods: We developed a UV-light microscopic method as a tool to visualize the zebrafish lens and we compared this with established microscopic methods, including confocal microscopy. Fluorescence microscopy with reflected light illumination and a UV-filter was used to image the lens of living, genetically modified zebrafish embryos (as early as 5 days post fertilization). Results: UV-light microscopy allowed complete visualization of the lens of the zebrafish embryo in vivo. Detailed cataractogenic structures in the lenses of genetically modified fish were detected and rapidly imaged. Discusssion: We present fluorescence microscopy with UV-illumination as a valuable tool to analyze detailed lens abnormalities in living zebrafish embryos. In vivo visualization is enabled by UV-light induced auto-fluorescence, analogous to slit lamp biomicroscopy used in human diagnostics. Our study showed this method has superior properties and suggests to be of use to study the pathogenesis of cataract in inborn errors of metabolism.

A-009

A-007 Spectrum of metabolic diseases identified by clinical whole exome sequencing 1

1

1

1

1

Lee H , Ko Y , Lee C , Lee J S , Kim H D , Lee J S

Complementary test for the diagnosis of Pompe disease Schenone A B 1, Frabasil J 1, Durand C 1, Bambara C 1, Sokn S B 1

1 1

Laboratorio Dr N A Chamoles, Buenos Aires, Argentina

1

Yonsei University College of Medicine, Seoul, Republic of Korea

A-008

BACKGROUND: Pompe disease (PD) is an autosomal recesive inborn error of metabolism due to the deficiency of acid alfa-glucosidase. Glycogen storage in the cells is the result of the enzyme deficiency. PD has a wide clinical spectrum, from the classic infantile form (IOPD) to the late-onset in adulthood (LOPD). The diagnosis could be done measuring the enzyme activity with fluorogenic substrate in dried blood spot (DBS). Pseudodeficiency was describe in this disease and showed in vitro enzyme activity in the pathological range. Blood film examination for the presence of periodic acid-Schiff (PAS)-positive lymphocyte (PPL) vacuoles was described to support the diagnosis of PD.The aim of this study is to evaluate the usefulness of blood films examination stained with PAS reagent to the diagnosis of IOPD and LOPD Methods: Peripheral blood films, DBS, leukocytes and DNA were collected from: 50 healthy children between 1 day and 1 year old and 50 healthy adults to be control group; 4 IOPD, 4 LOPD and 1 patient with pseudodeficiency. All of them were evaluated for PPL presence, which was expressed as percentage of PPL per 100 lymphocytes, counted by microscopic inspection. GAA activity was measured in DBS and leukocytes by a fluorometric method. Results: The %PPL obtained were: Control Group: healthy children 1–14 % (mean = 6.5), healthy adults 1–31 % (mean = 13.5). Patients Group: IOPD 46– 56 % (mean = 51.7), LOPD 27–37 % (mean = 32), pseudodeficiency patient: 19 %. All of them have DBS and leukocytes enzyme activity in the pathological range and mutations described as pathogenic. Discusssion: PPL is not usefull to detect patients with LOPD, the results obtained from this patients overlaps with adult healthy controls. Patient with pseudodeficiency showed PPL within the normal range. Patients with IOPD could be detected from healthy controls supporting the diagnosis of Pompe disease.

UV-light microscopy and its application to visualize detailed cataractogenic structures in the zebrafish lens

03. Newborn screening

Rumping L 1, Graaff, de A 2, Schellekens P A W 5, Tessasori F 2, Haaften, van G W 3, Houwen R H J 4, Jans J J M 1, Hasselt, van P M 1, Verhoeven-Duif N M 1

A-010 Withdrawn

Background: Next generation sequencing technique has become one of the popular tests for the diagnosis of unexplained neurodevelopmental deficit in childhood. Inborn errors of metabolism are a common cause of delayed development with unknown origin. Clinical whole exome sequencing was performed in 40 children showing symptoms of delayed development in order to make a confirmative diagnosis. Methods: We utilized the Trusight One sequencing panel providing comprehensive coverage of < 4,800 clinically known disease-associated genes. Sequencing was performed using the Illumina MiSeq. The sequenced reads were mapped to the human reference (UCSC hg19) with Burrows-Wheeler Aligner (BWA), and variants were identified with the Genome Analysis toolkit (GATK). Sequence variants were filtered according to various quality parameters. All reported variants were confirmed by Sanger sequencing. Results: Six of 40 patients were found to have different metabolic diseases that can cause neurologic symptoms including delayed development. These were, glutaric aciduria type 1, mucolipidosis II, hyperprolinemia type 1, congenital disorder of glycosylation, type Ik, pyruvate carboxylase deficiency, biotinidase deficiency. Discusssion: The above diseases are not common in the Korean population. NGS technique was very helpful for the diagnosis of rare metabolic diseases and those diseases were not suspected to be the reason for the patients’ symptoms. Rare metabolic diseases are even more difficult to diagnose in practice. Therefore, when a patient shows neurologic symptoms of unknown origin, NGS can be one of the techniques for making a definitive diagnosis.

S247

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 A-011

Classical PKU with unusual neonatal presentation Fingerhut R 1 2 4, Lehnherr N 5, Pfeifle V 5, Holland-Cunz S 5, Sluka S 1 2 4, Szinnai G 5, Huemer M 2 3 5, Rohrbach M 2 3 1 Swiss Newborn Screening Laboratory, Zurich, Switzerland, 2Childrens Research Center, Zurich, Switzerland, 3Div Metabol, Univ Childrens Hospital, Zurich, Switzerland, 4 Univ Childrens Hospital, Zurich, Switzerland, 5Univ Childrens Hospital, Basel, Switzerland

Background: Newborn screening (NBS) for phenylketonuria (PKU) using tandem mass spectrometry is performed in Zurich since 2005. Phenylalanine (Phe) and the ratio (Phe/Tyr) are sensitive and specific tests to detect PKU. The main clinical features of untreated PKU are developmental delay and intellectual disability, which become evident from the first weeks of life. Case Report: We describe a boy born at term from non-consanguineous Swiss parents with tetrahydrobiopterine (BH4) sensitive PKU with unusual neonatal presentation. The child presented with floppiness, irritability, recurrent bilious vomiting and failure to pass meconium until 32 h after birth, resulting in the clinical suspicion of an intoxication type metabolic disease such as maple syrup urine disease (MSUD) rather than PKU. Ketonuria or hyperammonaemia were not observed. Results: NBS performed on the 4th day of life revealed elevated Phe of 650 μmol/L, low Tyr of 30 μmol/L, and a Phe/Tyr ratio of 22. However, slightly elevated branched-chain amino acids initially supported the clinical suspicion of MSUD, but alloisoleucine was not detectable. Morbus Hirschsprung was suspected due to dilated intestinal loops and lack of intestinal gas in the anorectal region. On day 8 branched-chain amino acids had normalised; BH4-test and urinary pterin supporting the diagnosis of BH4 sensitive PKU. Dietary Phe restriction was initiated immediately. Discusssion: Hirschsprung’s disease was diagnosed by rectal suction biopsy and consecutive a pull through procedure with resection of rectosigmoid and colorectal anastomosis were performed successfully. However the patient continued to suffer with severe constipation as well as vomiting and impaired oral feeding. Postoperative therapy was difficult because of the interaction of both rare conditions, metabolic disease and intestinal motility disorder. Meanwhile, with the age of 10 months the situation got much better and he is developing mentally and physically.

Results: SCADD was confirmed by U-EMA in 5/11 (45 %) in period A and in 15/32 (47 %) pts in period B. In particular in period B, 63 % of cases (5/8 pts) with C4 ≥ 1 μmol/L and 42 % of cases (10/24 pts) with C4 < 1 μmol/L (false negative) were confirmed. U-EMA levels were extremely variable. All patients did not show any metabolic decompensation during follow-up. Discusssion: Our preliminary data showed that measuring free EMA with C4 levels reduces false negatives for SCADD at NBS, however it has doubled the number of recalls. We need to confirm our results with molecular analysis of ACADS gene in all U-EMA positive cases. A longer follow up is mandatory to evaluate the clinical impact of this condition in these patients and the opportunity to measure free EMA in our NBS program.

A-013

Ratio C8/C10 as a discriminative predictor for MCADD in NBS Glab-Jablonska E 1, Polawski T 1, Sykut-Cegielska J 1, Oltarzewski M 1 1

Screen Dep, Inst of Mother and Child, Warsaw, Poland

Background: Medium Chain Acyl Co-A Dehydrogenase Deficiency (MCADD) is the most frequent inborn error of fatty acids oxidation in newborn screening (NBS) in Poland. Tandem mass spectrometry (MS/MS) has been used by Institute of Mother and Child in NBS since 2003 year. The whole country was covered MS/MS screening in 2014 year. Methods: Routine dried blood spots from NBS were analyzed by MS/MS from period 2010–2014 years. Samples were taken at 3–5 day of life. We screened 1 112 520 newborns. We used API 3200 (AB Sciex), CTC autosampler and Shimadzu LC 20 pump. Blood samples were extracted in methanol containing stable amino acids and carnitines isotopes and derivatized with 3 N-HCl in nbutanol. Concentration were calculated using in-house software with calibrators prepared from blood enriched with known amounts of amino acids and carnitines. Results: We recalled 159 cases with high C6 (above 0.26 μmol/L), C8 (above 0.27 μmol/L) and C10 (above 0.45 μmol/L) levels. 27 MCADD cases were diagnosed (all confirmed by enzyme activity in lymphocytes, 0.9–16 %). Following ratios for MCADD parameters were selected: C8/C10, C8/C2, C8/C0, C8/C5, MC/Total (where MC is a sum C6 + C8 + C10 + C10:1 and Total is a sum of all measured acylcaritines). The most discriminative ratio C8/ C10 with cut off 1.4 allowed to reject 95 % of false positive results. Discusssion: The ratio C8/C10 used in our center allowed to lower the number of false positive results of MCADD what resulted in reduced financial costs and parent’s stress.

A-012 A-014 Free ethylmalonic acid (EMA) measurement for newborn screening of short chain CoA dehydrogenase deficiency (SCADD): preliminary results Baronio F 1, Righetti F 2, Bettocchi I 1, Ortolano R 1, Monti G 2, Pession A 1, Cassio A 1 1

2

NBS Reg Centre,Ped Unit,AOU, Bologna, Italy, NBS Lab, Neonat Unit, AOU, Bologna, Italy

Newborn screening may detect inappropriately high protein intake in newborn infants Maines E 1, Gugelmo G 1, Vincenzi M 1, Campostrini N 1, Teofoli F 1, Camilot M 1, Bordugo A 1 1

Inher Metab Dis Unit, NBS Center, AOUI, Verona, Italy

Background: Butyryl carnitine levels (C4) ≥1 μmol/L is utilized to screen short Chain CoA dehydrogenase deficiency (SCADD) in newborn screening (NBS) programs; suspicious cases are confirmed by the assesment of urinary ethylmalonic acid (U-EMA). In order to detect false negative results, we started measure both C4 and free EMA on Guthrie cards in all children from July 2015. The aim of this study is to evaluate the incidence of SCAD before and after introduction of free EMA dosage. Methods: We compared children recalled for C4 ≥ 1 μmol/L from July 2014 to April 2015 (period A) with children recalled for positive free EMA with C4 > 0.3 μmol/L from July 2015 to April 2016 (period B). All suspicious cases underwent U-EMA testing. 20/33.300 pts in period A and 44/33.540 in period B were recalled. Data of 11/20 patients of period A and of 32/44 of period B are available. Period B pts were subdivided for C4 levels: C4 ≥ 1 (8 patients) and C4 < 1 μmol/L (24 patients).

Background: Hypertyrosinaemia is a common finding in expanded newborn screening (NBS). It may be caused by inherited enzyme deficiencies in the catabolic pathway of tyrosine, but it is most often associated with common and benign transient hypertyrosinaemia of the neonate (TTN). Inappropriately high protein diet is a known risk factor for TTN and has been associated to metabolic acidosis, severe electrolyte imbalances and a clinical picture similar to tyrosinaemia type 1. Case Report: The patient was born full term. In the first 24 h of life she presented transient clotting abnormalities treated with plasma transfusion. NBS performed at 36 h of life was not eligible due to the transfusion. At 9 days of life the newborn was growing well with breast milk and was allowed home. At that time, NBS was normal. NBS was also repeated at 15 days of life due to low birth weight and

S248 it showed high Tyr, Phe and Met levels. Succinylacetone (SUAC) was normal. A severe liver disease was suspected. At admission, clinical examination was normal. Initial testing results were consistent with moderate metabolic acidosis with normal liver function tests. Blood Tyr was around 2000 μmol/L. SUAC was undetectable. The patient was put on a Phe/Tyr-free formula feed for 48 h. The acidosis quickly corrected. Blood Tyr, Phe and Met normalized. When Phe/Tyr-free milk was stopped, abnormalities have not recurred. Dietary history revealed that, after the discharge, the patient was fed breast milk associated with undiluted goat’s milk because her mother has not sufficient breast milk. Results: We described the case of a newborn with a positive NBS result for severe hypertyrosinaemia with also high Met and Phe levels secondary to goat’s milk consumption. Discusssion: NBS may detect inappropriately high protein intake, such as goat’s milk, preventing significant infant morbidity. A thorough medical history is important to rapidly identify the causes of such metabolic disturbances.

A-015

Biotinidase deficiency: evaluation of patients diagnosed with newborn screening

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 aim was to describe the profile of patients followed in the Department of Genetics HC-UFMG-NUPAD Methods: We classified the type of hyperphenylalaninemia for all patients as: mild, classical, BH4 deficient, permanent and transient hyperphenylalaninemia. The presence of severe mental retardation (totally dependent for activities of daily living) was evaluated in patients with late diagnosis. The diagnosis classification was defined by the results of examinations undertaken at the first visit (after 2014) or by confirmatory test (CT). In the first consultation all patients underwent examination for diagnosis of BH4 deficiency. Results: From 311 patients in treatment for phenylketonuria, 252 (81.0 %) are early diagnosis and 59 (19.0 %) are late diagnosis. From early diagnosis: 8 (3.2 %) have BH4 deficiency, 138 (54.8 %) classic phenylketonuria, 96 (38.0 %) mild phenylketonuria, 10 (4.0 %) permanent hyperphenylalaninemia. Regarding the late diagnosis: 38 (64.4 %) have severe mental retardation, 1 (1.7 %) has deficiency of BH4, 56 (94.9 %) classic phenylketonuria, 2 (3.4 %) mild phenylketonuria. From the total patients, 188 (60.45 %) underwent CT. Concerning age range: 11 (3.54 %) are infants, 54 (17.36 %) preschool children, 40 (12.86 %) students, 135 (43.41 %) adolescents, 71 (22.83 %) adults. Discusssion:There has been no case of a false negative screening test and all patients are being treated appropriately according to the diagnosis. The newborn screening program is essential for early detection of the disease and avoidance of sequelae.

Demirkol M 1, Cakar E 1, Bas K 1, Karaca M 1, Balci C 2, Gokcay G 1 1 Dep Ped Nutr and Metab, Istanbul Univ, Istanbul, Turkey, 2Tokat Goverment Hospital, Tokat, Turkey

04. Dietetics and nutrition

Background: Biotinidase deficiency (BD) (OMIM 253260) is an autosomal recessively inherited disorder. Newborn screening for BD is performed in many countries because the disorder can be treated effectively and inexpensively with biotin. Early diagnosis and treatment prevents disease related cutaneous findings, alopecia, hypotonia, seizures, acidosis, hearing and vision problem Methods: Patients diagnosed with BD between 1991 and 2015 were evaluated. Patients born before September 2008 were screened by our center, later the screening was performed by the National Newborn Screening of the Ministry of Health. BD is classified according to the plasma biotinidase activity as profound BD ( C/p.M1T (0.5 %) located in exon 1. The majority of mutant alleles were missense mutations-19 (55,9 %), besides 8 (23,5 %) - splice site, 4 (11.8 %) -nonsense mutations and 3 (8.8 %) deletions. Discussion: In summary, we established the mutation spectrum of PKU genotypes in the Moldavian population and identified a novel missense variation p.M1T plus found the rare missense allele p.Y268C in two Moldavian subjects that was recently found as a novel variation in a Romania patient.

A-021 Background: Patients with PKU have lower bone mineral density (BMD) as control healthy people. PKU itself and the phenylalanine (Phe)-low diet together can cause this pathophysiological alteration. However, there is no clear data, whether metabolic control can influence BMD’s change in adult PKU patients. Methods: In a monocentric study 79 adult PKU patients were enrolled. Bone mineral density (BMD) at the lumbar spine and femoral neck was repeatedly measured using dual-energy X-ray absorptiometry (DEXA) in a 4-year interval. Average yearly serum Phe, tyrosine (Tyr) were calculated. The correlation between the change in BMD and average Phe and Tyr concentrations were studied. Results: From the 79 PKU patients 44 were female (56 %). Mean age was 33.9 ± 6.4 years, mean Phe concentration was 625 ± 239 micromol/L, whereas mean Tyr concentration was 52 ± 18 micromol/L, the calculated

Spectrum and Outcome of Phenylketonuria (PKU) in India Jalan R A 1, Jalan A B 1, Kudalkar K V 1, Shinde D H 1, Joshi M M 1, Borugale M A 1, Shirke S M 1, Mahamunkar A P 1, Tawde R J 1 1

NIRMAN, Div of Biochemical Genetics, Navi Mumbai, India

BACKGROUND: PKU is a group of disorders of Phenylalanine metabolism including PAH defect and other pterin metabolism disorders. In absence of nationwide NBS programme or registry we do not have any incidence. It is believed that PKU is more common in south India.

S250 Objective To study the spectrum and outcome of symptomatically detected PKU of various types in India. Methods: This is a retrospective study of 19 patients over last 16 years. Biochemical analysis was performed, including plasma aminoacids, urine aminoacids, GC/MS of urine, urine Pterins, blood DHPR enzyme and in appropriate cases CSF Pterins and in some cases CSF Neurotransmitters. Results Out of 19(M = 12; F = 7), 14 had history of consanguinity(73.6 %). Classical PKU(10), GTPCH(AR) (3), PTPS def(1), DHPR Def(5). The youngest patient was 1.5 months and the eldest was 37 years old. The common clinical presenting symptoms were developmental delay(18), microcephaly(18), hypo pigmented hair and skin(12), seizures(7), dystonia(3) and Autism with ADHD(1). One child expired of unknown cause at the age of 4 months. 10 patients were lost for follow up and 9 were under therapy and regular follow up. Discusssion: Of 9 patients who are under follow-up, normal development was seen with no seizures and disappearance of hypo pigmentation(3), (2 Classical PKU and 1 DHPR Def), with gain in milestones and reduction in seizure frequency and dystonia(4) (1 Classical PKU, 2 GTPCH and 1 DHPR Def), and reduced seizure frequency but no change in behavior(2) (DHPR Def sibs). In India the PKU diets were not easily available and were very costly before 2014. Now one local company produces diet. Biopterin & Kuvan are not available. All the patients were treated with low protein diet and supplementation of AA formula. Those treated early and had good compliance, had better outcome. Even the 37 yrs old patient showed response to diet initially but later he could not continue due to financial constraints.

A-022

Bone mineral density in children, adolescents and young adults with phenylketonuria and hyperphenylalaninemia

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 A-023

PKU is still late diagnosed in Morocco: urgency to establish a systematic neonatal screening Dahri S 1, Talbaoui H 1, Kriouile Y 2, Agadr A 3, Chaouki S 4, Chabraoui L 1 1 Biochemistry laboratory, Univ Hospital, Rabat, Morocco, 2Pediatry service, Univ Child Hosp, Rabat, Morocco, 3Pediatry service Univ Military Hosp, Rabat, Morocco, 4Pediatry service Univ Hosp, Fes, Morocco

Background: PKU is an autosomal recessive disease caused by PAH deficiency. In the absence of screening and treatment it causes mental retardation. Methods: In our laboratory, since 1991 we have screened for PKU and other aminoacidopathies using TLC of plasma and urinary aminoacids. To confirm and follow up PKU we use a spectroflurimetric measurement of Phe. Results: We present a retrospective study of 135 Moroccan PKU patients almost diagnosed following clinical symptoms and that over 25 years. The estimated PKU frequency was of 1/12000. Majority of the patients were late diagnosed even in families presenting previous affected cases. Mean age of diagnosis was 5 years and mean phenylalanine level was 1427 μmol/l. Major symptoms were mental retardation, motor delays, speech disabilities (96 %) and convulsions (32 %). The patients diagnosed and treated between the ages of 4 and 12 months showed a subnormal psychomotor development without behavior problems, but they have some learning difficulties. Only four patients were diagnosed and treated from the neonatal stage. They showed normal neurodevelopment. Discusssion: PKU is a frequent aminoacidopathy in Morocco. Recently, the Moroccan Ministry of Health has adopted a newborn screening program for congenital hypothyroidism. We suggested introducing screening for PKU in the national program to treat precociously patients and prevent mental retardation in our country.

Leiva C A 1, Cornejo V 1, Bravo P 1 1

INTA, Univ of Chile, Santiago, Chile

Background: It has been reported that children with phenylketonuria (PKU) on strict diet has a higher risk of bone demineralization, which can lead to osteoporosis and fractures in various age groups. To investigate this, total, spine (L2-L4) and lumbar bone mineral density (BMD) were determined in 16 mild hyperphenylalaninemias (mHPA) (Phenylalanine range: 1–4 mg / dl), 16 classic PKU (2 to 12.5 mg / dl) and 16 age-matched control subjects (age range 6–23 years, of both sexes). Methods: A descriptive analysis was applied by z-score categories for variables and the Kruskal-Wallis statistical test was used. Results: There were no significant differences between groups in BMD (p < 0.05). Spine BMD between control and mHPA group were similar, in both cases 2 patients had BMD values lower than normal (Zscore < −1); 4/16 patients of PKU group had decreased values. The median z-score of spine BMD in the control group is −0.4, −0.3 in PKU group and 0.05 in mHPA group. In femoral neck BMD, 3/16 cases from PKU group had decreased values; unlike the control group they were all within the normal range (Z-Score −1 to +1) and in the mHPA group only one case had decreased values. The median Z-score of femoral neck BMD in the control group was 0.2; PKU - 0,3 and 0.65 mHPA. For total BMD; 2/16 patients in the control group and 1/16 of mHPA group had values below the normal range in the PKU group and all were within the acceptable range. The median total BMD Z-Score in control group was 0.5; 0.2 in PKU and 1.25 in mHPA. Discusssion: There were no significant differences between groups in BMD. This could be because the group with PKU has maintained an adequate follow-up that includes sustained contributions of calcium and vitamin D, provided by the special milk formula without FA and calcium supplements, from the neonatal period when they were diagnosed onwards. It is therefore important to monitor these patients in order to prevent future problems in the bone architecture.

06. Phenylketonuria: treatment, BH4

A-024

The first experience with a BH4 loading test for PKU patients in Kazakhstan Salimbayeva D N 1, Svyatova G S 1, Ormankyzy A 1, Musabalaeva G 1 1

Sceintific center of obstetrics, gynecol, Almaty, Kazakhstan

Background: Phenylketonuria is an autosomal recessive disorder caused by a deficiency of the enzyme phenylalanine-4-hydroxylase (PAH). PAH converts the essential amino acid phenylalanine into tyrosine using the cofactor tetrahydrobiopterin (BH4). In most parts of the world, including Kazakhstan, BH4 is prescribed to patients from 4 years of age onwards. Methods: We use a standard protocol of the 48-h BH4 loading test with 20 mg/kg. The 48-h BH4 loading test was conducted using 20 mg BH4/kg/ day and filter paper blood samples were collected at T = 0, 8, 16, 24 and 48 h. Patients with Phe blood concentrations below 400 μmol/L where supplemented with Phe (L-Phe in powder) until the test was finished. In this test, a reduction in Phe concentration of at least 30 % was considered as positive. 7 patients with classical PKU older than 4 years were included in the BH4 loading test. All patients had a permissible level of Phe, normal intellect and a constant low-protein diet with amino acid formula. We studied the PAH gene of all patients. Results: After the BH4 loading test, two patients were BH4-sensitive, three were partly BH4-sensitive and two had negative results. We analyzed the correlation between PAH genotypes and response to BH4. Two BH4sensitive patients have R408W/P281L genotype and unknown mutations. The genotypes of the partly BH4-sensitive group were R408W/P281L, R243Q/R243Q and R408W/unknown. Two patients with negative BH4sensitivity had R243Q/R243Q and IVS4 + 5G > T/ IVS4 + 5G > T genotypes.

S251

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 DISCUSSION: Thus, our first experience of the BH4 loading test for PKU patients in Kazakhstan showed that BH4 sensitivity of same genotypes was different. Consequently, the study of BH4-sensitivity in Kazakhstan will continue.

07. Sulphur amino acid disorders

A-025

Thromboembolic manifestations in classical homocystinuria

Results: Sulfite decreased mitochondrial mass and the activities of creatine kinase and citrate synthase, which were prevented by bezafibrate. Furthermore, sulfite decreased glutathione concentrations, and the activities of glutathione peroxidase (GPx), glutathione reductase, glutathione Stransferase (GST) and glucose-6-phosphate dehydrogenase. Bezafibrate attenuated the inhibition of GPx and GST activities. Finally, we verified that PGC1αimmunocontent in the nucleus was decreased by sulfite and that this effect was also prevented by bezafibrate. Discusssion: Our results show thatthe impairment of energy metabolism, antioxidant defense system and mitochondrial biogenesis induced by sulfitemaycontribute to the pathophysiology of the neurologic dysfunction observed in SO deficiency. These data also suggest that bezafibrate could be considered a promising therapy for this disease. Financial Support: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP IBN-Net, INCT-EN.

Ben Abdelaziz R 1 2, Nammouchi M 2, Ben Chehida A 1 2, Hajji H 1 2, Boudabous H 1 2, Ben Turkia H 1 2, Abdelmoula M S 1 2, Kaabachi N 1, Azzouz H 1 2, Tebib N 1 2 08. Other amino acid disorders 1

La Rabta Hospital, Tunis, Tunisia, 2Medical School, Universite Tunis Elmanar, Tunis, Tunisia A-027 Background: Classical homocystinuria (CH) is an autosomal recessive disorder of methionine metabolism caused by cystathionine beta synthase deficiency. It is associated with a high risk of thromboembolic (TE) complications. Methods: A retrospective multicenter study was conducted in Tunisian hospitals to report TE manifestations in CH patients and their management. Results: Twenty six patients with CH were reported between 1984 and 2016. Sixteen TE events were noted in 14 patients (54 %) at a median age of 15 years. In 8 patients, it was a presenting manifestation. Six of them had venous ischemic stroke and two had deep venous thrombosis of the lower limbs. In six patients, TE complications occurred while on treatment; five patients had venous stroke and one patient had a renovascular hypertension complicating a renal artery thrombosis. In one case the TE event (stroke) occurred after surgery without preparation. Thrombophilia tests were performed in 7 patients. Abnormalities (added to hyperhomocysteinemia) were found in one patient. On a low-protein (or low-methionine) diet, specific treatment and anticoagulation the outcome was favourable in most cases without major sequelæ. Discusssion: TE complications are frequent in CH especially in late treated patients and those with a poor metabolic balance. They are more frequently venous. A good metabolic balance and an appropriate preparation for surgery are necessary to avoid such life threatening complications.

A-026

Sulfite intrastriatal administration induces mitochondrial dysfunction and alters antioxidant defenses in rat striatum: protective effects of bezafibrate Grings M 1, Moura A P 1, Parmeggiani B 1, Alvorcem L M 1, Boldrini R 1, Motta M M 1, Pletsch J T 1, Cardoso G M 1, August P M 1, Matte C 1, Wyse A T S 1, Wajner M 1 2, Leipnitz G 1 Dept Bioq, ICBS, UFRGS, Porto Alegre, Brazil, 2HCPA, Porto Alegre, Brazil

1

Background: Sulfite oxidase (SO) deficiency is a recessive inherited disorder that can arise either from the deficiency of SO or a deficiency of its molybdenum cofactor synthesis. This disorder is characterized by the accumulation of sulfite in tissues and biological fluids, severe seizures and progressive encephalopathy. SO deficiency is often a fatal disease and there is no effective treatment for all phenotypes. Therefore, we investigated the in vivo effects of sulfite on antioxidant defenses, bioenergetics and PGC-1αimmunocontentin rat striatum, and the potential protectiveeffect of the PPAR receptor activator bezafibrate on these alterations. Methods: Thirty-day-old Wistar rats received a single intrastriatal injection of sulfite (2 μmol) and were euthanized 30 min after the administration. In some experiments, a pretreatment with bezafibrate (30 mg/kg/day) was performed by gavage during 7 days. Striatal homogenates were used to determine the biochemical parameters.

The effects of methylphenidate on processing speed and executive functioning in a 12-year old girl with tyrosinemia type 1: a case report Raets E 1, Eyskens F J M 1, Simons A 1 1

Div Metab Dis, Univ Mother Child Hosp, Antwerp, Belgium

Background: The patient was diagnosed with tyrosinemia type 1 at the age of one. From the start motor and language development were delayed. At the age of three the patient had a liver transplantation. Through the years teachers and parents described the patient as withdrawn and distracted. She has followed regular education but repeated the first grade. At the end of the fifth grade her school results were poor. Methods: At the age of twelve the patient presented with attention and concentration problems. She would easily lose things and had problems getting everything organized. An intelligence and neuropsychological investigation was performed and indicated a borderline intelligence. There were low average scores for fluid intelligence (Gf), crystallized intelligence (Gc) and visual processing (Gv) and low to average scores for working and short-term memory (Gsm). However, there were extremely low scores for processing speed (Gs) and executive functioning. Moreover these scores were significantly lower opposed to the other broad abilities. There were also problems with selective attention and attention fluctuations. As a result of these findings methylphenidate was initiated and a new investigation was performed 3 weeks after startup. Results: A significantly higher score was found for processing speed (Gs) and selective attention. Attention fluctuations were absent. Significantly higher scores were also found for executive functioning. There was no difference found for working and short-term memory (Gsm). Discusssion: One case of tyrosinemia type 1 has shown problems with processing speed and executive functioning. When administering methylphenidate, it shows a good improvement in processing speed and executive functioning which is clinically relevant. Future studies may investigate the effects of methylphenidate on processing speed and executive functioning in a larger group of patients with tyrosinemia type 1.

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The multiple faces of lysinuric protein intolerance with a novel mutation Ozaydin E 1, Gunduz M 2, Tural D 1, Altan H 1, Unal O 2 1 Infancy Service, Ank Child Hema-Onco Hos, Ankara, Turkey, 2Div Metab Dis,Ank Child Hema-Onco Hosp, Ankara, Turkey

S252 Background: Lysinuric protein intolerance (LPI) is an inherited defect of cationic amino acid transport caused by mutations in SLC7A7 encoding the y + LAT1 protein. We present a severe case with multiorgan involvement such as hemophagocytic lymphohistiocytosis, lung involvement and renal insufficiency. Case Report: Fifty-day old baby was referred to our hospital with hyperammonemia and hypoglycemia. There was first-degree consanguinity between parents. He was admitted to Infancy Service with vomiting and mild hyperammonemia. Metabolic investigations were performed. Urea cycle disorders were thought due to orotic aciduria. The lysine level was mildly high in urine but was evaluated as normal. Low protein diet was initiated. The samples for genetic analysis were sent. But his clinical findings deteriorated over time and hematologic abnormalities related with macrophage activation syndrome developed. The follow-up urine lysine levels elevated. Failure to thrive, hemophagocytic lymhohistiocytosis, orotic aciduria were thought to be related with LPI. The genetic analysis confirmed our diagnosis. The citrulline was added to the therapy. Respiratory insufficiency progressed to oxygen dependency. Bronchoscopy was performed and PAS(+) lipoproteinaceous material evidencing alveolar proteinosis was observed in bronchoalveolar lavage. The baby was diagnosed as LPI and pulmonary alveolar proteinosis. Unfortunately, he expired at 1 year of age with renal insufficiency and sepsis. Results: Urine lysine levels gradually increased (800, 1500, 4000, 8000 N:56– 200 μmol/L). Urine organic acid analysis revealed orotic aciduria. The molecular analysis detected homozygosity for c.478_499 + 11del33bp in SLC7A7 gene. Discusssion: Early diagnosis and therapy may be important in order to halt the progression of the disease but in this case the prognosis caused by a novel mutation was very poor. On the other hand, novel therapeutic approaches are needed to treat this severe multiorgan disorder.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 A-030

Behavioral responses in an animal model of Maple Syrup Urine Disease treated with tianeptine Scaini G 1, Morais FA 1, Abelaira H M 4, Reus G Z 4, Quevedo J 4, Schuck P F 2, Ferreira G C 3, Streck E L 1 Lab Bioenergetics, UNESC, Criciuma, Brazil, 2Lab IEM, UNESC, Criciuma, Brazil, 3Lab Neurochem, UFRJ, Rio de Janeiro, Brazil, 4Neurolab, UNESC, Criciuma, Brazil 1

Background: Maple Syrup Urine Disease (MSUD) is caused by deficiency of the complex branched-chain α-keto acid dehydrogenase leading to accumulation of the branched-chain amino acids (BCAA). Considering that previous reports suggest that MSUD patients are at high risk for chronic neuropsychiatric problems and neurological dysfunction, we investigated the effects of tianeptine administration, an antidepressant with proven effects on hippocampal plasticity, on depressive-like parameters in an animal model of MSUD. Methods: Wistar rats (7 days) received a pool of BCAA or saline twice a day for 21 days, and were also supplemented with tianeptina (15 mg/kg) once daily. Twelve hours after the last administration the rats were submitted to anhedonia and forced swimming test, before adrenal gland weight and nerve growth factor (NGF) levels were evaluated. Results: Our results demonstrate that in the MSUD in sucrose intake was significantly lower, while the adrenal gland weight and the immobility time were significantly higher. In addition, we showed a significant decrease in the protein levels of NGF in the hippocampus. Interestingly, the treatment with tianeptine prevents depressive-like parameters, and was able to prevent the decrease in NGF levels. Discusssion: This study demonstrates that tianeptine showed antidepressant effects in an animal model of MSUD, and this can be attributed to its action on the neurotrophin signaling pathway related to depression.

Pyroglutamic aciduria: A splice site mutation (IVS9 G > A) in GSS in a Tunisian child

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Kerkeni E 3, Ghedira N 3, Sakka R 1 3, Ben Hmida H 1, Ben Ameur K 1 3, Bizid M 1 3, Kaabachi N 2, Chioukh F Z 1 3, Monastiri K 1 3

Nonketotic hyperglycinemia: different and atypical manifestations of the disease

Dept Intens care neonat Med Teach Hosp, Monastir, Tunisia, 2Dept Biochem Teach Hosp LaRabta, Tunis, Tunisia, 3Res Unit 01-UR-08,14 Fac Medicine, Monastir, Tunisia

Galimberti C 1, Brambilla A 1, Pasetti M 1, Tursi S 1, Corbetta C 2, Ravazzani V 2, Pretese R 1, Gasperini S 1

1

Background: Glutathione synthetase (GSS) deficiency (OMIM 231900, 266130) is the most frequent defect among the rare autosomal recessive inherited disorders of glutathione metabolism. Typically, patients affected by the severe form of this disease develop anaemia, acidosis, progressive neurological symptoms and recurrent bacterial infections. The present study is aimed at performing the molecular characterization of pyroglutamic aciduria with glutathione synthetase deficiency in a consanguineous Tunisian newborn infant. Methods: A newborn girl, born to first cousins parents, with a family history of early deaths, was admitted to the intensive care unit, on the 3rd day of life, for vital distress. The diagnosis of pyroglutamic aciduria was suspected following the association of metabolic acidosis and hemolytic anemia. Mutational analysis of the entire coding and flanking intronic region of GSS gene was performed by direct sequencing. Results: Direct sequencing analysis showed a G to A substitution at the acceptor site of the intron 9 (IVS9 G > A) in homozygous state in the patient. Samples from parents were found to be carriers of the alteration. This mutation was at the origin of numerous aberrantly spliced RNA transcripts. It seems decrease the glutathione synthetase activity in cultured fibroblasts at 9 %. Discusssion: The identification of the causative mutation in the Tunisian patient here studied will now facilitate treatment, prenatal diagnosis and genetic counselling in his at-risk families.

1 Dept of Ped, MBBM Found S Gerardo, Monza, Italy, 2Regional Newborn Screening Lab, Milano, Italy

Background: Nonketotic hyperglycinemia (NKH) is characterised by a defect in the glycine clivage system leading to the accumulation of glycine in all body tissues, enclosed blood, urine and cerebrospinal fluid (CSF). The classic neonatal form presents within few days of life with poor feeding, lethargy, hypotonia, hiccups, apnoea, convulsion and coma. Atypical forms with milder disease and later onset have been reported. Case Report Patient (pt) 1 was born from consanguineous Turkish parents and developed a severe encephalopathy, seizures and apnoeic attacks within few hours of life. Respiratory support and Phenobarbital were started. Pt2 was born from unrelated Italian parents, presented in neonatal period with lethargy and poor feeding without seizures. Pt3, a 4-year-old boy with severe psychomotor delay without epilepsy, was born from non-consanguineous Philippine parents. Hypotonia, poor feeding and psychomotor delay were evident in the first year of life. Results Diagnosis of NKH was made in all three patients: Pt1 had plasma glycine 1708 μmol/l (normal range: 200–600) in the neonatal period; CSF glycine was not tested; at 16 months vagus nerve stimulation was performed with control of seizures. Pt2 plasma glycine was 588 μmol/l, CSF glycine 78 μmol/l (normal range:0-10 μm/l), CSF/plasma glycine ratio 0.14. She improved greatly with Na benzoate and dextromethorphan but from the 7th month she developed seizures with progressive neurological deterioration and started Phenobarbital and Levetiracetam with good control of seizures. Pt3

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 plasma glycine was 729 μmol/l, CSF 48 μmol/l and CSF/plasma glycine ratio 0.065. Discussion Clinical presentations of NKH are very heterogeneous ranging from neonatal life-threatening convulsive encephalopathy to milder neurologic impairments like in the last pt without epilepsy. All patients with severe neurological involvement, even without seizures, must be tested for NKH.

A-032

Nutritional monitoring of patients with maple syrup urine disease Lopes J C C 2, Kanufre V C 1, Cruz A F 1, Maioli T U 2, Arantes R R 1, Valadares E R 1 1 HC, Fed Univ Min Ger, Belo Horizonte, Brazil, 2Dept of Nutr, Fed Univ Min Ger, Belo Horizonte, Brazil

Background: The purpose of this study is to describe the nutritional status of children with maple syrup urine disease (MSUD) treated in a public hospital service specialized in Inborn Errors of Metabolism in Minas Gerais, Brazil. Methods: Five children with MSUD were included in the study, all diagnosed late, aged 5 to 16 years, 2 males and 3 females. Anthropometric data such as height, weight, arm circumference, tricipital skinfold and subscapularis were measured. The data were classified by BMI/age index (WHO). Body composition was assessed by arm muscle area (AMA) and classified by Lohman standard. The fat mass was measured by the sum of tricipital skinfolds and subscapular skinfolds and classified by NCHS standard. The protein intake was measured by the 24-h recall. All evaluations were performed at scheduled consults. Results: Two patients were classified with thinness and the others with eutrophia. The assessment of AMA has shown that one patient had mild deficit and one had severe deficit of muscle mass. Regarding evaluation of fat mass, one patient presented severe deficit of fat. The assessment of protein intake showed that the three patients who consumed higher amounts of protein in comparison to the literature recommendation, had no muscular mass deficit, while the two patients that consumed the recommended amount had some degree of mass deficit. Discusssion: Children with MSUD are at nutritional risk because of the severity of the disease, as well as food restriction in treatment. The regular nutritional assessment and monitoring must be conducted to maintain growth and optimal development without affecting their amino acid profiles. The literature recommendations should be used, however adapted to the clinical evolution of each patient.

09. Urea cycle disorders

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Clinical, laboratory data, molecular features and outcome of 14 citrullinemia type 1 patients. Zaman T 1 2, Moarefian S H 2, Behnam B 3, Rahmanifar A 2, Nagel M 4 1

Metab Div Dis,Tehran Univ of Medical Sc, Tehran, Iran, Islamic Republic of, Iranian National Research Unit, Tehran, Iran, Islamic Republic of, 3div of Medical Gen and Biotech,IUMS, Tehran, Iran, Islamic Republic of, 4Weisswasser Laboratory, Weisswasser, Germany 2

Background: Citrullinemia type1 (CTLN1) is an autosomal recessive urea cycle disorder caused by a defect in the argininosuccinic acid synthase (ASS1) gene located on chromosome 9q34.1 whichcontains 16 exons. More than 93 mutations have been described with heterogeneous manifestations from hyperammonemia in the early neonatal period (classic-early onset) to neurodisability during childhood,

adulthood, pregnancy (non classic-late onset) or even asymptomatic. The phenotypic diversity remains unexplained. Methods: This is a retrospective study on 14 CTLN1 patients (2008–2014). Sequence analysis of the ASS1 gene confirmed the diagnosis. Results: 14 patients:7 boys and 7girls; 8 families: 6 consanguineous.11/ 1 4: e a rl y on s e t se v e re n e o na t al : 7 c a s e s p re s e nt e d: po o r feeding,lethargy at 1–3 days; citrulline; 1147–3871 μmol/L,blood ammonia;3.4–10 times normal; 2/7 with onset on the 1 st day, deceased before treatment on the 2 nd day and 5/7 deceased despite treatment at mean age of 10.4 days, 2/11; onset at 3 rd day, ammonia: 7.3 times normal, developed well after treatment but deceased in hyperammonemic attack at 3.5& 10 months.2/11 neonatal; onset at 1 st and 9 th, ammonia level 150 survived but with developmental delay in one and speech delay in another (follow up 3–6 years).2/14;infantile form :one, onset at 8 months; referral & treatment at 14 month developed well after treatment and just has speech delay and the second;onset at 10 month with seizure and developmental delay deceased at 17 years.1/14 asymptomatic; referred for growth delay with citrulline; 1298 μmol/L. responded well . Sequencing analysis in classic; Gly 390 Arg (c.1168 G > A (Gao,et al.,2003) the most common world mutation(4 classic),M376 T (2 classic),R279X (3 classic), S29T,G110A,(het),S341P(homo) (2 classic);R 403 A (infantile), G 389A;fs (asymptomatic). Discusssion: Citrullinemia 1 has different phenotypes. The most important prognostic factors are the mutation type, the ammonemia level and early treatment.

A-034

Autism spectrum disorder as a clinical presentation of undiagnosed urea cycle defects Wilson W G 1, Black E D 1 1

Univ Virginia Child Hosp, Charlottesville, Virginia, United States

Background: Autism spectrum disorders (ASD) can have a variety of underlying causes, but urea cycle disorders have not been commonly reported. We report two unrelated boys with ASD who were later found to have carbamoyl-phosphate synthetase (CPS) deficiency (Patient 1) and ornithine transcarbamylase (OTC) deficiency (Patient 2). Case Report: Patient 1, now age 12 years, presented at age 4 years with developmental delay and ASD. There was increased plasma glutamine, decreased citrulline, and normal urine orotate. Blood ammonia was 423 umol/L. Enzymatic analysis of liver tissue confirmed CPS deficiency. Patient 2 died at age 15 years following an influenza-like illness complicated by hyperammonemia, coma and death. He had a long history of ASD and had been clinically evaluated at several institutions. Lab testing at age 8 years showed a mild elevation of plasma glutamine and urine orotate on one occasion, with normal plasma citrulline and blood ammonia. When hospitalized at age 15 years, orotate and glutamine were elevated, and citrulline was undetectable. Blood ammonia concentration was 223 umol/L, and increased to 559 within 24 h, just prior to death. OTC deficiency was suspected. Although no mutation testing was done on him, his mother was found to be heterozygous for a pathogenic mutation in OTC. Results: Both patients with ASD had previously unsuspected urea cycle disorders. Discusssion: These two patients were found to have undiagnosed CPS deficiency and OTC deficiency, respectively. Current recommendations for the evaluation of children with ASD tend to emphasize molecular testing (fragile X testing, chromosomal microarrays and/ or some type of “next generation” gene sequencing), but do not stress testing for inborn errors of metabolism. While inborn errors of metabolism may not be common causes of ASD, they include conditions that might be amenable to dietary modification or pharmacotherapy.

S254 A-035

Argininosuccinic aciduria: from identification to management of paucisymptomatic late onset forms Paquay S 1, Desnous B 2, Paviolo M 1, Imbard A 3, Pichard S 1, Benoist J F 3, Schiff M 1 Ref Cent Metab Dis, Robert Debre Hosp, Paris, France, 2Dep Pediatr Neurol, Robert Debre Hosp, Paris, France, 3Dep Biochemistry, Robert Debre Hosp, Paris, France 1

Background: Argininosuccinic aciduria, the second most common urea cycle disorder (UCD) is due to argininosuccinate lyase (ASL) deficiency. Neonatal onset forms are characterized by hyperammonemic comas. Late onset ASL patients exhibit variable symptoms ranging from intermittent hyperammonemia to unspecific psychomotor delay, behavioral and cognitive disturbances. Systemic hypertension, brittle hair or liver fibrosis are specific findings compared with other UCDs that can occur independent from hyperammonemia. We report two atypical late onset forms of argininosuccinic aciduria. Case Report and Results: Patient 1 presented with myoclonic epilepsy and mild psychomotor delay at the age of 3. Clinical examination revealed hepatomegaly that prompted to perform a metabolic workup. The latter found argininosuccinic acid (ASA) accumulation in body fluids without hyperammonemia or liver dysfunction. ASA level in cerebrospinal fluid (CSF) was 3.6 fold higher than in blood. Patient 2 presented with acute episodes of apnea, cyanosis and hypertonia at age 3 months. Clinical findings revealed mild hepatomegaly and hypotonia. High ASA level was detected in blood and urine, without hyperammonemia. Low protein diet and arginine supplementation were introduced, resulting in decreased blood ASA levels in both patients who exhibited moderate neurodevelopmental delay. Discusssion: Nonspecific symptoms may occur in late onset ASL patients and time for metabolic screening is a challenge. In particular, myoclonic epilepsy is an unusual initial presentation. Increased ASA level in CSF combined with reported nitric oxide (NO) deficiency despite arginine supplementation may contribute to neurotoxicity. While protein restriction and arginine supplementation can decrease hyperammonemia if present and limit blood ASA accumulation, the impact of treatment on the neurological disease remains questionable. Recent data suggest that ASL patients might benefit from NO supplementation.

A-036

Identification of three novel mutations in patients with citrullinemia type 1 Arslan N 1, Kose E 1, Unal O 3, Gunduz M 3, Haberle J 4, Bulbul S 2 1 Div Metab Dis, Dokuz Eylul Univ Hosp, Izmir, Turkey, 2 Div Metab Dis, Kirikkale Univ Hosp, Kirikkale, Turkey, 3 Ankara Children’s Hem Oncology Div Metab, Ankara, Turkey, 4 Div Metab Dis,Uni Children’s Hosp, Zurich, Switzerland

Background: Citrullinemia type 1 is an autosomal recessive genetic disorder that is caused by mutations in the argininosuccinate synthetase 1 (ASS1) gene. Here, we reported molecular genetic analysis of our citrullinemia type 1 patients and their clinical characterizations. Methods: The study group consisted of 10 patients (1 female, 9 males) who were treated with the diagnosis of citrullinemia type 1 from three centers in Turkey. Results: The current age of patients was 64.1 ± 60.7 months (min: 1 month, max: 18 years) and the mean age at diagnosis was 449 ± 917 days (min: 3 days, max: 8 years). In four patients, a

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 homozygous c.1168G > A mutation was detected. All patients with homozygous c.1168G > A mutation diagnosed in newborn period with the clinical presentation of classic neonatal onset form. In three patients homozygous c.257G > A, homozygous IVS11 + 49C > T and homozygous 1085G > T mutations were revealed. Clinical presentation was compatible with late onset form of the disease in patient with homozygous IVS11 + 49C > T mutation. In three patients compound heterozygous novel mutations (c.1168G > A, c.490G > C; c.1168G > A,c.869 T > C; c.1168G > A;c.1165A > C) were identified. Citrullinemia was diagnosed in patient with compound heterozygous (c.490G > C, c.1165A > C) novel mutation when he was 26-monthold. He was born at full term after an uneventful pregnancy to healthy, third degree–related parents. The other two patients with n o v e l c o m p o u n d h e t e r o z y g o u s ( c . 11 6 8 G > A , c . 8 6 9 T > C ; c.1168G > A, c.1165A > C) mutations were diagnosed on the first month of life as neonatal onset form and were born to nonconsanguineous parents. Discusssion: In our study, consistent with the literature, a correlation was found between homozygous c.1168G > A mutation and classic neonatal onset form. Because the rarity of the disease, further molecular genetic investigations are required for analyzing genotypephenotype correlations of citrullinemia patients.

10. Organic acidurias: branched-chain

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Different clinical course of two Korean maple syrup urine disease patients with novel mutations of BCKDHB gene Kim Y M 1, Park Y S 1, Nam S W 1, Kim G H 2, Yoo H W 2, Cheon C K 1 1

Dep Pediatr, Pusan Nat Univ Child Hosp, Yangsan, Republic of Korea, 2Med Genet Centr, Asan Med Cent, Seoul, Republic of Korea Background: Maple syrup urine disease (MSUD) is a rare inborn error disorder caused by dysfunction of the branched-chain-alpha-ketoacid dehydrogenase (BCKDH) complex. Mutations in four genes are associated with maple syrup urine disease: BCKDHA, BCKDHB, DBT, and DLD. Here, we report two MSUD patients having novel mutations in BCKDHB gene. Case Report & Results: A 10-day-old girl visited our hospital due to an elevated leucine level in her newborn screening test. A plasma amino acid analysis showed high levels of isoleucine (467.8 μmol/L), leucine (179.7 μmol/L), and valine (385 μmol/L), respectively. Alloisoleucine was also increased at the level of 39.9 μmol/L. Genetic testing identified heterozygosity for two missense mutations in BCHDHB gene: c.506A > G (p.Y169C) and c.790G > A (p.E264K). These two mutations have not been reported previously. She has been treated with BCAA restricted diet and thiamine, and shows normal development and growth at the age of 17 months. A 16-year-old girl visited our clinic and this subject had already been diagnosed with MSUD by biochemical findings during the neonatal period. Her older sister with MSUD died during neonatal period. Although she treated with a BCAA-restricted diet and thiamine supplements, she encountered several metabolic crises and her growth and mental status were severely retarded. She needed multiple antiepileptic drugs for control of epilepsy. Genetic analysis idntified two deleterious mutations in BCKDHB gene, c.853C > T (p.R285*) and c.583_586del (p.Y195fs), respectively. The latter mutation was a novel mutation. Discusssion: Our cases showed different phenotypes, depending on genotypes, in MSUD patients and three novel mutations of BCHDHB gene identified in this study. Despite the fact that there are few reports of Korean MSUD patients, the molecular genetic analysis can help to predict severity of disease and management of the disease.

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Influenza A (H1N1) Virus-Associated Acute Pancreatitis and Posterior Reversible Encephalopathy Syndrome (PRES) in a Patient with mut0 Methylmalonic Acidemia (MMA) Unal O 1, Gunduz M 1, Koc N 1, Bayrakci U 3, Genc H 2, Yaman A 4, Ademhan D 1 1 Ankara Children’s Hem Oncology Div Metab, Ankara, Turkey, 2Ankara Children’s Hem Oncolog Div Neurol, Ankara, Turkey, 3Ankara Children’s Hem Oncolog Div Nephro, Ankara, Turkey, 4Ankara Children’s Hem Oncolog Div Gastro, Ankara, Turkey

Background: Survival in MMA has improved over time due to increased knowledge and experience. However, all phenotypes of MMA are still characterized by periods of intermittent metabolic decompensation, usually associated with intercurrent infections. Pancreatitis is another well-known complication of MMA. Case Report: Here we present an episode of H1N1-associated acute pancreatitis and PRES in a patient with mut0 MMA, and our management. Results: A 7.5 -year-old boy with mut0 MMA was admitted due to vomiting and metabolic acidosis. High fever occurred and H1N1 was found to be positive. Continuous veno-venous hemofiltration (CVVH) was started for resistant metabolic acidosis. Severe abdominal pain began and abdominal ultrasonography showed large fluid collection. Diagnostic laparotomy was performed and pancreas was seen to be severely edematous and hyperemic. Hemodialysis treatment was continued for a month. He needed total parenteral nutrition, then enteral nutrition via jejunal tube along with pancreatic enzyme replacement therapy. A pseudocyst developed and was drained with cystogastrostomy. Then a seizure, visual loss and blood pressure fluctuations occured. Cranial MRI was compatible with PRES. Anticonvulsant treatment was started, clinical manifestations of PRES improved but he needed long term treatment for seizures originating in the occipital lobe. He recovered from all complications and discharged after 3 months. Discusssion: Association of H1N1 and acute pancreatitis has been reported in very few patients. PRES after acute pancreatitis is another very rare association reported in the literature. Clinical manifestations of acute pancreatitis after H1N1 infection and PRES after acute pancreatitis occured in our patient. He was treated succesfully with CVVH, then long term hemodialysis, careful parenteral and enteral nutrition via jejunal tube, pancreatic enzyme replacement therapy and anticonvulsant treatment.

A-039

Disorders of isoleucine degradation: diagnostic problems in two patients Sredzinska M 1, Rokicki D 1, Pajdowska M 1, Kozlowski D 1, Bogdanska A 1, Piekutowska-Abramczuk D 1 1

The Childrens Memorial Health Institute, Warsaw, Poland

Background: Deficiency of beta-ketothiolase (acyl-CoA acetyltransferase1), caused by mutations in the ACAT1 gene, is a well-defined rare organic aciduria affecting ketone body metabolism and the isol e u c i n e c a t a b o l i c p a t h w a y. H S D 1 0 d e f i c i e n c y, 2 - m e t y l - 3 hydroxybutyryl-CoA dehydrogenase (MHBD), caused by mutations in the HSD17B10 (HADH2) gene on chromosome X (milder phenotype in females), is relatively rare defect of isoleucine degradation. We present two clinical cases with 2 methyl-3 hydroxybutyric acid (HMBA) excretion differentiated by molecular analysis.

Case Report: 1.Clinical onset at age of 1.5 yrs occurred as ketoacidosis crises brought on by acute infection with vomiting, dyspnea, lethargy followed by coma. Due to severe metabolic acidosis, intravenous fluids with glucose and bicarbonate were administered. Urinary organic acids profile (OA) showed significantly elevated HMBA, but only small amounts of 2-methylacetoacetate and tiglylglycine. Recurrent ketoacidosis was also observed. Initial blood spot acylcarnitine profile revealed slightly increased tiglylcarnitine, but subsequent control analyses were normal. Sequencing of ACAT1 gene was performed, which revealed a homozygous c.890C > A(p.Thr297Lys) mutation. 2.An 8 yrs old girl presenting mild, slowly progressive encephalopathy, with extrapyramidal signs. Clinical onset at age of 1.5 yrs was observed as: psychomotor retardation, speech delay, difficulties in walking and irritability. MRI of the brain revealed a basal ganglia involvement. OA profile revealed slightly elevated HMBA and tiglylglycine. Levels of HMBA remained stable during long period of time. Acylcarnitine levels were normal. Molecular studies confirmed MHBD by finding a heterozygous mutation, c.751A > T(p.lle251Phe), in the HSD17B10 (HADH2) gene. Results and Discussion: HMBA is a marker of inborn errors of isoleucine degradation, but molecular investigations are still necessary to establish proper diagnosis, especially in mild clinical phenotype patients.

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Insulin resistant hyperglycemia in methylmalonic acidemia decompensation in a 1 year old child Chavez Lopez E 1, Redecillas S 2, Lorite R 2, Ortega Lopez J 3, Valenzuela I 3, Arranz Amo J A 4, Del Toro M 5 1 Div Ped Neur, Vall dHebron Univ Hosp, Barcelona, Spain, 2Div Gast and Nutr, Vall dHeb Univ Hosp, Barcelona, Spain, 3Div Ped Care Unit, Vall dHeb Univ Hosp, Barcelona, Spain, 4Clinic Lab, ValldHeb Univ Hosp, Barcelona, Spain, 5Div Met Dis, Vall dHeb Univ Hosp, Barcelona, Spain

Background: Hyperglycemia mimicking diabetic ketoacidosis has been reported as a rare association of methylmalonic academia (MMA). In a few neonatal cases, hyperglycemia is insulin resistant and has a fatal outcome. We report a patient with MMA and insulin resistant hyperglycemia which led to death at the age of 12 months. Case Report: A 1 year old boy born to consanguineous parents was diagnosed with MMA in the neonatal period. He was treated with diet, carnitine, isoleucine and cobalamin. Despite treatment he presented with failure to thrive and multiple decompensations with acidosis which required frequent admissions. At the age of 11 months he was admitted with a new acidotic episode with skin manifestations resembling acrodermatitis enteropathica. Blood analysis showed lactic acidosis, hyperammonemia, thrombocytopenia, anemia and high levels of methylmalonic acid. Treatment and diet were modified and carglumic acid and antibiotic therapy for skin lesions were added. His metabolic status worsened as did the skin lesions and on the tenth day of admission he was transferred to the pediatric intensive care unit. He developed hyperglycemia up to 250 mg/dl initially treated with insulin. Investigations were performed: pancreatic enzymes, antibodies against pancreatic islets, blood cultures, infection screening; all with negative results. Results: Despite aggressive treatment with insulin and glucose restriction glycemia reached 410 mg/dL and the patient died. Discusssion: Insulin resistant hyperglycemia is an exceptional and severe manifestation of MMA usually presenting in the neonatal period. Our patient presented with this fatal complication in a decompensation at the age of 12 months.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Discusssion: On the light of the course of the disease of the first patient, we consider this mutation as pathognomonic definitely.

Propionic acidemia with severe decompensation despite normal newborn screening results 11. Organic acidurias: others Olsson D 1, Zetterstrom R H 2, Nergardh R 1, Heidenborg C 1, Lajic S 1, Nordenstrom A 1 A-042 1

Dept Women Child Health Karolinska Inst, Stockholm, Sweden, 2Cent inh metab dis, Karolinska Univ Hosp, Stockholm, Sweden Background: The risk of decompensation in mild forms of propionic acidemia must be considered. Propionic acidemia was included in the Swedish newborn screening program in late 2010. Case Report: We report on a patient with normal newborn screening results who was diagnosed with propionic acidemia after a severe decompensation at 2 years of age. Prior to diagnosis the patient had a history of episodic vomiting, preference for low protein foods and failure to thrive but was otherwise considered to be healthy and with a normal psychomotor development. Following a 30 h period of inter-current illness with vomiting and reduced food intake the patient became drowsy and unresponsive and was admitted to hospital. The patient displayed clinical signs of dehydration and was unresponsive to visual and vocal stimuli. Results: Initial laboratory findings indicated a severe metabolic acidosis with pH 6.96, anion gap 25, glucose 1.2 mmol/l and ammonia 197 micromol/l. After 3 days of intensive care treatment including hemodialysis the patient recovered and could later be discharged from the hospital displaying no obvious signs of sequelae. Plasma acylcarnitines, urinary organic acids and mutation analysis confirmed the diagnosis of propionic acidemia. Discusssion: This case report emphasizes that supposedly less severe cases of propionic acidemia may risk life-threatening decompensations.

A-139

Two patients with neonatal-onset MSUD in Slovakia with different clinical course. Brennerova K 2, Bzduch V 2, Behulova D 1, Skodova J 1, Krajciova A 3, Hikkelova M 3 1 Dep Lab Med,Univ Child Hosp, Bratislava, Slovakia, 2Ist Paed Dep Univ Child Hosp, Bratislava, Slovakia, 3Alpha medical, GenLab, Bratislava, Slovakia

Background: MSUD is a rare IEM of branched amino acids with neurological symptomatology. Two patients with neonatal-onset MSUD were diagnosed at last 16 years in Slovakia. Case Report: Neurological symptoms of the first child were appeared on the 5th day of life and progressed to coma with severe hypertonic syndrome. Diagnosis of MSUD was established on the 10th day of life on the basis of the biochemical analysis; the leucine concentration in plasma was 2600 μmol/l. Initiation of adequate treatment led to a normalisation of neurological status of the child. The patient is compound heterozygote for the mutations of BCKDHB gene (exone 1: c.93_103delGGCGGGGCT, exone 5: c.515G?A; second mutation has not yet been described). The patient has required strict low leucine diet and four times haemodialysis in severe metabolic crisis during 16 years of life. Second patient was admitted to hospital because of apathy and lack sucking at the age of 2 weeks of life. MSUD diagnosis was determined by biochemical analysis and validated by the molecular genetics with mutations in BCKDHB gene (c.526A>T and c.853C>T). Over the next 4 years, the patient was on the dietary treatment without metabolic crisis. Results: We described two patients from Slovakia with neonatal onset MSUD with different clinical course. Both are compound heterozygotes in BCKDHB gene, one of the mutations was not yet described.

Internal quality control (IQC) of qualitative organic acid analysis using numerics allows improved monitoring Urbano Blanco G 1, Fitzsimons P E 1, Mayne P D 1 1

Dep Clin Bio, Temple St Child Univ Hosp, Dublin1, Ireland

Background: Medical laboratories are responsible for ensuring that test results are fit for clinical purpose by defining and fulfilling the required analytical performance goals. To define and standardize these goals can be particularly challenging in qualitative assays. We describe a method to define the acceptability limits for urinary qualitative organic acids analyzed by GC-MS. Methods: Each batch of patient samples for organic acid analysis is extracted in parallel with a commercial, lyophilized urine IQC reconstituted with a known concentration of suberylglycine. Each sample and control has two internal standards added: Heptadecanoic acid (HA), used to compare the relative abundance of each peak for chromatogram interpretation, and heptanoylglycine used to assess sensitivity of the assay. Post GC-MS analysis, selected compounds in the IQC chromatogram are labeled and a report with the abundances of each compound generated. This report is exported as a CSV (comma-separated values) file to an Excel© sheet containing formulas to calculate the abundance of each compound as a percentage of HA. Mean+/− 2SD intervals are created for the selected compounds. Results: Numerical intervals determining acceptability of the batch were established. These values enable plotting relevant compounds using Levey-Jennings charts, or to objectively compare the performance of two or more GC-MS analyzers. Monitoring of hexanoyl and suberylglycine gives confidence to asymptomatic MCADD detection and appear to be sensitive indicators of GC column deterioration allowing preventative maintenance planning. Discusssion: This method of IQC recording using numerics improves the monitoring of the GC-MS performance over time, allows the user to set limits for batch acceptance and to compare performance of multiple analyzers in an objective manner which are requirements for ISO 15189 accreditation. This method may also be used to calculate the measurement uncertainty for this qualitative assay.

12. Carbohydrate disorders

A-043

Detection through newborn screening versus clinical symptoms of galactosemia: phenotype and genotype of two cases Lopez-Uriarte A 1, Torres-Sepulveda M R 1, Hinojosa-Amaya A B 1, RuizHerrera M C 1, De la Rosa-Marban E 1, Sanchez-Pena A 1, Martinez-Garza L E 1 1

Depart Genetica, Univ Autonom Nuevo Leon, Monterrey, Mexico

Background: Galactosemia is caused by deficiency of enzymes GALK, GALT or GALE. Measurement of total galactose (TGal) in newborn screening programs (NBS) have made early detection possible, allowing prompt intervention with improvement of life quality. Here

S257

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 we present the clinical and molecular characterization of two cases (A & B). Case Reports: A) A term newborn male of non-consanguineous parents with a healthy 5 yr old brother was identified as having galactosaemia by TMS newborn screening. Breast feed was discontinued and supplemental soy feeds commenced. Currently the child is 4 yrs, asymptomatic with galactose-free diet, vitamin D and calcium. Ophthalmological and neuropsychological evaluations are normal. B) A 3 week old male born in another Mexican state developed jaundice, abdominal distention and feed refusal on day 7. He developed late neonatal sepsis, liver failure and cholestasis and was unresponsive to treatment. He was transferred to our hospital, galactosaemia diagnosed with implementation of dietary therapy; cataract in right eye diagnosed at 3 month old. Now the child is 3 yo shows mild psychomotor retardation, anthropometry 10th percentile. Results: A. TGal 24 h life: 22 mg/dL (NV 1.3–10). 2nd sample (4 d) 21 mg/dL. 3rd sample 62.2 mg/dL (24 h of standard milk). Beutler test: normal. Sequencing of GALT and GALE no reported mutations. Sequencing of GALK: compound heterozygous, one variant non previously reported.B. TGal: 98.8 mg/dL. GALT sequencing: homozygous mutation. Discusssion: Due to the morbidity and mortality of untreated galactosemia it is important to include its detection in NBS. NBS Information to pediatricians of major manifestations could improve early detection avoiding complications. It is important to establish the enzymatic defect in order to know the prognosis.

A-044

Pompe Disease and Hirschsprung disease: A case report of a rare association Kasapkara C 1, Ozbay-Hosnut F 2, Evirgen-Sahin G 2, Ardicli B 3, Kilic M 1, Aksoy E 4 1

Div Metab Dis, Dr Sami Ulus Child Hosp, Ankara, Turkey, 2Div Ped Gastroent, Sami Ulus Child Hosp, Ankara, Turkey, 3Div Ped Surgery, Sami Ulus Child Hosp, Ankara, Turkey, 4Div Ped Neurology, Sami Ulus Child Hosp, Ankara, Turkey

A-045

Exercise induced fatigue: McArdle disease in an adult patient with myopathy Aydogdu S 1, Uskudar Cansu D 2, Kilic Yildirim G 1, Erdogan B 3 1 Dept of Ped Metab, Osmangazi Uni Med Fac, Eskisehir, Turkey, 2Dept of Rheumatol, Osmangazi Uni Med Fac, Eskisehir, Turkey, 3Dept of Pathol, Eskisehir Public Hosp, Eskisehir, Turkey

Background: McArdle disease (glycogen storage disease type V) is a rare autosomal recessive disorder due to myophosphorylase deficiency by myophosphorylase gene (PYGM) mutations. It is the most common muscle glycogenosis in adults. Characteristic findings are exercise intolerance, myalgia, stiffness, sometimes rhabdomyolysis and myoglobinuria which are relieved by rest. The diagnosis is established by muscle biopsy for histochemical and biochemical analysis and/or DNA analysis. Case Report: We report a case of 58-year-old female patient with the history of exercise intolerance, fatigue and weakness which improved after a little rest since childhood. The intensive exercise worsened these symptoms. She had eleveted serum CK and normal LDH levels and she was diagnosed as polymyositis since 5 years. The first muscle biopsy showed non-specific findings. Because elevated levels of CK had not improved despite medical treatment, the second muscle biopsy was performed. Subsarcolemmal and intermyofibrillar PAS-positive vacuoles filled with glycogen led us to consider glycogen storage disease. Results: Specific myophosphorylase staining was negative and the genetic analysis showed homozygosity for the PYGM c.808 C > T (p.R270*) mutation. We started creatine monohydrate supplementation with moderate aerobic exercise. Because she has diabetes mellitus, we could not recommended a carbohydrate-rich diet and sucrose before exercise. Discusssion: The clinical symptoms of McArdle disease usually begin in childhood or young adulthood with exercise intolerance. Symptoms deteriorate with age and diagnosis is often delayed. It is pointed out that McArdle disease should be considered in the differential diagnosis of unexplained myopathies in adults with persistently elevated CK levels.

A-046 Background: Pompe disease (PD) is a rare, inherited autosomal recessive metabolic disorder caused by the deficiency of the lysosomal acid alpha-glucosidase (GAA) enzyme. The prevalence of PD ranges from 1:40,000 to 1:300,000 births and depends on geographic and ethnic factors. Clinical manifestations may vary from a rapidly progressive disabling disease with cardiomegaly, hepatomegaly, weakness, generalized hypotonia, and death within the first year of life, to a mild presentation characterized by slowly progressive myopathy predominantly involving the skeletal muscles. Currently, the administration of the recombinant GAA(ERT) is being used with promising results. Hirschsprung disease is a developmental disorder of the enteric nervous system that is characterized by absence of ganglion cells in the distal intestine, and it occurs in approximately 1 in every 500,000 live births. Case Report: Herein we present an 11-month old boy admitted to our hospital with complaints of psychomotor retardation, constipation and macroglossia. This child was the third child born at full-term to a consanguineous couple through normal delivery without complications, with appropriate weight and length for gestational age. Clinical history revealed chronic constipation and delayed passage of meconium in the newborn period. The diagnosis of HD was corrected and right transverse loop colostomy and bridge operation was done. Results: The activities of alfa glucosidase with inhibition 0.22 (0.9–7.2) nmol/spot 21 h and at pH 3.8 0.38 (1.5–10) nmol/spot 21 h. Molecular genetic work-up exhibited compound heterozygous c. [896 T > C]; [1655 T > C] mutation and verified the diagnosis. ERT, 20 mg/kg alglucosidase alpha was started every 2 weeks. Discusssion: This is the first report of co-existence of Pompe disease and Hirschsprung disease, and in consideration of the prevalence of each disease this association is a very unusual event.

Coexistence of Glycogen Storage Disease Type III and Haemophilia B in Two Brothers Bulut D F 1, Yilmaz B S 1, Kor D 1, Shin Y S 2, Leblebisatan G 5, Tumgor G 4, Gonkek S 1, Yuksel B 3, Mungan H N O 1 1 Cukurova Univ, Div Ped Metab and Nutr, Adana, Turkey, 2Div Metab Dis, Univ Child Hosp, Munich, Germany, 3Cukurova Univ, Div Ped Endoc, Adana, Turkey, 4 Cukurova Univ, Div Ped Gastro, Adana, Turkey, 5 Cukurova Univ, Div Ped Hemato, Adana, Turkey

Background: Glycogen storage disease (GSD) type III, is an autosomal recessively inherited disorder, characterized by fasting hypoglycemia, hepatomegaly, growth retardation, progressive myopathy and cardiomyopathy. It is caused by a deficiency in glycogen debranching enzyme encoded by the AGL gene, located on chromosome 1p21. There is no specific treatment, other than symptomatic management of hypoglycemia and dietary intervention. Haemophilia B (HB) is an X-linked recessive bleeding disorder, caused by the mutations of coagulation factor IX (F9) gene. FIX replacement therapy using plasma-derived protein or recombinant protein significantly reduces bleeding. Case Report: 18 months-old and 3 months-old brothers admitted to our hospital with complaints of hypoglycemic convulsions and hepatomegaly. Parents were non-consanguineous. After excluding the other probable causes of hypoglycemia and hepatomegaly, a liver biopsy was performed. It was compatible with glycogen storage disease. Frequent feeding and consumption of uncooked corn starch were recommended. After 4–5 years follow-up, high serum creatine kinase levels were observed.

S258 Results: Genetic analysis revealed homozygosity for the known p.Q667* (c.1999C > T) mutation within the AGL gene. Both of the patients complained that they have frequent gum bleedings and epistaxis. As prothrombin time was normal and activated partial thromboplastin time was prolonged, HB was the suspected diagnosis. Further studies showed both patients have low Factor IX (1–5 %) levels. Molecular analyses of F9 gene of both brothers are ongoing. Discussion: We would like to highlight the coexistence of two unrelated disorders, GSD type III and HB, that can cause bleeding diathesis. Although the bleeding tendency can be a part of GSD III with severe liver dysfunction, other causative diseases should be kept in mind.

A-047

Sinus bradycardia revealing hypertrophic cardiomyopathy in glycogen storage disease type III. Ben Chehida A 1 2, Mansouri H 1, Ben Abdelaziz R 1 2, Hajji H 1, Boudabous H 1 2, Abdelmoula M S 1 2, Ben Turkia H 1 2, Azzouz H 1 2, Tebib N 1 2 1 La Rabta Hospital, Tunis, Tunisia, 2Medical School, Universite Tunis Elmanar, Tunis, Tunisia

Background: Limited data are available regarding heart rhythm abnormalities in GSD III. Only few series have reported electrocardiography (ECG) findings and, in most cases, ventricular hypertrophy is the predominant feature. Case reports suggested that arrhythmia could occur. To our knowledge, sinus bradycardia has not been reported before in GSDIII patients. Case Report: Here, we report sinus bradycardia as the first sign of cardiac involvement in an 18-years-old Tunisian GSDIII patient. Results: The patient presented to our pediatric department with hypoglycemia, hepatomegaly and seizures, when 9 months old. Debrancher enzyme deficiency was confirmed by an enzymatic assay performed on a frozen liver biopsy. He followed a diet therapy based on frequent meals and uncooked starch. He had normal motor milestones, but baseline CPK levels were at 1210 UI/l. Further electromyography was normal until the age of 7 years, when his EMG showed a myopathic pattern, while he had no muscular clinical complaints. Serial echocardiograms were normal until the age of 10 years, when he was temporarily lost for follow-up. At the age of 18 years, an electrocardiogram found sinus bradycardia and electric features of left ventricular hypertrophy. Echocardiography confirmed hypertrophic cardiomyopathy, without evidence of heart failure. Electrophysiological assessment over 24 h was normal. Discusssion: To our knowlege, this is the first report of sinus bradycardia in GSDIII. Sinusal tachycardia or arrythmia were described in few cases. Longitudinal follow-up with serial 12-lead ECGs in patients with GSD III are necessary to examine the heart rhythm, perform additional tests and prescribe pharmacological treatment, when needed.

13. Disorders of fatty acid oxidation and ketone body metabolism

A-048

A single paediatric centre experience of L-carnitine supplementation in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) Batten W A 1, Chronopoulou E 2, Pierre G 2 1 Dep Pharm, Bristol Royal Hosp Child, Bristol, United Kingdom, 2Dep Paed Met Dis, Bristol Royal Hosp Chi, Bristol, United Kingdom

Background: MCADD is the most common type of fatty acid oxidation disorder in the UK. L-carnitine is a co-factor in the oxidation of fatty acids in mitochondria and eliminates excess medium chain fatty acids. L-carnitine supplementation in MCADD is controversial with no consistent findings in publications.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Methods: Records of 35 patients in our paediatric centre with MCADD were analysed. Results: Of 35 patients treated for MCADD, 13 patients (37 %) received L-carnitine. The most common age group treated was 6– 10 years (n = 6). The mean dose of L-carnitine = 77.7 mg/kg/day; range 45–100 mg/kg/day; most common dose = 100 mg/kg/day (n = 6). 92 % of patients received the 300 mg/mL liquid. Of the 13 patients, 1 had low free carnitine but was asymptomatic; 8 were symptomatic with low levels; 3 were symptomatic with borderline low levels and 1 had no reason documented. All symptomatic patients improved with therapy. Those who stopped supplementation on correction (n = 3), all restarted due to return of symptoms with falling levels. 2 of these patients have a vegetarian diet, a reason for low free carnitine. 2 patients had their doses reduced from 100 to 50 mg/kg/day due to side effects, particularly fishy body odour exacerbated by hot weather. 1 patient complained of the same effect, but the dose remained the same. Discusssion: The majority of patients in this centre are not treated with L-carnitine. The reason for treatment is mainly low free carnitine associated with symptoms. Clinical and biochemical improvement was observed with treatment. The most common dose prescribed was 100 mg/kg/day; with the observed side effect of a fishy body odour also being the most common reason for dose reduction. The most commonly prescribed preparation is a 300 mg/mL licensed liquid. Problems with availability and palatability reduce the usage of licensed tablet preparations.

A-049

Medium-chain acyl-CoA dehydrogenase activity in correlation with acylcarnitines in dry blood spot of the patients with MCAD deficiency detected by newborn screening Sykut-Cegielska J 1, Polawski T 1, Jablonska E 1, Taybert J 2, Kowalik A 1, Oltarzewski M 1 1 Screening Dep, Instit Mother and Child, Warsaw, Poland, 2Met Out Clinic, Instit Mother and Child, Warsaw, Poland

Background: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial fatty acid oxidation and usually is included in the panel of diseases detected by MS/MS newborn screening (NBS). Aim: To evaluate a clinical relevance of MCAD activity and acylcarnitine profiles in cases identified by NBS in our centre. Methods: MCAD activity was measured in isolated lymphocytes using octanoyl-CoA as a substrate. Amount of formed 2-octanoyl-CoA was determined by HPLC UV–VIS. Results: In 2010 we set up an assay of MCAD activity as a confirmation test for suspicion of MCAD deficiency, based on NBS results. Until now 35 such cases were detected with MCAD activities in the range of 0–18.5 % of mean control value with median value of 1.3 %. Additionally two asymptomatic siblings from two families were detected as affected ones. In the first dry blood spot (collected at the third day of life) mean (range) acylcarnitine values were as follows: C6 – 1.23 μmol/l (0.37–6.28), C8 – 7.55 μmol/l (1.31– 24.93), C10 – 9.05 μmol/l (0.17–2.81), C8/C10 – 9.05 (1.8–18.63). Interestingly significantly lower values were obtained in the second dry blood spot: mean (range) C6-carnitine was 0.39 μmol/l (0.10– 0.87), C8-carnitine – 1.86 μmol/l (0.26–10.91), C10-carnitine – 0.20 μmol/l (0.05–0.42), but not the ratio C8/C10 – 8.69 (1.42– 18.43). Discusssion: MCAD activity correlates the best with C8/C10 ratio (correlation coefficient −0.74 by Pearson method), particularly from the first NBS filter paper. Less prominent C6-C10-carnitines concentration in the subsequent assays may be due to regular feedings responsible for better metabolic balance. Conclusion: MCAD activity measurement though not routinely used in NBS until now, seems to be simple and useful way (along to DNA analysis) to confirm diagnosis of MCAD deficiency and evaluate a risk of clinical onset for NBS positive cases.

S259

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 A-050

A-052

10-year-follow-up of 3 patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) treated with heptanoate (C7)

Prenatal findings and autopsy examination in a newborn with multiple acyl-CoA dehydrogenase deficiency

Zlamy M 1, Pichler K 1, Michel M 1, Scholl-Buergi S 1, Karall D 1 1

Dep Pediatrics, Med Univ Innsbruck, Innsbruck, Austria

Background: Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is an autosomal recessive disorder. In patients with LCHADD the accumulation of toxic intermediates of β-oxidation causes immediate symptoms and long-term complications. Therapy with heptanoate (C7) utilizes the anaplerotic effect to enhances ATP production. Methods: We retrospectively evaluated clinical and biochemical parameters of 3 patients treated with heptanoate and a fat-defined diet. All patient have confirmed LCHADD and are homozygous for the common mutation c.1528G > C. Results: The treatment dose of heptanoate equates 0.6–0.8 g/kg/d. Patients treated with heptanoate appeared clinically more stable than before therapy. Patient 1 showed no more CK peaks after heptanoate was started. Patient 2 still showed episodes of rhabdomyolysis but the associated CK concentrations were lower than before. Patient 3 showed laboratory signs of hepatopathy during infections, without any significant changes of the CK concentrations. It is unclear if this stabilization is due to the anaplerotic effect of heptanoate or less infection rates with increasing age. Discusssion: Further prospective placebo-controlled studies and in-vitro models are needed to evaluate the exact impact of heptanoate supplementation and to clarify the cellular mechanisms in the use of heptanoate.

A-051

Severe rhabdomyolysis and dilated cardiomyopathy: unusual clinical presentation of primary carnitine deficiency Madeo A 1, Guemann A S 1, Iserin F 2, Arnoux J B 1, Brassier A 1, De Lonlay P 1 1 Div Metab Dis, Necker Hosp, Paris, France, 2Div Ped Cardiol, Necker Hosp, Paris, France

Background: Systemic primary carnitine deficiency (CDSP) is an autosomal recessive disorder due to a defect in the carnitine transporter OCTN2, leading to urinary carnitine wasting and low plasma carnitine levels. This results in defective fatty acid oxidation and fat accumulation in liver, skeletal muscle and heart. The spectrum of clinical manifestations includes hypoglycemia, hyperammonemia, hepatic failure, dilated cardiomyopathy (DCM) and muscle weakness. Here we describe the case of a child presenting with unusual severe rhabdomyolysis associated to critical DCM and metabolic decompensation. Case Report: His medical history was unremarkable, except for slight fatigability and occasional muscular pain after exercise. Motor development was normal. At the age of 5, during a flu-like syndrome, he developed a severe rhabdomyolysis at 220,000 U/L, with hyperkalemia and transient renal failure, requiring hemodialysis. He had a severe DCM (EF 10 %) and dysrhythmia with 3 cardiac arrests, requiring a ventricular assist device. He also had hypoglycemia, hyperammonemia (887 μmol/L) and transient hepatic failure. Results: Acylcarnitine analysis and organic acids chromatography were normal, but under carnitine supplementation. Muscle biopsy showed myocyte atrophy, myocyte surcharge and a possible gamma-sarcoglycane deficiency. Respiratory chain study was normal. Finally, a new test without carnitine supplementation showed a marked reduction of free (2.3 μmol/L) and total (3.1 μmol/L) carnitine, contrasting with normal urinary carnitine. Carnitine supplementation at 200 mg/kg/day was started. DCM had a favorable evolution. Discusssion: Severe rhabdomyolysis, especially if associated to DCM, is one of the possible clinical manifestations of CDSP. Moreover, this case widens the classical distinction between the early metabolic and the late cardio-myopathic presentation, since this 5 year-old child had a “mixed” hepatic/metabolic and cardiomyopathic phenotype.

Yildiz Y 1, Sirma Dokuzboy R 2, Talim B 4, Yigit S 3, Ceylaner S 5, Sivri H S 1, Tokatli A 1, Coskun T 1, Dursun A 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 2Dept Pediatr, Hacettepe Univ Child Hosp, Ankara, Turkey, 3Div Neonatol, Hacettepe Univ Child Hosp, Ankara, Turkey, 4Ped Path Unit, Hacettepe Univ Child Hosp, Ankara, Turkey, 5Intergen Genetic Diagnosis Center, Ankara, Turkey 1

Background: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a disorder of energy metabolism caused by defects in electron transfer flavoprotein or its oxidoreductase. Some neonatal-onset MADD cases may present with congenital anomalies. Here, we present a neonate with prenatal findings of MADD, who died on the fourth day postpartum, with his autopsy findings. Case Report: A male infant with prenatal ultrasonographic findings of anhydramniosis, enlarged, polycystic kidneys and cardiomegaly was born at term via C-section. He required resuscitation and was intubated. He had depressed, wide nasal bridge, low-set ears, rocker-bottom left foot, hypospadias, cystic, dysplastic kidneys and hypertrophic cardiomyopathy. Sweaty feet odor emerged soon after protein and lipid-containing parenteral nutrition was initiated. Free carnitine level was decreased with elevated C5 and C5DC while urine contained increased glutaric acid, 2-hydroxyglutaric acid, ethylmalonic acid and glycine conjugates, suggesting MADD. He died 84 h after birth despite cessation of protein and lipids, and initiation of glucose, riboflavin, carnitine administration and peritoneal dialysis. Results: Autopsy examination revealed diffuse cystic renal dysplasia, cardiomegaly and biventricular hypertrophy. Lipid accumulation was detected in cardiac and skeletal muscle, liver and kidney. Other pathological features were pulmonary hypertension, pneumonia, intraalveolar, adrenal and periventricular hemorrhage. Sequencing of ETFDH gene revealed a novel homozygous frameshift mutation (c.1524_1524delA; p.K509Nfs*16). Discusssion: Differential diagnosis of neonatal-onset MADD can include chromosomal diseases and autosomal recessive polycystic kidney disease, but sweaty feet odor strongly suggests MADD. This patient already displayed findings of MADD during the prenatal period. Perhaps, earlier suspicion and treatment of MADD may have altered the outcome in this case.

A-053

First Slovak patients affected by two primary defects in mitochondrial energy metabolism Behulova D 1, Ostrozlikova M 1, Holesova D 1, Sebova C 1, Saligova J 3, Potocnakova L 3, Skoknova M 4, Bzduch V 2, Brennerova K 2, Skodova J 1, Pereckova J 1, Dluholucky S 5, Knapkova M 5, Gorova R 6, Ostrovsky I 6, Lisyova J 7, Chandoga J 7 1 Dept Lab Med, Univ Child Hosp, Bratislava, Slovakia, 21st Dept Pediat, Univ Child Hosp, Bratislava, Slovakia, 3Univ Child Hosp, Kosice, Slovakia, 4Dept Neonat Intens Med, Univ Child Hosp, Bratislava, Slovakia, 5Neonat Screen Centre, Univ Child Hosp, Banska Bystrica, Slovakia, 6Chem Inst, Fac Nat Sci, Comenius Univ, Bratislava, Slovakia, 7Inst Hum Biol Gen Clin Gen, Univ Hosp, Bratislava, Slovakia

Background: In humans, mitochondria generate up to 95 % of ATP utilizing three main pathways: tricarboxylic acid cycle, oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO). Single inherited defect in any of these pathways results usually in serious disease. First Slovak patients affected simultaneously by two primary disorders in mitochondrial energy metabolism are reported.

S260 Methods: We evaluated clinical, biochemical and molecular characteristics of children suffering from two disorders of energy metabolism in mitochondria. Results: Over the period of the last 4 years, 5 children (4 boys and 1 girl) affected by two defects in OXPHOS and/or FAO were identified. Romani patients came from East Slovakia. ATP-synthase deficiency due to TMEM70 deficiency and SCADD were detected in 3 cases, MCADD and SCADD in 2 cases. TMEM70 deficient/SCADD patients presented after birth with typical severe life-threatening symptoms, lactic acidosis and hyperammonaemia. Organic acid profile revealed elevated excretion of 3-methylglutaconic and ethylmalonic acids. One child died during the first day of life, two boys are handicaped at the ages of 8 months and 4 years. Diagnosis of two FAO defects MCADD/SCADD in 2 patients was based on typical acylcarnitine profiles in newborn screening and pathognomonic patterns in followup organic acid investigation. At present both children are without neurologic impairment at the ages of 6 months and 3 years. Children affected with TMEM70 deficiency were homozygous for the common mutation c.317-2A > G, MCADD patients were homozygotes for the frequent mutation c.985A > G. Most of the 5 reported SCADD patients were homozygotes or compound heterozygotes for mutations c.310_312delGAG and c.1138C > T. Discusssion: Further studies are necessary to evaluate metabolic interactions in patients with the coincidence of primary energy metabolism defects in mitochondria. Supported by Projects ITMS 26240220007 and ITMS 26240220086.

A-054

Thermolabile long-chain ketoacyl-CoA thiolase variant of MTP deficiency presenting with a myopathy-only phenotype Cole D S 1 2, Olpin S 3, Ferdinandusse S 4, Moat S J 2 1

Cardiff University, Cardiff, United Kingdom, 2Univ Hosp Wales, Cardiff, United Kingdom, 3Sheff Child Hosp, Sheffield, United Kingdom, 4Lab GMD, AMC, Amsterdam, Netherlands Background: Mitochondrial trifunctional protein (MTP) deficiency may present with a wide array of clinical phenotypes. These include a severe infantile form with cardiomyopathy, an intermediate form presenting in childhood with hepatic manifestations, and a milder neuromyopathic phenotype. Here we report a new myopathy-only phenotype, with a specific thermolabile defect in the long-chain ketoacylCoA thiolase (LCTH) domain of MTP. Case Report: The patient became symptomatic at 10 years, with myalgia precipitated by exercise. She had several episodes of rhabdomyolysis, some requiring intensive care, over the following years. There was no hypoglycaemia; precipitants included prolonged exercise, alcohol, and intercurrent illness. Following the birth of her first child, she developed rhabdomyolysis. Her subsequent pregnancies were successfully managed with intravenous dextrose. She has developed progressive problems with chronic myalgia and worsening exercise tolerance. She has no neurological or cardiac signs or symptoms. Results: Creatine kinase was normal between attacks. Minor elevations in long-chain acyl- and hydroxy-acylcarnitines were found. Fibroblast fatty acid flux studies and CPT2 enzymology were normal. Gene panel testing revealed compound heterozygosity for mutations in the HADHB gene. Fibroblast enzyme activities revealed a thermolabile defect in LCTH. Nerve conduction studies were normal, mild retinopathy was noted on slit-lamp examination but electroretinograms were normal. Cardiac investigations were normal. Discusssion: This case demonstrates the clinical phenotype of a previously unreported thermolabile defect in LCTH. Diagnosis was difficult on biochemical grounds as the findings were subtle and not confirmed on fatty acid flux studies. The case emphasises the importance of genetic investigation and confirmatory enzymology. Importantly the phenotype exhibited predominantly myopathic disease with no evidence of neurological involvement apart from mild retinopathy.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 A-055

From exercise intolerance to functional improvement: multiple acyl-CoA dehydrogenase deficiency in an adult patient Kilic Yildirim G 1, Aydogdu S 1, Uskudar Cansu D 2, Erdogan B 3 1 Dept of Ped Metab, Osmangazi Uni Med Fac, Eskisehir, Turkey, 2Dept of Rheumatol, Osmangazi Uni Med Fac, Eskisehir, Turkey, 3Dept of Pathol, Eskisehir Public Hosp, Eskisehir, Turkey

Background: Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD-glutaric aciduria type 2) is an autosomal recessive disorder that is mainly caused by electron transfer flavoprotein dehydrogenase (ETFDH) gene mutations. It is one of the common type of lipid storage myopathies. Patients often manifest as symmetrical proximal, axial muscle weakness and difficulty in lifting the neck; sometimes dysphagia, mastication deficits and respiratory insufficiency. Diagnosis is based on urinary organic acid profile and the acylcarnitine pattern; is must be confirmed with ETFDH gene analysis. Patients show a dramatic response to riboflavin. Case Report: A 29-years old female patient presented with an acute loss of head and neck control. She had a thyroid surgery history before a few mounths ago. The first physical examination was normal except muscle weakness in the neck and proximal limbs. Serum creatine kinase (CK), transaminases and lactate dehydrogenase (LDH) levels were elevated. Electromyography was normal, the first and second muscle biopsies showed non specific findings and inflammatory myositis was considered. Muscle weakness progressed and laboratory findings were’nt normalised for 7-year follow-up. She was admitted to the hospital two times with lung infection during this period. At the age of 36, a third muscle biopsy was performed and showed lipid storage myopathy. The findings of acylcarnitine and urine organic acid analysis were compatible with MADD. Results: Molecular genetic analysis showed a novel p. S515I (c.1544G > T) homozygous mutation in the ETFDH gene. She dramatically responded to riboflavin treatment. Discusssion: Were report a novel mutation in the ETFDH gene in an adult patient with late-onset MADD, characterised with difficulty in lifting the neck as early symptom. As a treatable metabolic disorder, late-onset MADD should be considered in the differential diagnosis of unexplained myopathies in adults.

14. Mitochondrial disorders: nuclear encoded, disorders of pyruvate metabolism and the Krebs cycle

A-056

Neuroradiological findings in children with mitochondrial disorders Zubarioglu T 1, Kiykim E 1, Cansever M S 2, Aktuglu-Zeybek A C 1, Yalcinkaya C 3 1 Div Nutr Metab Dis, Ist Univ Cer Med Fac, Istanbul, Turkey, 2Cent Lab, Ist Univ Cer Med Fac, Istanbul, Turkey, 3Div Neur, Ist Univ Cer Med Fac, Istanbul, Turkey

Background: Mitochondrial diseases are characterized by a broad clinical and genetic heterogeneity that leads to diagnostic difficulties. Brain MRI appears as a useful guide in diagnosis of neurometabolic disorders. Evaluation of different neuroradiological findings in association with clinical and laboratory features should also be helpful to get the definite diagnosis of mitochondrial disorders. In this study, various combinations of MRI lesions in cases of respiratory chain deficiency and known mutations were summarized. Methods: Ten patients in whom the diagnosis of mitochondrial disease was made by measurement of respiratory chain complex activities and/or molecular analysis enrolled in this study. All the patients were symptomatic and had

S261

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 abnormal neurologic findings in physical examination. Data of brain MRI features of the patients were collected retrospectively. Results: Pathologic MRI findings were listed as bilateral and symmetrical brainstem lesions, cerebrocerebellar atrophy, periventricular and lobar white matter signal alterations, cavitating leukoencephalopathy, polymicrogyria, bilateral and symmetrical basal ganglia lesions, diffuse leukoencephalopathy, corpus callosum hypo/agenesis and posterior spinal cord involvement. One of the 10 patients had a normal brain MRI. Discusssion: Mitochondrial disorders should be listed in differential diagnosis of various neurometabolic diseases due to wide clinical and genetic heterogeneity. Recognition of different brain MRI features should be helpful to overcome the diagnostic difficulties. It should also be remembered that a normal MRI could not exclude the diagnosis of mitochondrial disease.

A-057

An ethylmalonic encephalopathy case, mimicking meningococcemia. Kilic M 1, Dedeoglu O 2, Kesici S 3, Gocmen R 4, Yuksel D 2 1 Div Metab Dis, Sami Ulus Child Hosp, Ankara, Turkey, 2Div Pediatr Neurol, Sami Ulus Child Hosp, Ankara, Turkey, 3Intensiv care unit, Sami Ulus Child Hosp, Ankara, Turkey, 4Div Pediatr Radiol, Sami Ulus Child Hosp, Ankara, Turkey

Background: Ethylmalonic encephalopathy (EE) is a rare autosomal recessive disorder characterized by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhea, with fatal outcome early in life. EE is caused by mutations in ETHE1, a mitochondrial protein in sulphide (H2S) detoxification. Case Report: A 10-month-old girl was admitted to hospital because of fever, rash, poor feeding, respiratory difficulty. She was in shock and encephalopathy and immediately intubated. Initial diagnosis of meningococcemia and septic shock was considered and symptomatic treatment was started. Her physical examination showed significiant petechiae, acrocyanosis, microcephaly, axial hypotonia and developmental delay. There was first degree cousin marriage and two previous abortions in the family. In laboratory findings non-ketotic hyperglycemia, glycosuria, severe metabolic and lactic acidosis with increased anion gap were detected. Serum pyruvate and ammonia levels were normal. Tandem mass spectrometry showed increased C4-C5 acyl-carnitines. Urinary organic acid analyses showed increased succinic, fumaric, 2-ketoglutaric, ethylmalonic acid. Brain MRI revealed abnormal signal hyperintensity involving the bilaterally symmetric middle cerebellar peduncles and basal ganglia in T2 and Flair series. Brain proton MR spectroscopy showed a lactate peak in the areas of altered MRI signal. Results: Homozygous c.554 T > C; p.L185R mutation was found in ETHE1 gene. Therapy with riboflavin, coenzyme Q10, metronidazole, acetylcysteine was given. The treatment outcome was good but she remained oxygen dependent with mild developmental delay. Discusssion: EE should be thought of in cases of encephalopathy with recurrent petechial skin lesions and acrocyanosis. In addition to COX and SCAD deficiency, other EE signs are explained by the accumulation of H2S, including damage of endothelial cells and vasodilation, which account for the petechiae and the acrocyanosis.

Background: Deficiency of SUCLA2 results in Leigh’s or a Leigh-like syndrome with onset of severe hypotonia in early infancy, severe muscular atrophy, and sensorineural hearing impairment. Lactate levels can be normal or moderately elevated. Here we present an infant, severely affected with mitochondrial disorder caused by a homozygous mutation in SUCLA2 gene. Case Report: A 2-year old male infant was admitted to hospital for lack of independent sitting and walking. Physical examination showed failure to thrive, mild dysmorphism, nystagmus, axial hypotonia, developmental delay, muscular atrophy and decreased deep tendon reflexes. There was first degree cousin marriage and history of sibling death, abortion and intrauterine exitus. Serum creatine kinase level was high and septal hypertrophy was seen in echocardiography. Laboratory findings showed mild elevated levels of serum lactate and pyruvate. Tandem mass spectrometry showed mildly increased alanine, C3, C14:2 levels, while urinary organic acid analyses showed mildly increased succinic, glutaric, adipic, 3-OH isovaleric and methylmalonic acids. Muscle biopsy was nonspecific but OXPHOS enzymes were not studied yet. Brain MRI revealed bilateral abnormal signal hyperintensity involving the basal ganglia in T2 series. Results: A novel homozygous c.433_434delA mutation was found in SUCLA2 gene by exome sequencing. Mitochondrial vitamin cocktail therapy was given. He is 5 years old now and the treatment outcome is poor with severe developmental delay. Discusssion: So far,less than 25 patients with a diagnosis of SUCLA2 gene defect were described in the literature. Even though MMA is useful marker of decreased succinyl-CoA ligase activity, it is not so specific. Clinical exome sequencing has become an increasingly popular diagnostic tool in patients with heterogeneous genetic disorders.

A-059

Neonatal congenital lactic acidosis with pyruvate carboxylase deficiency in an Omani neonate AL Hashmi N 1, Othman M 1 1

child health dep metab u, Royal hopsital, Muscat, Oman

Background: Pyruvate carboxylase (PC) is a key enzyme for gluconeogenesis and anaplerotic pathways in brain. PC deficiency is a rare autosomal recessive neurometabolic disorder with three described characteristic presentations. Case Report: We report a patient with a typical clinical and neuroradiological presentation. She presented with neonatal lactic acidosis. She since the third day of life started to develop vomiting, hypothermia, lethargy, irritability, hypoglycemia and severe metabolic acidosis. During admission a progressive deterioration was observed. Despite different attempted therapies patient died during the neonatal period. Results: The diagnosis was confirmed by PC sequencing which showed PC (sequencing) homozygous mutation c.2278C > T (p. Arg760Trp). Discusssion: PC deficiency need to be consider among the differenital diagnosis of patient with congenital anomlies, lactic acidosis and hyperammoniemia .

15. Mitochondrial disorders: mtDNA A-058 A-060 A mitochondrial disorder patient with a novel mutation in SUCLA2 gene Kilic M 1, Kilic E 3, Ozgul R K 4, Kavak P 5, Yuceturk B 5, Demirci H 5, Aksoy A 2, Sagiroglu M S 5

Long-term developmental trends of pediatric mitochondrial disease: The five stages of developmental decline

1 Div Metab Dis, Sami Ulus Child Hosp, Ankara, Turkey, 2Div Pediatr Neurol, Sami Ulus Child Hosp, Ankara, Turkey, 3Div Pediatr Genetic, Hem-Onco Child Hosp, Ankara, Turkey, 4Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 5Bilgem, TUBITAK, Kocaeli, Turkey

Eom S 1, Lee Y M 2 Yonsei Univ Coll of Med, Epilepsy Res In, Seoul, Republic of Korea, 2Yonsei Univ Coll of Med, Dep of Ped, Seoul, Republic of Korea 1

S262 Background: Children with encephalopathy experience many limitations in daily life, although little is known about the long-term developmental outcome of pediatric patients with mitochondrial diseases (MDs). This study aimed to evaluate the clinical course and long-term developmental trends in pediatric patients with MDs. Methods: Data of 53 pediatric patients were obtained from hospital records based on an MD diagnosis and the results of developmental evaluations using the developmental quotient (DQ). Additionally, disease-related clinical variables were reviewed, and clinical progress was determined through observation. Subgroup analyses by epilepsy severity, syndromic diagnosis, diffuse brain atrophy, and clinical rating were performed. The pre-diagnostic, diagnostic, and post-diagnostic evaluations were compared using repeatedmeasures ANOVA. Results: Using the DQ, significant differences were observed in children’s developments at the first pre- and second post-diagnostic evaluations in the diffuse brain atrophy subanalysis. According to the clinical progress, the improving/static group presented a significantly higher DQ level than the mildly/severely deteriorating group at the pre-diagnostic and diagnostic states. Onset age of first symptom positively correlated with the developmental functioning level post-diagnosis. From the first pre-diagnostic to the sixth postdiagnostic evaluation, an overall decline in development was observed. Follow-up revealed consistent patterns of significant developmental deterioration of DQs during lead time, whereas no significant differences after diagnosis were observed. Discusssion: Despite the small patient number and the overall cohort not being followed-up, our findings highlight the value of DQ as a predictor and measure of long-term functioning in children with MD. Moreover, physician’s confirmation of diagnosis and initiation of treatment may shorten the lead time to diagnosis and alleviate developmental regression.

A-061

New variation in D-loop of mitochondrial DNA in non alcoholic fatty liver disease Kamfar S H 1, Saidijam M 1, Yadegarazari R 4, Houshmand M 2, Hasrak K 3 1 Hamadan University of Medical Sciences, Hamadan, Iran, Islamic Republic of, 2NIGEB, Tehran, Iran, Islamic Republic of, 3baqiyatallah el azam hospital, Tehran, Iran, Islamic Republic of, 4Kermanshah University of Medical Science, Kermanshah, Iran, Islamic Republic of

Background: Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of disorders characterized by the accumulation of triglycerides. Mitochondria play a central role in hepatic lipid metabolism and several studies support a role of mitochondrial dysfunction in development of NAFLD. It has been suggested that mitochondrial DNA (mtDNA) mutations might be effective in development of several diseases. mtDNA is known for high mutation rates caused by lack of protective histones, inefficient DNA repair systems, and exposure to mutagenic effects of oxygen radicals. Alteration in the noncoding displacement (D) loop of mitochondrial DNA are present in many diseases like NAFLD. Thereforeby focusing on alterations in mitochondrial D-loop, we investigated a link between mtDNA and NAFLD. Methods: The DNA from blood of 33 NAFLD patients and 153 healthy controls fromdifferent ethnicities were investigated to determine sequence variations in the mitochondrial D-loop region. Polymerase chain reaction (PCR) and DNA sequencing methods were used to detect possible variation in the mitochondrial D-loop. Results: Although the polymorphisms atdifferent loci in mitochondrial Dloopwere not significantly correlated with NAFLD, but the new insertion C16220 was significantly (12.1 %) present in patients compared to control (0 %) samples (p < 0.05). There was no significant difference (p > 0.05) of C variations, including C7TC6, C8TC6, C9TC6 in D310 mitochondrial DNA between patients and control samples Discusssion: Our study suggest that C16220 insertion may have an indirect role in development of NAFLD. Keywords: non alcoholic fatty liver disease, mitochondrial DNA displacement loop, C16220 insertion

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 A-062

Clinical features of a child homoplasmic for the MT-ATP6 m.9176 T > G mutation, presenting with an abnormal newborn screen consistent with multiple carboxylase deficiency Akroyd R M D 1, Knoll D 2, Wilson F 5, Potter H 3, Van Karnebeek C D M 4, De Hora M 2, Glamuzina E 1 1 Nat Metab Service, Starship Child Hosp, Auckland, New Zealand, 2Nat Test Centre, Lab Plus, Auck Hosp, Auckland, New Zealand, 3Mol Gen, ChCh Hosp, Christchurch, New Zealand, 4Div Biochem Dis, BC Child Hosp, UBC, Vancouver, Canada, 5 Radiology Dept, Starship Child Hosp, Auckland, New Zealand

Background: Elevated 3-hydroxyisovalerylcarnitine (C5OH) detected by tandem mass spectrometry (MS/MS) is used as a NBS test for multiple carboxylase deficiency (MCD) in New Zealand. A case of m.8993 T > G related Leigh-like disease was previously reported to present with this biochemical signature on NBS. Incidental diagnosis of a mitochondrial disease on NBS has ethical implications if it does not improve outcome. Case Report: Clinical review of a now 8 month old girl with severe faltering growth due to m.9176 T > G mutation diagnosed in the work up of an abnormal ENBS result consistent with MCD. Results: The case presented at 24 h with tachypnoea, mild acidosis and poor feeding. Day 2 ENBS revealed C5OH of 1.33 umol/L ( C) in the MT-TV gene coding for tRNA valine. The patient has been followed-up with anti-acidosis therapy, allopurinol for increased uric acid levels in addition to fat-rich diet. The metabolic parametres after 1 year follow-up seems to be better with stabilization of the left ventricular hypertrophy. Discusssion: The novel mutation in our patient in MT-TV gene expands the genotypic spectrum and confirms MT-TV gene as a hotspot for mitochondrial cardiomyopathy.

A-065

Leigh-like syndrome caused by mutation m.3308 T > G in the MT-ND1 gene Krylova T D 1, Itkis Y S 1, Mikhaylova S V 2, Zakharova E Y 1 1 FSBI Res Cent Med Gen, Moscow, Russian Federation, 2Dep Med Genet, Chil Clin Hosp, Moscow, Russian Federation

Background: Leigh syndrome is the most common pediatric phenotype of mitochondrial disease. This disorder is genetically and clinically heterogeneous. More than 75 mutations, both in nuclear and mitochondrial genomes, were described to cause the syndrome. We present a 13-year-old male with exotropia, spasticity, distonia, psychomotor retardation. The MRI shows bilateral lesions in the basal ganglia. Now the male is wheel-chaired person. His 11-year-old sister is affected only with ataxia and increased fatigability. Methods: Sequencing of the whole mtDNA was performed on Ion Torrent. To measure the oxygen consumption in patient’s and his sister’s fibroblasts we applied protocols with intact and permeabilized cells on the Oxygraph-2 k (Oroboros corp., Austria). The level of FGF-21 in serum was detected by ELISA kit. Results: We have found a transition m.3308 T > G in the first codon of MT-ND1 gene in homoplasmic state in blood and urine of both sibs. Their mother has the same substitution only in urine (in the homoplasmic state). Flux Control Rations in intact cells R/E, netR/E (p < 0,05) were higher compared to the control samples. In permeabilized fibroblasts we observed differences in CI + II/E, Rot/E (p < 0,05) between groups. FGF-21 was high in the patient’s serum (2000 pg/ml), but the sister has showed normal serum FGF-21 level ( G mutation was reported and associated with the sudden infant death. Our results show the mutation m.3308 T > G can be associated with biochemical abnormalities of OXPHOS and FGF-21 level leading to Leigh-like syndrome. Also it demonstrates a higher penetrance of the mutation in male.

16. Disorders of purines, pyrimidines, nucleic acids and porphyrias

A-066

Dihydropyrimidine dehydrogenase (DPD) deficiency: first case in the Czech Republic Bartl J 1, Martincova O 1, Kovacikova T 1, Krijt J 1, Jahnova H 1, Peskova K 1 1

Inst Inher Metab Dis, Gen Univ Hosp, Prague, Czech Republic

Background: Dihydropyrimidine dehydrogenase (DPD) deficiency is a rare, autosomal recessive disorder of pyrimidine metabolism showing large phenotype variability. Enzyme deficiency results in accumulation of uracil and thymine. Homozygous and heterozygous mutation carriers are endangered by toxic effect of oncologic therapy with 5fluorouracil (5-FU), also metabolized by DPD. Case Report: 18 years aged female was examined for chronic mild cognitive impairment. Due to MRI finding of cerebellar peduncle demyelinization and specific oligoclonal bands in CSF diagnosis of multiple sclerosis was assessed by neurologists and treatment with glatiramer acetate was launched. However, a broad metabolic screening was conducted contemporary. Results: Biochemical investigation revealing increased urinary excretion of uracil 141 mmol/mol creat. (controls < 25) and thymine 256 mmol/mol creat. (c. < 10) and elevated plasma concentrations of uracil 18 μmol/L (c. < 5) and thymine 16 μmol/L (c. < 1) suggested DPD deficiency. In cooperation with foreign laboratory decreased DPD activity in patient’s fibroblasts and two pathogenic mutations of the DPYD gene [c.1905 + 1G > A] [c.1057C > T] in trans were subsequently found. Discusssion: The first DPD deficiency patient from the Czech Republic is presented. Next course of disease will most likely show if the neuropsychological symptoms described in this patient are caused by DPD deficiency or multiple sclerosis. Biochemical diagnostics on metabolites level is easy, fast and reliable tool for disease detection, and moreover important if oncological chemotherapy in the concerned family members is necessary. Supported by: MZ CR – RVO VFN64165 and OPPK CZ.2.16/3.1.00/24012.

S264 A-067

A molybdenum cofactor deficiency case presenting with status dystonicus

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Discusssion: Genetic testing should focus primarily on LDLR, and subsequently on PCSK9 and APOB. The Greek population is genetically homogeneous, which allows for a quick molecular diagnosis of the disease. Cascade screening is feasible in the Greek population and will certainly facilitate the identification of additional patients.

Kilic Yildirim G 1, Carman K B 2, Aydogdu S 1, Yarar C 2, Kiral E 3 1 Dept of Ped Metab, Osmangazi Uni Med Fac, Eskisehir, Turkey, 2Div Ped Neurol, Unit Osmangazi Med Fac, Eskisehir, Turkey, 3Dept Ped Intensive Care, Univ Osmangazi, Eskisehir, Turkey

Background: Molybdenum cofactor deficiency (MoCD) is a rare metabolic disorder characterized by severe and rapidly progressive neurologic damage caused by the functional loss of sulfite oxidase. Status dystonicus (SD) is also a rarelife-threatening movement disorder, characterized by the development of increasingly frequent or continuous severe episodes of generalized dystonic spasms and requires urgent management. Here we describe a 10-month-old male patient whit Molybdenum cofactor deficiency presenting with status dystonicus who was treated with high dose of baclofen. Case Report: The patient was the second child of nonconsanguinous parents. He had seizures during the postpartum first day and severe truncal hypotonia, poor head and motor control and swallowing dysfunction over the first months of life. He was admitted to the intensive care unit because of SD. On physical examination spasticity, opisthotonus, microcephaly, neuromotor and growth retardation were detected. Biochemical test results were normal, except low serum uric acid. Dipstick test for urinary sulfite was positive. Lens dislocation wasn’t determined. Results: Genetic studies revealed a novel IVS1-1G > A (c.2S1-1G > A) homozygous mutation in the MOCS1 gene which has not been previously reported to the best of our knowledge. Diazepam and baclofen treatment were started, but opisthotonus position improved after high doses of baclofen. Discusssion: MoCD should be considered in any child with seizures in the presence of hypouricemia. This is the first patient with MoCD presenting with SD to the best of our knowledge. The effectiveness of high dose baclofen treatment was shown in status dystonicus in this patient.

17. Peroxisomal, sterol, bile acid, lipid and lipoprotein

A-068

Genetic causes of monogenic Familial Hypercholesterolemia in the Greek population: Past, present and future

A-069

Dysmorphic Facial Features and other Clinical Characteristics in Two Patients with Peroxisomal Biogenesis Factor 1 (PEX1) Gene Mutations Gunduz M 1, Unal O 1 1

Ankara Children’s Hem Oncology Div Metab, Ankara, Turkey

Background: Peroxisomal disorders are a group of genetically heterogeneous metabolic diseases related to dysfunction of peroxisomes. Dysmorphic features, neurological abnormalities and hepatic and gastrointestinal dysfunction can be presenting signs of peroxisomal disorders. Dysmorphic features may include craniofacial dysmorphism, skeletal abnormalities, shortened proximal limbs, calcific stippling of epiphyses and renal cysts in different disorders linked to peroxisomal dysfunction. Case Report: Here we presented dysmorphic facial features and other clinical characteristics in two patients with PEX1 gene mutation. Follow up periods were 3.5 years and 1 year in these patients. Results: Case I was a 1-year- old girl referred with neurodevelopmental delay, bilateral hearing loss and visual problems. She had bilateral hearing loss and hepatomegaly. Ophthalmologic examination suggested septo-optic dysplasia. Cranial magnetic resonance imaging (MRI) showed nonspecific gliosis of subcortical and periventricular deep white matter. Case II was a 2.5-year-old girl referred for global developmental delay and elevated liver enzymes. She had bilateral nystagmus and hepatomegaly. Ophthalmologic examination findings were consistent with bilateral nystagmus and retinitis pigmentosa. Cranial MRI was normal. Dysmorphic facial features including broad nasal root, low set ears, downward slanting eyes, downward slanting eyebrows, epichanthal folds were found in both patients. Very long chain fatty acid analyses were suggestive of peroxisomal disorders in both patients. Molecular genetic analysis of PEX1 gene detected novel homozygous IVS1-2A > G mutation in Case I and homozygous p.G843D (c.2528G > A) mutation in Case II. Discusssion: Neurologic, hepatic and gastrointestinal abnormalities are common clinical findings in different genetic abnormalities associated with peroxisomal dysfunctions. Dysmorphic features can also be prominent signs.

A-070

Mollaki V 1, Drogari E 1 1

Metabolic Unit,Univ Child Hosp Ag Sophia, Athens, Greece

Background: Familial Hypercholesterolemia (FH) is a leading cause of premature atherosclerosis, which is underdiagnosed worldwide. Genetic defects in LDLR, APOB and PCSK9 genes cause FH, and confirmation of a gene defect is essential for an indisputable diagnosis of the disease. The present study aimed to review the genetic defects that have been characterized in FH patients of Greek origin and define an effective, future strategy for molecular diagnosis. Methods: A literature search was performed in MEDLINE and EMBASE on genetic studies with FH patients of Greek origin. Results: To date, no APOB and PCSK9 mutations have been found in the Greek population, although it must be noted that only a small number of patients has been screened for PCSK9 mutations. In total, 41 LDLR defects have been characterized. Studies that used Sanger sequencing show that six common LDLR mutations, 1646G > A (p.Gly546Asp), c.858C > A (p.Ser286Arg), c.81C > G (p.Cys27Trp), c.1285G > A (p.Val429Met), c.517 T > C (p.Cys173Arg), and c.1775G > A (p.Gly592Glu), account for >80 % of all mutations. Founder mutations exist only in a subpopulation in North West Greece and the Greek Cypriot population, likely because of geographic isolation, but not in the general population.

Phenotypic and molecular characterization of peroxisomal diseases in Tunisian patients Chioukh F Z 1 4, Ben Ameur K 1 4, Ben Hmida H 1 4, Kerkeni E 4, Bizid M 1, Kaabachi N 2, Cheillan D 3, Vanier M T 3, Monastiri K 1 4 1 Dept Intens care neonat Med Teach Hosp, Monastir, Tunisia, 2Dept Biochem Teach Hosp LaRabta, Tunis, Tunisia, 3Lab Bioch Genet, Lyon, France, 4Res Unit 01-UR-08,14 Fac Medicine, Monastir, Tunisia

Background: Zellweger syndrome (ZS) is a severe manifestation of disease within the spectrum of peroxisome biogenesis disorders. Affected patients with ZS typically die after few weeks from apnea, respiratory failure, or infective complications. Methods: This is a retrospective study of ZS patients admitted in the Intensive Care and Neonatal Medicine Department of Monastir, Tunisia between 2010 and 2015. All patients had measurements of the plasma concentrations of very long chain fatty acids (VLCFA) and enzymatic assay of peroxisomal metabolism. Results: Eight cases were included in the study. In one case, prenatal diagnosis using cultured cells derived from placental cells was positive. Recurrence of the disease was observed in three families. All patients had a characteristic

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 phenotype of distinctive facial features, pronounced hypotonia, and poorly controlled seizures. Ventilatory support was needed in two cases. Cholestasis and hepatic injury were observed in three cases. Deaths occurred after a few days in two cases because of respiratory distress. ZS was confirmed by elevated VLCFA. Genetic study was done in one case. Molecular analysis was done in two cases and by whole exome sequencing for one case identifying a homozygous p.N457Y mutation in the HSD17B4 gene. Discusssion: ZS is a serious disorder with multiple congenital anomalies that is progressive and fatal. Despite the lack of treatment options, prompt diagnosis of ZS is important for providing appropriate symptomatic care, definitive genetic testing, and genetic counseling.

A-071

Two siblings with neonatal-onset Dubin-Johnson Syndrome and hypothyroidism harboring a novel mutation in the ABCC2 gene Kor D 1, Seker Yilmaz B 1, Bulut F D 1, Ceylaner S 2, Topaloglu A K 3, Onenli Mungan N 1 1 Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Intergen Genetic Lab, Ankara, Turkey, 3Div Ped End, Univ Cukurova, Adana, Turkey

Background: Dubin-Johnson Syndrome (DJS) is a rare autosomal recessive disorder that causes conjugated hyperbilirubinemia without elevation of liver enzymes. It is a generally benign disorder, but recurrent jaundice episodes triggered with catabolic processes can be observed. DJS is rarely diagnosed in the neonatal period. Case Report 1: The second child of consanguineous parents, a 12 days-old-male infant was referred to our clinic for jaundice. His prenatal, perinatal and family history for intrauterine infections, hematological and other inherited disorders were all unremarkable. His growth parameters were normal, organomegaly was not detected. The total bilirubin was normal and direct bilirubin levels were 4 and 1.5 mg/dl, respectively. Other hematologic and biochemical investigations included liver function tests which were normal. The patient had mild primary hypothyroidism and L-thyroxin supplementation was started. In the 4-year follow-up, direct and total bilirubin levels increased during infection episodes. Based on these data the suspected diagnosis of DJS. Currently, he is 4 years old with normal growth, development and liver function tests. Case Report 2: The older brother admitted to our hospital with the complaints of abdominal pain and constipation at the age of 4 years old. The bilirubin levels were not screened. All of the laboratory parameters were in normal ranges except thyroid function tests. Primary mild hypothyroidism was treated with L-thyroxin. He was otherwise healthy to the age of 9 years when his brother was diagnosed as DJS. He is now 13 years old without any health problems. Results: DJS was confirmed with the molecular analysis showing a novel homozygous mutation IVS29 + 1G > A (c.4146 + 1G > A) on ABCC2 gene in the two siblings. The parents were heterozygous for this mutation. Discussion: Here we report these 2 siblings with neonatal-onset DJS due to a novel ABCC2 mutation and comorbidity with primary hypothyroidism.

18. Lysosomal disorders: mucopolysaccharidoses, oligosaccharidoses

A-072

Multi-system lesions in siblings with mucopolysaccharidosis type VI and effects of galsulfase treatment. Lobzhanidze T V 1 1

City Clinical Hospital 64, Moscow, Russian Federation

Background: Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disease caused by decreased synthesis or function of the enzyme Nacetylgalactosamine-4-sulfatase (arylsulfatase B).

Case Report: We report the characteristics of two adult male siblings with MPS VI. Both are homozygous for the R152W mutation. Diagnoses occurred at 26 and 20 years. Both have mild growth retardation, joint contractures, impaired vision, decreased pulmonary function, cardiomegaly, aortic valve stenosis, aortic and mitral valve insufficiency, and spinal stenosis and cord compression. The elder sibling also has significantly decreased range of motion in his extremities, mitral valve stenosis, tricuspid valve insufficiency, and hearing loss. The elder and younger siblings are 152 and 164 cm tall and weigh 55 and 65 kg, respectively. Enzyme replacement therapy (ERT) with galsulfase was initiated at 27 years of age for the elder sibling and is ongoing. Results: After 9 months of ERT, urinary glycosaminoglycan excretion decreased threefold; after 24 months, normal levels were achieved. Liver and spleen sizes decreased, weight increased by 4 kg, mobility of extremity joints improved, and spine flexion and extension amplitude increased. ECG, Echo-CG, and pulmonary function tests showed no significant changes. ERT was well tolerated. Considering the presence of neurological symptoms and spinal cord compression at the Th12-L1 level, laminectomy was performed at 28 years of age, with subsequent prosthetic repair. After the surgery, the patient experienced increased exercise tolerance and reduction of pain in the lumbar spine and numbness sensation in the lower extremities. Discusssion: Both siblings have the same R152W mutation and experienced multi-system lesion. The combination of ERT and surgical correction of spinal stenosis benefited the elder sibling. We hypothesize that the younger sibling could benefit from ERT as well. Conflict of Interest declared.

A-073

A case of alpha-mannosidosis with a novel mutation Kasapkara C 1, Kilic M 1, Ceylaner S 2 1 Div Metab Dis, Dr Sami Ulus Child Hosp, Ankara, Turkey, 2Intergen Genetic Center, Ankara, Turkey

Background: Alpha-mannosidosis is a rare lysosomal storage disorder with a heterogeneous clinical presentation that is inherited in an autosomal recessive pattern. Deficient alpha-mannosidase activity leads to lysosomal accumulation of mannose-rich oligosaccharides. It occurs in approximately 1 of 500000 live births and can be caused by 40 different mutations in the gene MAN2B1 located on chromosome 19. Alpha mannosidosis has been suggested to have three clinical types. We describe a case with type 2 alpha-mannosidosis. Case Report: He was born at term to first cousin parents. This 4 years and 9 months old boy was admitted to our hospital because of coarse facial appearence and speech delay. He showed frequent respiratory infections, coarse facies, mild hypertrophy of left ventricle and sensorineural hearing deficit. We confirmed the diagnosis by enzyme assay. Peripheral blood Alpha mannosidase enzyme level was 2.2 (NV: 100– 800) umol/g/h . Molecular analysis of MAN2B1 gene revealed novel homozygous p.A95P (c.283 G > C) mutation. Results: Bone marrow transplantation is an option in the first decade of life which makes early identification of affected patients critical. A promising therapeutic approach for an effective treatment of alphamannosidosis is the application of recombinant human LAMAN (rhLAMAN) into the bloodstream, designated as enzyme replacement therapy (ERT). The clinical trials are underway for ERT. We considered HSCT as a therapeutic approach in this patient. Discusssion: Alpha-mannosidosis is a genetic disorder of metabolism characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and mental retardation. At present, three clinical types have been suggested. Most patients belong to clinical type 2 as in our patient. These disorders, although rare, should be considered in the approach to a child with dysmorphism, developmental delay, skeletal deformities, and visceromegaly.

S266 A-074

An atypical patient with Hunter syndrome Bettocchi I 1, Ortolano R 1, Baronio F 1, Bertola F 2, Pession A 1, Cassio A 1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Results: Decompression surgery with laminectomy of C1 and C2 vertebrae was performed, resulting in expansion of the cervical cord width from 5 mm to 17 mm at its narrowest point and in rapid disappearance of pain and pyramidal signs, allowing the patient to walk independently. Discussion: Craniocervical decompression surgery may be associated with morbidity and mortality, and its indications, timing and surgical approaches are still topics of debate. However, successful and timely decompression may significantly improve the quality of life in MPS patients.

1 NBS Reg Centre, Ped Unit,AOU, Bologna, Italy, 2Hum Mol Genet, Univ Milano-Bicocca, Monza, Italy

A-076 Background: Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an X-linked lysosomal storage disorder caused by the deficit of iduronate 2sulfatase (IDS), an enzyme involved in the glycosaminoglycans (GAGs) degradation. Case Report: We report the case of a 5-year-old boy who was diagnosed with MPS II at 3 months of age. The main clinical features were: coarse face, eyelid ptosis, epicanthus, small nose, hypoplastic tip,anteverted nostrils, long filter, high arched palate, large and coarse ears, short and wide neck, truncal hypotonus, ligamentous laxity, brachymetatarsia, finger clinodactyly. Dosage of urinary GAG revealed high urinary glycosaminoglycans (GAG):660 mg/g creat (n.v. < 50), chondroitin solfate ++, dermatan solfate ++++, heparan solfate ++. Iduronate sulfatase activity in leukocytes was extremely low: 1.8 nM/mg/4 h (n.v. 59–77). A brain MRI, performed at 15 months, showed white matter lesions, moderate ventricular expansion, and gliosis. At the age of 18 months the patient started enzyme replacement therapy (ERT) with idursulfase (Elaprase®) at the dosage of 0.5 mg/kg body weight per week. ERT was well tolerated and safe. At last control the patient shows severe mental retardation, short stature, normal cardiac function, joint stiffness and bilateral hypoacusia due to repeated ear infections (hearing aids at 18 months). Despite ERT, hepatomegaly and upper-respiratory tract infections did not improve. Results: Sequencing of the IDS gene revealed the deletion of exons 1–7, extending to regions Xq28 e Xq27.3, removing the entire pseudogene IDS2 and genes FMR1 and AFF2. Discusssion: The particular severity of the patient phenotype suggested to verify a potential “contiguous gene syndrome” . Therefore we will verify the presence of pathological copy number variations (CNVs) in the genome, by the array CGH (aCGH) technology.

A-075

Adult Mucopolysaccharidosis Type VI Patient with Severe Cervical Cord Compression at Diagnosis Sivri H S 1, Bilginer B 2, Ozgen Mocan B 3, Yildiz Y 1, Dursun A 1, Tokatli A 1, Coskun T 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey, 2Dept Neurosurgery, Hacettepe Univ Hosp, Ankara, Turkey, 3Dept Radiology, Hacettepe Univ Hosp, Ankara, Turkey 1

Background: Stenosis of the craniocervical junction is one of the most serious complications of mucopolysaccharidoses. Although it is commonly a late finding, it may be present at diagnosis, especially if there is considerable diagnostic delay. Case Report: Here, we present 25-year-old male and 18-year-old female siblings with consanguineous parents, who presented with progressive walking difficulties. She had pain and limited range of motion in shoulder and hip joints, but could walk independently, while he had undergone multiple hip surgeries and been wheelchair-bound for the last 2 years. Physical examination was remarkable for distinct coarse facies, corneal opacities, joint contractures, short stature and and normal mental capabilities, consistent with mucopolysaccharidosis type VI (MPS VI), which was confirmed by urine glycosaminoglycan electrophoresis, plasma arylsulfatase B levels and ARSB gene sequencing. The older brother also had hyperactive knee jerk and Achilles’ reflexes and bilateral clonus, suggesting compression of the pyramidal tract. Craniospinal magnetic resonance imaging revealed severe compression of the spinal cord at the craniocervical junction.

A case of mucopolysaccharidosis type VII presenting with gingival hypertrophy. Joost K 1, Tomberg K 4, Saks K 2, Hein H 3, Roht L 1, Zordania R 1 1 Tartu Univ Hosp, Gen Dept, Tallinn, Estonia, 2Tallinn Child Hosp, Tallinn, Estonia, 3Central Lab of Health Board, Tallinn, Estonia, 4North Estonia Med Cent, Tallinn, Estonia

Background: Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is a very rare lysosomal storage disease caused by mutations in GUSB gene which lead to deficient b-glucuronidase activity. Clinical presentation varies from severe fetal hydrops to milder form with later onset and normal intelligence. Objective: to present the clinical problems and diagnostic process in a patient with MPS VII. Case Report: The girl was admitted to the hospital at the age of 3 years due to the gingivitis of unknown etiology. Clinically the patient was otherwise without any problems. In peripheral blood smear prominent dark-purple granules in granulocytes, in some lymphocytes and monocytes and the same morphologic finding in bone marrow granulocytes of all stages of maturation referred to possible metabolic storage disorder. Differential diagnosis in patient included Chediak-Higashi syndrome (CHS), mucopolysaccharidoses and several immunodeficient conditions. Results: CHS was excluded by LYST gene sequencing. Analysis of urinary glycosaminoglycanes (GAGs) revealed moderate elevation of urinary GAGs – 33,4 mg/mmol cr (ref. < 15) and two-dimensional electrophoresis showed nonspecific changes. As clinical problems were not specific to any of the known pathologies, multigene approach was applied- 6 genes which are known to be associated with the basophilic granulation were analyzed. This revealed 2 heterozygous mutations in GUSB gene: c.511G > A (paternal) and c.1880G > A (maternal). Confirmatory biochemical analysis from skin fibroblasts revealed deficient b- glycuronidase activity- 0,6 nmol/h/mg (ref. 78– 270). Discusssion: MPS VII may present with moderate urinary GAG elevation and non-specific electrophoreti profile and minimal storage symptoms- therefore high clinical suspicion should be maintained. Complete work-up of mucopolysaccharidoses is indicated in patients with characteristic morphologic findings in leukocytes.

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Cranial and Spinal Magnetic Resonance Imaging in Mucopolysaccharidoses Olgac A 1, Damar C 3, Alimli A 2, Derinkuyu B 2, Oztunali C 2, Ucar M 2, Boyunaga O 2, Tumer L 2 1 Div Ped Metab Dis,Gazi Univ Hosp, Ankara, Turkey, 2Div Ped Rad, Gazi Univ Hosp, Ankara, Turkey, 3Div Ped Rad, Gazi Univ Hosp, Ankara, Turkey

Background: The mucopolysaccharidoses (MPS) represent a group of inheritable, clinically heterogeneous lysosomal storage disorders, in which progressive accumulation of glycosaminoglycans (GAGs) can affect organs and tissues all over the body, including the skeletal and neurological systems. We aim to discuss the neuroimaging findings in MPS patients that are being followed in our clinic.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Methods: The cranial and spinal magnetic resonance imaging (MRI) findings of 25 MPS patients (5 MPS Type 1, 3 MPS Type 2, 4 MPS Type 4, 6 MPS Type 4, 7 MPS Type 6) were analysed. Results: Cranial MRI findings detected in all patients were hydrocephalus and J shaped sella. Cerebral atrophy, white matter signal changes and perivascular spaces were commonly encountered in MPS Type 1 and 3 patients, in addition to these findings, calvarial thickening was also seen in MPS Type 3. The most common finding in MPS Type 4 patients were spinal canal stenosis at the level of C1-2 vertebrea. White matter signal changes, perivascular spaces and spinal canal stenosis were also detected in MPS Type 6 patients. Discussion: MPS’s are chronic progressive and multisystemic lysosomal storage diseases. Although typical facial appearance and skeletal features eases the recognition of the disease, in subtypes with less prominent skeletal involvement, diagnosis may be delayed. Especially in patients with unexplained neurological complaints, findings of neuroimaging studies such as J shaped sella and hyrocephalus may be helpful in the diagnostic process.

A-078

Methods: We evaluated retrospectively six patients’ clinical presentation, physical examination and laboratory findings. The diagnosis was confirmed by molecular and/or filipin staining test. Results: Mutations on NPC1 gene were identified in the 6 patients, median age 5.4 years (range 2–13 years). All but one were admitted with jaundice in neonatal period. In addition to jaundice, the most prominent physical finding was splenomegaly whereas no neurological or dysmorphic findings were present. The mean values of laboratory on the presentation were; ALT 44 ± 40.1 U/L (16–132), AST 183.6 ± 147.7 U/L (69–502), GGT 217.2 ± 75.8 U/L (98– 315), total bilirubin 8.6 ± 4.6 mg/dL (0.4–15.9), direct bilirubin 5.6 ± 4.2 mg/ dL (0.3–12.2) and in all of them cholestasis had resolved by 6–9 months. All patients were put on miglustat therapy when the first neurological symptom presented (mean age of 3.7 ± 2.9 years). Two of them died at 3 and 3.5 years of age despite miglustat therapy. Discusssion: The infantile NP-C disease presentation usually includes unexplained neonatal cholestatic jaundice that is not so easy to diagnose at this period. Neonatal cholestasis with splenomegaly and lack of portal hypertension signs should raise the suspicion of NP-C. Neurologic symptoms are uncommon in this early period but patients should be followed carefully for the first signs of hypotonia and developmental delay.

De novo mutation in a patient with attenuated form of MPS Type II A-080 Kiykim E 1, Zubarioglu T 1, Cansever M S 1, Aktuglu Zeybek A C 1 1

Ist Univ Cerr Med Fac Div Nutr Metab, Istanbul, Turkey

Background: Mucopolysaccharidosis type II (MPS II), is a rare X-linked recessive disorder caused by a deficiency of the lysosomal enzyme iduronate-2sulfatase (IDS), leading to the progressive accumulation of glycosaminoglycans (GAGs) in several organs. The phenotypic spectrum of MPS II varies, with two typical clinical forms. The severe form is responsible for approximately 75 % of all MPS II patients, the attenuated form presents a slower progression of the disease, usually marked by fairly normal intelligence, hepatosplenomegaly, joint contractures and conductive and sensorineural hearing loss. Case Report: A 17 years old male patient was admitted to our clinic with complains of joint contractures. He was the third child of nonconsanguineous parents and his prenatal, natal, and postnatal history was uneventful. His physical examination revealed dolichocephaly with mild facial dysmorphism. He had hepatomegaly and joint stiffness especially distal interphalangeal joints and dysostosis multiplex was detected on X-ray. There is no corneal clouding or mental retardation (Iq:110) were detected. Results: The patienty had low iduronate-2-sulfatase activity and a novel mutation p.D275G (c.824A’G) on IDS gene detected. The segregation analysis was compatible with de novo mutation as it did not show in parents. Discusssion: In this case a de novo and novel mutation results with attenuated form of MPS type II was defined.

19. Lysosomal disorders: sphingolipidoses

A-079

Niemann-Pick Type C: a rare cause of neonatal cholestasis Caltepe G 1, Eren E 1, Demirbas F 1, Kalayci A G 1 1

Div Ped Gastroenterol, Ondokuz May Univ, Samsun, Turkey

Background: The clinical spectrum of Niemann-Pick type C (NP-C) is extremely heterogeneous ranging from a prenatal severe presentation with hydrops fetalis in utero to an adult-onset chronic neurodegenerative disease. NP-C also causes neonatal cholestasis without any neurological symptoms in early infancy. Early diagnosis is essential so that therapy with miglustat can be initiated as soon as neurological symptoms appear in order to slow the progression of the neurological damage. Here we report our experience with early-onset NP-C patients.

Combined Treatment of Enzyme Replacement Therapy and Substrate Deprivation Therapy in Two Chronic Neuronopathic Gaucher Type III Patients Ozer I 1 1

Div Metab Dis, Medeniyet Univ Child Hosp, Istanbul, Turkey

Background: Enzyme replacement therapy (ERT) is not effective in Gaucher Type III (GDIII). Substrate deprivation therapy (SDT) was combined with ERT in two GDIII patients and its success is discussed. Case Reports: First patient (β-glucosidase: 0.48 nmol/mg protein/h (nv: 5.4– 16.8); GBA gene:1172 T > C, homozygous mutation) presented at the age of 10 months and treatment was started with imiglucerase (40 u/kg/dose). Because of the onset of epilepsy at the age of 4 yrs and deafness at the age of 9 yrs, imiglucerase was increase to 60 u/kg/dose. As the symptoms persisted, a combined treatment of imiglucerase (40 u/kg/dose once a month) and miglustat (400 mg/day oral) was started at the age of 12 years. After 6 months, epilepsy seizures ceased; hepatosplenomegaly (HSM) resolved. Following a 3-month period during which the patient did not take the therapy, convulsions recommenced and she was hospitalized with status epilepticus. When her anticonvulsant was changed and the combined therapy was restarted regularly, the convulsions were stopped again. Second patient (β-glucosidase: 1.68 nmol/mg protein/h (nv: 5–18); GBA gene: L444P homozygote mutation). Symptoms started at the age of 7 months and treatment was started with imiglucerase (40 u/kg/dose) at the age of 15 months. The patient presented at the age of 7 years with epilepsy and the same combined therapy was started. In the first 12-month period during which the treatment was administered regularly, the convulsions were brought under control and HSM resolved. When the patient’s treatment was interrupted for 3 months, she developed HSM, thrombopenia, and vaginal hemorrhage. Results: With the combined therapy, HSM of both patients recovered completely, their neurologic findings were stabilized whereas all the findings deteriorated in non-treatment periods. Discusssion: Our experience with combined ERT and SDT shows that it may be beneficial in GDIII.

A-081

Gaucher disease with lung involvement in Algerian children. Hadji A 1, Sokhal S 1, Benali Khoudja N 1, Smati L 1, Hassoun F 1, Boukari R 1 1

Div Ped Dis, Univ Med, Algiers, Algeria

S268 Background: Pulmonary involvement has been described in all types of Gaucher disease (GD) but it is considered as relatively rare manifestation, restricted to patients with other severe manifestations. There are reports suggesting that homozygosity for L444P mutation in the GBA gene is associated with a substantial risk for developing primary pulmonary disease in GD. Methods: We describe our experience in 7 of 16 Algerian Gaucher disease patients in our clinic who presented with pulmonary signs or symptoms. The study included 7 GD patients with lung involvement who had complete data set including clinical features, chest radiography and high-resolution computed tomography (HRCT); pulmonary function tests (PFT), oxygen saturation (SaO2), and flexible bronchoscopy and bronchoalveolar lavage (BAL). Echocardiography was performed to exclude pulmonary hypertension and/or intra pulmonary shunts. Results: 7/7 (3 boys, 4 girls) 15 months old to 14 years old had symptomatic pulmonary involvment with chronic cough or infections. All of them are homozygous for the L444P mutation. Chest Xray in all patients showed diffuse interstitial infiltration in both lung fields; HRCT showed interlobular septal and intralobular interstitial thickening and bilateral ground-glass appearance without signs of respiratory infection. One of the patients had atelectasis. BAL analysis showed Gaucher cells in 3 patients. A 15-month-old child died of pneumonia despite enzyme therapy. With the diagnosis of progressive pulmonary involvement in Gaucher disease, we increased the dosage of imiglucerase from 60 to 80 U/kg/2 weeks in one patient. Discusssion: Primary lung disease in children homozygous for the L444P mutation in the GBA gene emerges as a significant clinical manifestation of GD with unclear response to ERT.

A-082

Acid sphingomyelinase deficiency (Niemann-Pick disease type A/B) in the Czech Republic Jahnova H 1, Poupetova H 1, Vlaskova H 1, Sikora J 1, Honzik T 1 2, Zeman J 1 2 1 Inst Inherit Metab Dis, Charles Univ, Prague, Czech Republic, 2Ped Dep, Charles Univ Hosp, Prague, Czech Republic

Background: Niemann-Pick type A/B disease is a rare autosomal recessive lysosomal disorder caused by mutations in the SMPD1 gene. As a result, acid sphingomyelinase deficiency (ASMd) develops. Original division into two clinically distinct forms, acute neurovisceral (type A) and chronic purely visceral (type B), proved to be inaccurate. In fact, ASMd phenotype constitutes a clinical continuum. Methods: Our observational retrospective study presents clinical and laboratory data of the Czech patients diagnosed with ASMd between the years 1971 and 2015 Results: ASMd was identified in 25 Czech patients from 20 families (19 males, 6 females). Only 4 patients, aged 2, 4, 20 and 51 years, are currently alive and followed up. Five patients were classified as type A, two patients manifested as type B, the rest of patients expressed the intermediate phenotype characterized by combination of visceral and neuropsychiatric symptoms after 2 years of age. All patients developed splenomegaly or hepatosplenomegaly, 6 lung disease, 13 cherry-red spot, 14 psychomotoric retardation/decline, 8 peripheral neuropathy, 6 epilepsy, 4 psychiatric disease. Uniquely, one patient suffered from severe valvular heart disease. Elevated chitotriosidase activity (median 836 nmol/ml/h) proved to be useful laboratory marker (performed in 9 patients). Analysis of the SMPD1 gene was performed in 23 patients. The prevalent mutation was p.Q294K, which was found (homo- or heterozygously) in 10 patients, all presenting with the intermediate phenotype. Discusssion: The birth prevalence of ASMd in the Czech Republic is expected to be 1:335 000. Most of the Czech patients presented with variable visceral and neuropsychiatric symptoms manifested from early infancy to late adulthood. Awareness of the extreme clinical variability of ASMd can contribute to early diagnosis and efficient family counselling. Grant RVO VFN 64165.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 A-083

Fabry disease and thrombocytopenia- is there a causal relationship? Stepien K M 1, Sharma R 1 1

Mark Holland Metab Unit, SRFT, Salford, United Kingdom

Background: Fabry disease (FD) (alpha-galactosidase A deficiency) is a lysosomal storage disease of globotriaosylceramide (Gb-3) occurring in cells of reticuloendothelial system of the bone marrow, lymph nodes, spleen and liver. Splenomegaly or thombocytopaenia is not regarded as a phenotypic manifestation of FD. We report three cases of thrombocytopaenia in a cohort of more than 200 patients with Fabry disease. Case Report & Results: Case 1- 62-year old female with FD (GLA gene: p.C90R) diagnosed in 2007 currently treated with Enzyme Replacement Therapy (ERT). She has been known to have predominantly vascular renal involvement on renal biopsy (eGFR 39 ml/min) and left ventricular hypertrophy. Platelets were gradually decreasing from 119 to 48 × 10^9/L over the last 2 years that was not associated with splenomegaly. Haemoglobin varied between 111 and 126 g/L and monocytes were raised at 1.7 × 10^9/L and she remains under the care of Haematologist. Case 2- 57-year old male with FD (GLA gene: p.R301Q) diagnosed in 2002 currently treated with ERT. He has left ventricular hypertrophy and implantable loop recorder. Renal functions are stable (eGFR >90 ml/min). Platelets fluctuated between 149 and 122 × 10^9/L over the last 6 years. Haemoglobin is 146 g/L and no other blood cell counts were affected. Case 3- 28-year old female with FD (GLA gene: p.R301Q) p.E7X) remains asymptomatic (eGFR > 90 ml/min). Her platelets ranged between 20– 50 × 10^9/L, haemoglobin 109 g/L, White Cell Count 3.9 × 10^12/L and lymphocytes 1.3 × 10^9/L. Panhypogammaglobulinaemia has also been identified. She is under the care of Haematologist and Immunologist. Discussion: The prevalence of peripheral blood cytopaenias other than anaemia in FD patients may be higher than previously recognised. Thrombocytopaenia despite no splenomegaly may be a relatively frequent manifestation of FD. However, it requires expert investigation to exclude other common haematological causes of cytopaenia.

A-084

The effect of Enzyme Replacement Therapy on lipid profile in patients with Fabry disease. Stepien K M 1, Jovanovic A 1, Hendriksz C J 1 1

Mark Holland Metab Unit, SRFT, Salford, United Kingdom

Background: Fabry disease, an X-linked genetic condition caused by alphagalactosidase deficiency, is associated with increased accumulation of glycosphingolipids in tissues and premature vascular disease. Hypercholesterolaemia (total cholesterol >4 mmol/L) is another recognised risk factor for cardiovascular disease in Fabry disease. Lipid profile in this condition has not been well defined but it is thought to be affected by cellular glycosphingolipids accumulation and subsequent inhibition of apoA-Imediated cholesterol efflux. As a result HDL-cholesterol may be raised above 1.2 mmol/L. The aim of this study was to assess the influence of Enzyme Replacement Therapy (ERT) with recombinant alpha-galactosidase A on lipid profile in patients with Fabry disease. Methods: From the cohort of patients with Fabry disease we selected 30 patients to analyse their lipid profile before and after the ERT. Fasting lipid profile was measured once a year as part of their annual blood test. We excluded patients who were treated with statins, had diabetes mellitus or alcohol excess. Changes in total cholesterol and HDL-cholesterol after alphagalactosidase treatment (1 mg/kg fortnightly) were analysed using two-tailed paired t-test. Results: Nine patients were taking statins. 21 patients (13 males, 8 females) were included in the analysis. The mean age at diagnosis of Fabry disease was

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 25.7 years (range 1–51 years) and the mean age at the onset of symptoms was 17.6 (range 2–51). 5 patients were smokers. Mean total cholesterol before and after ERT was commenced was 4.83 (SD ± 1.08) and 4.58 (SD ± 1.68) respectively (p = 0.735). Mean HDL-cholesterol before and after ERT was commenced was 1.57 (SD ± 0.38) and 1.68 (SD ± 0.34) respectively (p = 0.059). Discusssion: Fabry disease is associated with raised total cholesterol and HDLcholesterol. Although statistically non-significant, there was a noticeable decrease in total cholesterol and a marked rise in HDL-cholesterol after the longterm ERT. Conflict of Interest declared.

A-085

Memantine in the treatment of forgetfulness in Niemann Pick C disease Stepien K M 1, Bell L 1, Gabrielides A 1, McNelly B 1, McLoughlin M 1, Brocklehurst S 1, Hill A 1, Sharma R 1 1

Mark Holland Metab Unit, SRFT, Salford, United Kingdom

Background: Niemann Pick C (NP-C) is a progressive neurodegenerative condition caused by mutations in the genes NPC1 and NPC2. Patients with NPC may exhibit an inability to focus, forgetfulness, learning difficulties, slurred speech and loss of speech, as well as depression and even hallucinations. So far no drugs have shown to be effective in the treatment of poor memory. Memantine is a medication acting on the glutamatergic system by blocking NMDA receptors. It is a novel class drug used in Alzheimer’s disease and in dementia with Lewy bodies. It has been associated with a small positive effect on cognition, mood, behaviour and the ability to perform daily activities in moderate to severe Alzheimer’s disease. The aim of this project was to assess the effectiveness of memantine in our NPC patients’ cohort. Methods: We extracted Mini Mental Score Examination (MMSE) results in all patients with NPC. We analysed MMSE results in patients who are taking memantine and compared them with control group (patients who are not taking memantine). Results: Among 21 patients with NP-C, 5 were treated with memantine at variable dose (10 or 20 mg) for median 28 months (range 12–54). Among them, 3 patients had MMSE completed before they started memantine therapy and 2 of them had MMSE completed repeatedly. The median MMSE score before memantine therapy was started was 24 (range 23–25) and the median MMSE score was 26 (23–29) within a year since the drug was started. Among 14 control patients, 3 had MMSE completed repeatedly. Among these 3 patients, the score remained stable within a year (median 14 (range 9–20) vs. 18 (range 10–19) within 12 months). The medication is generally well tolerated. Discusssion: Potentially memantine may be helpful in treatment of NP-C dementia. Longer term follow up of our patients’ cohort is required to appreciate if memory remains stable, if not better, on memantine therapy.

A-086

Late onset GM1 gangliosidosis mimicking spinocerebellar ataxias and later extrapyramidal disorders Kostalova E 1, Poupetova H 1, Vlaskova H 1, Zumrova A 2 3 Inst Inher Metab Dis, Gen Univ Hosp, Prague, Czech Republic, 2Cen Hered Atax, Motol Univ Hosp, Prague, Czech Republic, 3Dept Child Neurol, Motol Univ Hosp, Prague, Czech Republic 1

Background: GM1 gangliosidosis is an autosomal recessive lysosomal storage disorder due to beta-galactosidase deficiency caused by mutations in the GLB1 gene. Mutations in the GLB1 gene cause both GM1 gangliosidosis and Morquio B disease which are allelic variants with phenotypic overlap. Patients with late onset GM1 gangliosidosis phenotypes usually present after the first year of life with progressive neurodegenerative impairment and mild or no skeletal deformities.

Case Report: The girl presented with unsteady gait after the age of 18 months. Brain MRI at 5 years of age showed mild cerebellar atrophy and white matter abnormalities. Between 5 and 14 years of age her gait became more ataxic. She had speech difficulties. Dystonia appeared at 15 years of age. MRI scans at 14 and 17 years of age detected basal ganglia signal abnormalities suspected of pantothenatekinase associated neurodegeneration, but pathogenic mutations in the PANK2 gene have not been identified. The 17-year-old girl had short stature, sternal protrusion, extrapyramidal and pyramidal signs, ataxia, dysarthria, dysphagia, oculomotor and cognitive abnormalities. Results: The elevation of plasma chitotriosidase activity was marker of lysosomal storage disease. The combination of neurodegeneration and skeletal abnormalities was suspected of GM1 gangliosidosis/Morguio B disease in spite of normal pattern of oligosaccharides and glycosaminoglycans in urine. The diagnosis was confirmed by low beta-galactosidase activity in leukocytes (5 nmol/h/mg, normal range 95–272) and in dried blood spot. The direct sequencing revealed compound heterozygosity for mutations c.464 T > G (p.L155R) and c.1657dupA (p.M553Nfs*32) in the GLB1 gene. Discusssion: GM1 gangliosidosis should be considered in the differential diagnosis in patients with cerebellar and/or extrapyramidal signs, especially in combination with skeletal abnormalities. Supported by MZCR-RVOVFN64165, MZCR-RVOFNM00064203/ 6005.

A-087

Homozygosity for p.R496H mutation in type 1 Gaucher disease. Irun P 1 2, Andrade-Campos M 1 2, Mingot-Castellano M E 3, Giraldo P 1 2 4 1 CIBERER, Instituto de Salud Carlos III, Zaragoza, Spain, 2Instituto Investigacion Sanitaria Aragon, Zaragoza, Spain, 3 Hospital Regional Universitario Malaga, Malaga, Spain, 4FEETEG, Zaragoza, Spain

Background: Type 1 Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the GBA gene. The R496H mutation has been reported previously only in combination with a different mutation on the other allele and classified as a very mild mutation. However, there are discordant conclusions between studies: some concluding that symptoms will most likely not develop in combination with N370S and others indicating disease manifestations including bone deformations. No neuronopathic disease has been reported even when R496H is in combination with a severe or null allele. The aim of this study is to describe clinical and biological characteristics of patients homozygous for R496H Methods: Diagnosis of GD was based on the determination of glucocerebrosidase activity (GC). Genotype was determined by amplifying the entire GBA gene by long PCR, which avoid amplification of pseudogene, followed by nested PCR and Sanger sequencing. Plasma chitotriosidase activity (ChT) was evaluated using a fluorogenic substrate and CCL18/PARC concentration by ELISA Results: In the Spanish GD Registry, we have identified two families whose GD index cases are homozygous for R496H. Family studies have allowed us to identify one more case in homozygosity and three carriers. The index cases are two women from unrelated families diagnosed with type 1 GD after the age of 40 years having GC activity below 26 % of healthy controls, mean ChT activity (5,479 ± 790 nmol/mL/h) and mean CCL18/PARC (622 ± 337 ng/mL). The third case detected in familiar studies has normal biomarkers values and GC (22 % of a healthy control). Only one patient, having bone disease with bone crisis and osteonecrosis in right femoral head, is under ERT and needs joint replacement. The other two patients are asymptomatic and out of therapy. In the two families there are relatives with different neurological disorders (Parkinson disease and dementia) Discusssion: The present study shows that the wide clinical variability of GD is also present in patients homozygous for p.R496H

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284

Ciana G 1, Ciana G 1, Sechi A 1, Dardis A 1, Malini E 1, Zanin I 1, Romanello M 1, Brondani G 2, Cattarossi S 1, Macor D 1, Bembi B 1

morbidities across patients. For patients with chronic visceral ASMD, earlier age at diagnosis was associated with greater morbidity. Analgesics, antibiotics, and respiratory drugs were the most commonly used medications. Discusssion: This chart review demonstrates significant disease burden and healthcare use in patients with ASMD. Diagnosis is often significantly delayed in patients with chronic ASMD. Funded by Genzyme Corp. Conflict of Interest declared.

1 Reg Coord Centre Rare Dis,Acad Hosp, Udine, Italy, 2Dep Diagn Imag Emerg Radiol, Acad Hosp, Udine, Italy

A-090

A-088

Long term efficacy and safety of velaglucerase in six patients with type 1 Gaucher disease previously treated with imiglucerase

Background: The temporary shortage of imiglucerase in 2009–10 forced drug dose reduction for patients with Gaucher disease type 1(GD1). In our experience, this was well tolerated by most patients, but a few complained of asthenia and bone pain. In 6 of our severely affected adult patients, it was possible to switch to velaglucerase under the aegis of a compassionate use programme administered by the Italian Agency for Drugs . Methods: The 6 adult patients included 3 females and 3 males. 4 patients had been splenectomized, 1 of whom had undergone liver transplantation. They received velaglucerase every other week at a dosage equal to that of imiglucerase administered before the shortage (ranging from 60 to 120 U/Kg/month). Data on blood count, bone pain, energy, abdominal and femoral magnetic resonance imaging and lumbar spine DEXA scores were collected retrospectively from clinical records, from the time of the drug switch for up to 44 months. Results: At the end of the follow up, all patients reported less asthenia, while bone pain improved in 3/6 patients. Hemoglobin increased more than 1.5 g/dl in one patient after 36 months, and remained stable (normal) in the other 5. Platelet counts decreased more than 15 % from baseline in 2 previously splenectomized patients (who started with values > 300,000/mm3), and remained stable in all the others. Liver and spleen volumes, bone imaging, and DEXAT scores did not change significantly. No side effects were reported. Discusssion: As recently shown by Elstein et al. (2015) in a longer follow up, velaglucerase was safe and able to maintain stability of the main indicators of the disease burden.

A-089

A retrospective natural history study of patients with acid sphingomyelinase deficiency (Niemann-Pick disease type A and B). Cox G F 1, Clarke L A 2, Giugliani R 3, McGovern M M 4 1 Sanofi Genzyme, Cambridge, United States, 2University of British Columbia, Vancouver, Canada, 3Medical Genetics Service, HCPA, Porto Alegre, Brazil, 4 Stony Brook Univ School of Medicine, Stony Brook, United States

Background: Acid sphingomyelinase deficiency (ASMD), also known as Niemann-Pick disease (NPD) type A and B, is a rare lysosomal storage disorder with a spectrum of severity ranging from fatal, infantile neurovisceral disease (NPD A) to an intermediate chronic neurovisceral form (NPD B variant) and a least severe chronic visceral form (NPD B). Methods: Burden of disease was assessed by abstracting clinical and healthcare use data from the medical records of 100 patients with infantile neurovisceral (13 %), chronic neurovisceral (6 %), and chronic visceral (81 %) ASMD. Results: Most patients were female (62 %), Caucasian (68 %), and from the Brazil (46 %) or US (41 %) sites. Median age at diagnosis was 0.6 yr (range 0.6– 1.7), 2.9 yr (range 0.4–42.8), and 6.0 yr (range 0.5–40.1) for infantile neurovisceral, chronic neurovisceral, and chronic visceral AMSD, respectively. Height/length and weight Z-scores decreased markedly over the first 5 years of life for all three forms. Developmental delay (92 %, 33 %, 15 %) and regression (54 %, 33 %, 1 %) were more common in the infantile and chronic neurovisceral than in the chronic visceral form, respectively. Overall, 54 % of patients underwent at least one surgical procedure. Patients with infantile neurovisceral disease had higher annual rates of hospitalizations (2.23, 0.42, 0.41/yr) and emergency room visits (0.72, 0.07, 0/yr) than patients with chronic neurovisceral or visceral ASMD. Platelet and WBC counts decreased over time in patients with chronic ASMD. Hepatic and respiratory disorders were the most common

GLB1 gene mutations: clinical, molecular, neuroradiological and neuropsychological presentation in three unrelated Italian patients. Fischetto R 1, Vendemiale M 4, Di Cuonzo F 3, Sesta M 1, Cornacchia D 4, Settembre M 4, Masciopinto M 1, Tummolo A 1, Lillo V 1, Burattini G 1, Piccinno E 1, Caciotti A 2, Morrone A 2, Papadia F 1 1 Mal MetabGenet Osp Ped, Bari, Italy, 2Lab Gen Mol Neurosc Osp Meyer, Firenze, Italy, 3Ist Neurorad Policl, Bari, Italy, 4Serv Psic Osp Ped, bari, Italy

Background: GM1 gangliosidosis (OMIM #230500) is an autosomal recessive disease characterized by a deficiency of the lysosomal enzyme β-galactosidase, encoded by the GLB1 gene. GLB1 mutations reduce or eliminate the activity of β- galactosidase, leading to substrate accumulation in many tissues, particularly the brain. Three clinical forms, based on age of symptoms onset and disease severity, have historically been used to classify patients as infantile, juvenile and adult forms. We present three Italian isolated patients with GLB1 gene mutations and different clinical presentation and outcomes. Case Report: Patient 1: presented in the 5th month of life with bronchopneumonias, hypotonia, organomegaly. GLB1 gene analysis revealed homozygous mutation c.1736 G > A (p.Gly579Asp). Central nervous system dysfunction: MRI showed generalized hypomyelinization, leading rapidly to spasticity, deafness, blindness and epilepsy. Integrated communication of the diagnosis was necessary to facilitate family adaptation to the evolution of the illness . Patient 2: presented at 9 months of age with vertebral dysplasia, normal anthropometry and neurodevelopmental milestones. GLB1 gene analysis identified compound heterozygosity for c.602G > A and c.841C > T mutations. MRI showed hyperintensity in the caudate nucleus. Miglustat was commenced at the 16th month of age. Parents were in denial of the diagnosis and tended to consider the disease a normal condition. Patient 3: presented at 36 yrs of age with a previous clinical diagnosis of GM1 juvenile form. Epilepsy, severe dystonia, absent ambulation and speech, and retinal red spots were present. GLB1 gene identified a compound heterozygosity for c.1736 G > A (p.Gly579Asp) and c.602 G > A (p.R201H) mutations. MRI showed generalized atrophy. Parents had already recognised the severity of the illness. Results: We observed, in our patients, different clinical, neuroradiological and neuropsychological outcomes and used markers for clinical progression, according to Tifft et al. (2015). Discusssion: We suggest early integrated communication of the diagnosis for developing adequate defensive strategies.

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GM1 gangliosidosis: a rapid diagnosis using THE enzyme measurement as confirmatory test in next-generation sequencing. Niezen-Koning K E 3, Van Veen-Hof I H 3, Van Spronsen F J 1, Derks T G J 1, Heiner-Fokkema M R 3, Diemen C C 2, Kerstjens W S 2, Sinke R J 2, SikkemaRaddatz B 2, Wijmenga C 2, De Koning T J 2 4 1

Beatrix Child Hosp,Univ Groningen, UMCG, GRONINGEN, Netherlands, Dept Genetics, Univ Groningen, UMCG, GRONINGEN, Netherlands, 3Lab Metab Dis, Univ Groningen, UMCG, GRONINGEN, Netherlands, 4Beatrix Child Hosp,Univ Groningen, UMCG, GRONINGEN, Netherlands 2

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: In modern clinical laboratory diagnostics more and more next generation sequencing (NGS) procedures are being used to diagnose inborn errors of metabolism. We present a case in which NGS indicated GM1 gangliosidosis. Quick confirmation was done with urine oligosaccharide analyses and enzyme activity measurements. In this way the diagnostic process could be further speeded up in this severely ill patient. Case Report: A patient, with only hepatosplenomegaly and severe dilated cardiomyopathy, was born from consanguineous Syrian parents. The patient died at age 3 months due to cardiac failure, shortly after the diagnosis was made. Whole genome sequencing and analysis of 2800 CGD related genes with subsequent phenotypic filtering of variants on HPO terms had been performed. Measurement of beta-galactosidase enzyme activity in leukocytes was used as the confirmatory test. Results: We identified a homozygous mutation (c.176G > A; p.R59H) in CLB1. In parallel abnormal oligosaccharides showed a pattern suggestive of GM1. Beta-galactosidase showed to be deficient (residual enzyme activity 1 %). Within a week there was a diagnosis and with this approach at least 2 weeks were gained in the diagnostic process. Discusssion: Classical biochemical tests and enzymatic assays in combination with NGS can further reduce the time to diagnosis in severely ill children. Moreover, instalment of a supportive therapy can be brought forward then. Furthermore, to assist NGS quick confirmation via enzymology is needed to get inside in the pathogenicity of the mutation involved. Our case, even though the patient died, is such example.

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Phenotypic and genotypic spectrum of Niemann-Pick A, B and C Disease: 2-year-experience of a local hospital

Background: Krabbe disease (KD) is a rare autosomal recessive lysosomal storage neurodegenerative disease, caused by a deficiency of galactocerebrosidase. In the most frequent infantile presentation (85–90 %) symptoms begin before 6 months of age. Methods: Retrospective review of the medical records of KD patients followed in CHUC Inborn Errors of Metabolism Reference Centre: 7 patients from 7 different families. Results: Three patients had the classical infantile presentation, with symptom onset between 2 and 4 months of age. Irritability, poor head control and developmental delay were the main features. Clinical deterioration was rapid, with seizures, feeding difficulties and death after 10–20 months. Two children had late-infantile forms, presenting at 6 months and 3-year-old with axial hypotonia and developmental delay/regression, progressing to spastic tetraparesis and optic atrophy, with typical symmetrical demyelination and corticospinal tract involvement in brain MRI.They are alive at 12 and 3-yearold respectively, with very poor quality of life. In the oldest patients symptoms started at 14 and 27-year-old (juvenile/adult forms) with spastic paraparesis and typical central nervous system MRI lesions. They are 40 and 33-years-old respectively, progressed to optic atrophy, and the oldest is tetraparetic. Family history was irrelevant in all cases. Enzymatic activity in leukocytes and/or fibroblasts was very low, but slightly higher in the third group. Mutational analysis identified 7 distinct genotypes (6 in compound heterozygosity), with 3 novel mutations. Discusssion: All patients had typical clinical manifestations. However, lateonset forms were predominant, supporting recent evidence that their proportion in KD may be underestimated. Typical brain MRI changes were present at clinical onset in late-onset forms, being an important clue for diagnosis. Awareness of this rare disorder is an important issue, since early diagnosis is crucial, as an effective therapy emerges.

Kilic M 1, Kasapkara C S 1 20. Lysosomal disorders: others 1

Div Metab Dis, Sami Ulus Child Hosp, Ankara, Turkey

Background: Niemann Pick (NP) A and B result from the deficient activity of acid sphingomyelinase, a lysosomal enzyme encoded by the SMPD1 gene. The central defect in NP type C is in intracellular trafficking of lipids. Up to now pathogenic variants in the NPC1 and NPC2 genes have been associated with this disorder. Methods: A total of six patients with Niemann-Pick A, B and C disease were studied. The diagnoses were confirmed with enzyme and/or molecular analyses. Results: The number of Niemann-Pick A, B and C patients were respectively 4/1/1. Hospital admission age ranged between 1 month and 7 years. Male/Female ratio was 3/3. Consanguinity rate was 4/6. Two of six patients (Niemann-Pick type A and Niemann-Pick type C) died before 2 years of age. Clinical findings were mongolian spots, coarse face, hepatosplenomegaly, liver involvement (hypertransaminasemia, cholestasis, coagulopathy, ascites, edema, jaundice), hyperlipidemia, macular dystrophy, cherry red spots, recurrent lower respiratory tracts infections, hypotonia, developmental delay, lung involvement and pancytopenia. Molecular testing identified homozygous mutations, c.416 T > C, c.573delT, c.967A > C, c.1267C > T, c.1327C > T in SMPD1 gene and c.352 G > T in NPC2 gene, respectively. The worst prognosis was in patients with NPC2 gene defects while the best in NP-B patients. Discusssion: Niemann-Pick disease should be kept in mind in the differential diagnosis of systemic symptoms with progressive brain, liver, spleen and lung involvement.

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Late-onset Krabbe disease might be more common than anticipated— Review of seven patients from a Portuguese Centre Duraes J 1, Carvalho J 1, Costa C 1, Caseiro C 2, Laranjeira F 2, Macario M C 1, Diogo L 1, Garcia P 1 1 Inb Err Metab Ref Cen, Cen Hosp Univ Cbr, Coimbra, Portugal, 2Inst Med Gen Jac Mag, Cent Hosp Porto, Porto, Portugal

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Late-infantile Metachromatic Leukodystrophy in a Palestinian girl: Report of a novel mutation in ARSA gene Dweikat I M 1, Alawneh I 1, Ashab M 2 1 Metabolic Unit, An-Najah Univ, Nablus, Palestine, 2Pediatric Dep, Makassed Hosp, Al-Quds, Palestine

Background: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal disorder caused by mutations in ARSA gene resulting in a deficiency of the enzyme arylsulfatase A (ASA). The late-infantile variant of MLD is the most frequent and severe phenotype. The aim of the study is to report a novel mutation in ARSA further expanding the genotype of this disorder Methods: Case study of a 30-month old Palestinian girl who presented with the typical phenotype of late-infantile MLD (psychomotor regression, progressive quadraparesis, ataxia, dysphagia, weakness with areflexia). Brain magnetic resonance imaging (MRI) showed the characteristic pattern. Genomic amplification of the whole ARSA gene exons and direct sequencing was performed on DNA extracted from peripheral blood sample Results: T2-weighted MR image demonstrates bilateral confluent areas of high signal intensity in the periventricular white matter with posterior predominance and sparing of the subcortical U fibers. Genomic amplification of the whole ARSA gene exons and direct sequencing performed on DNA extracted from peripheral blood sample showed that the patient is homozygous for the novel mutation R86G (p.Arg86Gly, c.256 C > G) in exon 2 and homozygous for the mutation T393C in exon 7 of ARSA gene. R86G mutation is a novel mutation detected for the first time in MLD patients. However, several other disease causing mutations such as R86W and R86Q were documented at the same codon. T393C mutation has been found to have a mild functional effect. Family segregation analysis confirmed that both parents and two siblings were heterozygous for the two mutations and the other two siblings were normal

S272 Discusssion: Since cloning of ARSA cDNA and gene, More than 150 mutations have been reported. Our report further expands the genetic spectrum of late-infantile MLD

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The three clinical cases of Cystinosis in Ukrainian patients Samonenko N 1, Shkurko T A 1, Olkhovych N V 1, Ivanova T 1, Pichkur N A 1

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Anti-failure medications were started as a standard management for heart failure. ERT with algucosidase alpha started for her at age of 13 months. Results: The patient is now at 3 years of age . All of her anti-failure medications were discontinued by the age of 2 years. Last echocardiography showed mild left ventricular hypertrophy with normal left ventricular function with a normal EF of 58 %. The frequency of her respiratory infections became far less over time. She tested negative for neutralizing antibodies against the ERT. Discussion: Better response to ERT in CRIM positive patients with Pompe disease has already been shown in several studies. Our patient has shown notable improvement with ERT and clinical evidence ofsustained response.

1

Cen Orph Dis Nat Child Hosp Ohmatdyt, Kyiv, Ukraine

Background: Nephropathic cystinosis is an inherited lysosomal storage disorder caused by defective transport of the amino acid cystine out of lysosomes. The stored cystine is poorly soluble and crystallizes within the lysosomes of many cell types, leading to widespread tissue and organ damage. Nephropathic cystinosis is further subdivided into infantile and late-onset (intermediate cystinosis). Symptoms are usually restricted to kidneys (eg, less severe form of Fanconi syndrome, proteinuria) and eyes (eg, photophobia). Nonnephropathic cystinosis is considered a benign variant and is usually diagnosed by an ophthalmologist treating patients for photophobia. Case Report: There are three cases of patients with cystinosis. The first two patients are siblings. Older brother 6 years old fell ill at the age of 1 year with the presentation of Fanconi renal syndrome. He had vomitingin the first 5 months of life, and from after year he had Fankoni syndrome with hypophosphatemia, metabolic acidosis, hypophosphatemic rickets, increasedrenal excretion of glucose, amino acids, grows retardation. In four years he formed renal failure. His brother had first symtoms at 10 month: proteinuria, glucosuria, hypophosphatemia and hypophosphaturia. Both demonstrated typical phenotype: blond hair and blue eyes. Photophobiaand cornealcrystals there are not found. Diagnosis was established by DNA analysis in gene CTNS. The third patient showedgrowthretardation, malabsorption syndrome, vomiting, metabolicacidosis, photophobia. The hypophosphatemia started after 4 years of illness with glucosuria and aminoaciduria. Results: The specific therapy was assignedwith good results. Discusssion: Cystinosis is a progressive disease with different clinical manifestations and affects many organs and systems. So that early correct diagnosis is very important for start of specific therapy.

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A Favorable response to enzyme replacement therapy in a patient with infantile Pompe disease associated with positive cross reactive immunological material status Al Mugheri A H H 1, Al Thihli K S S 1 1

Gen Dev Med, Sult Qab Univ Hosp, Muscat, Oman

Background: Pompe disease is an autosomal recessive glycogen storage disorder leading to generalized hypotonia, feeding difficulties, cardiomyopathy leading to early death in its most sever classic infantile form if left untreated. Enzyme replacement therapy (ERT) has resulted in decreased disease progression and improved survival. Among the most important factors affecting response to therapy is the cross reactive immunological material (CRIM) status. Here, we describe a favorable response of a CRIM-positive patient with Pompe disease treated with ERT at Sultan Qaboos University Hospital. Case Report: A girl at 9 months old was referred for evaluation of developmental delay,hepatomegaly,hypotonia and hypertrophic cardiomyopathy. Her past medical history then was significant for recurrent chest infections requiringmultiple admissions. Echocardiography showed Hypertrophic nonobstructive cardiomyopathy,with dilated left ventricle with moderate to severe reduction in ventricular systolic function,with an Ejection Fraction (EF) of 32 %. She was found to have low alpha glucosidase enzyme activity. GAA gene sequencing showed homozygous c.2015G > A. mutations confirming the diagnosis of Pompe disease. She tested positive for CRIM.

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Awareness of the family physicians on lysosomal storage diseases Kose E 1, Bulbul S 2, Arslan N 1 1 Div Metab Dis, Dokuz Eylul Univ Hosp, Izmir, Turkey, 2Div Metab Dis, Kirikkale Univ Hosp, Kirikkale, Turkey

Background: Because of the changing nature of the Inherited Metabolic Diseases, specially the Lysosomal Storage Diseases (LSDs), diagnosis is frequently delayed. Identification of affected patients depends mostly on the awareness of the physicians. As part of a project conducted to increase the awareness on IMDs, a preliminery study that was presented in SSIEM 2015 showed that, the knowledge of the physicians in general especially the family physicians was not in the desirable level. Due to the treatment chance for some LSDs early diagnosis is important. Therefore, this study was specified on the knowledge of LSDs of the family physicians, those first to face these patients. Methods: A questionnaire probing general knowledge on LSDs and a case of Gaucher Disease (GD, the most frequently seen LSD in Turkey) was prepared. It was sent via e-mail to 230 family physicians and only 73 (31.7 %) replied. Results: The mean age of the family physicians was 36.1 ± 8.4 years and the mean working years as a family physician was 5.5 ± 4.8 years. 90.4 % did not work at any metabolic disease department and 72.6 % have not seen any LSD patient before. To the question “Do you know any LSD?” 35.6 % answered “No”. When it was asked them to score their knowledge between 1 and 10, the mean score was 2.9 ± 1.6. Only 47.6 % could write at least one LSD and the given GD case was correctly diagnosed by only 5.4 % of the group. Discusssion: It is important for all clinicians to have knowledge about these rare disorders inorder not to underestimate the patients who attends with variaty of nonspecific symptoms. We believe the importance of informing the family physicians who are the first to see these patients. The results of the study gave us way how to work with the clinicians.

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The case of Pompe disease with late onset of the Russian girl athlete. Savostyanova E 2, Godina E 2, Silaeva L 2, Pushkov A 1 1 Scientific Center of Children’s Health, Moscow, Russian Federation, 2Univ Physical Education Sport Youth Tour, Moscow, Russian Federation

Background: Pompe disease is a rare multisystem genetic disorder that is characterized by absence or deficiency of the lysosomal enzyme alphaglucosidase (GAA). Pompe disease is a single disease continuum with variable rates of disease progression and different ages of onset. The late-onset Pompe disease forms are subtle and may go unrecognized for years. Methods: Screening for Pompe disease was performed among 500 Olympic Reserve School athletes. The level of the alpha-1,4

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 glucosidase activity was measured by tandem mass spectrometry in all patients enrolled in the study. All coding exons and adjacent intronic regions of GAA gene were examined by Sanger sequencing if the level of enzyme activity was decreased. Results: In two cases, the analysis of alpha-1,4 glucosidase activity showed a decreased enzyme activity - lower than the cut-off value of 1.62 micromoles/liter/hour. In a 14 year old girl we demonstrated two polymorphisms c.1726G > A and c.2065G > A in the homozygous state, which were already described in patients with decreased enzyme activity but without clinical manifestations of Pompe disease. The second patient, a 16 year old girl, was a compound heterozygote for mutations c.-3213 T > G described in patients with late-onset Pompe disease and the less severe c.2014C > T (p.Arg672Trp). Interestingly, at the time of the survey no clinical manifestations of Pompe disease were present. Discusssion: Late onset Pompe disease is a slowly progressive disease, thus, as we have shown, screening for this nosology can also reveal patients among subjects at presumed low risk.

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Results Case 1: An 11-year old girl presented with hepatosplenomegaly, growth failure, abdominal and bone pains. Diagnosis of Gaucher disease (GD) type 1 was made with enzymatic analysis (low β-glucosidase, elevated chitotriosidase) and Gaucher cells in bone marrow aspirate. Treatment with Imiglucerase is provided since 2014. Case 2: A 3-day old newborn presented with cholestasis, thrombocytopenia and hepatosplenomegaly. Enzymatic and molecular analysis confirmed the diagnosis of GD type 2. The child was homozygous for two mutations p.[H294Q];[D448H]. The clinical course showed progressive liver deterioration (severe cholestasis, ascites, portal hypertension) and death occurred at age of 2.5 months. Only symptomatic treatment was provided. Case 3: A 7-month old infant was referred for hepatosplenomegaly and facial dysmorphia and diagnosed with late-infantile Niemann -Pick type C disease. Genetic studies showed homozygous mutation c.2819C > T in NPC1 gene. There was progression of hepatosplenomegaly and psychomotor deterioration. Treatment with miglustat is expected to be initiated. Discussion: All presented patients showed unique clinical course and outcome. Neonatal cholestasis is an extremely rare presenting sign of GD type 2. Case 3 presents the first child in Macedonia diagnosed with Niemann-Pick type C. Establishing diagnosis of LSDs is very difficult because of the unavailability of specific investigations in our country. However, LSDs should be considered in the diagnostic approach of hepatosplenomegaly.

Farber disease: a case report with a novel mutation Onenli Mungan N 1, Bulut F D 1, Seker Yilmaz B 1, Kor D 1, Ceylaner S 2

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1 Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Intergen Genetic Lab, Ankara, Turkey

Infantile sialic acid storage disease: a novel mutation in the SLC17A5 gene

Background: Farber disease is a very rare lipogranulomatosis with heterogeneous clinical features due to the deficiency of the enzyme acid ceramidase. Subcutaneous nodules, joint swelling, contractures, hoarseness, and neurological involvement are the frequent signs and symptoms. Mutations in ASAH1 gene lead to the enzyme deficiency and the clinical findings. Here we report the second Farber case of our Metabolic Center with the same novel mutation. Case Report: A 3 months old female infant admitted to the hospital with complaints of subcutaneous nodules, restlessness, and inadequate nutrition. Parents were first degree cousins and her two cousins died with same complaints without a specific diagnosis. She had moderate hypotonia, corneal clouding, nystagmus, umbilical hernia, subcutaneous nodules, and a large mongolian spot. Hydrocephalus was detected in cerebral computerized tomography. Results: Mutation analysis revealed a novel homozygous mutation p.C31F (c.92G > T) on ASAH1 gene which is compatible with classic Farber disease. Her parents are heterozygous for the mutation. Interestingly, this mutation had only been described in our other Farber case. Discusssion: Here we report a case of Farber lipogranulomatosis, as it is a very rare disorder, and the mutation detected in this case has only been described in our other Farber case who had the similar signs and symptoms and died at the age of 12 months.

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Kor D 1, Seker Yilmaz B 1, Bulut D 1, Bisgin A 3, Incecik F 2, Onenli Mungan N1 1

Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Div Ped Neu, Univ Cukurova, Adana, Turkey, 3Div Genetic Dis, Univ Cukurova, Adana, Turkey Background: Infantile sialic acid storage disease (ISSD) is an autosomal recessive lysosomal storage disorder caused by mutations in the SLC17A5 gene and characterized by the accumulation of covalently unlinked (free) sialic acid in multiple tissues. The clinical features are severe developmental delay, coarse facial features, cardiomegaly, and hepatosplenomegaly. Death usually occurs in early childhood. Diagnosis of ISSD is based on clinical findings, biochemical assays of sialic acid and molecular analysis. Management is symptomatic and supportive. Here we report a patient with a novel homozygous mutation in the SLC17A5 gene. Case Report: A 17 month-old-male patient, a first child of consanguineous parents, presented with failure to thrive, psychomotor retardation, hypotonia, coarse face, large Mongolian spots, and hypothyroidism. His routine hematologic, biochemical and metabolic tests were within normal limits except for secondary hypothyroidism. Urine oligosaccharide analysis showed markedly increased excretion of free sialic acid (948,8 μmol/mmol creatinine). Results: These clinical and biochemical results indicated ISSD and the diagnosis was finally confirmed by molecular analysis of SLC17A5 gene, showing the homozygous p.G209E (c.626G > A) mutation. This mutation has not been reported previously. The parents were heterozygous for the same mutation. Discusssion: We report this case to emphasize considering ISSD in the differential diagnosis of other more common lysosomal storage diseases.

Lysosomal storage diseases in Macedonian children– report of three cases referred for hepatosplenomegaly Kostovski A 1, Zdraveska N 1

21. Lysosomal disorders: treatment, enzyme replacement therapy

1

Univ Child Hosp, Medical Faculty, Skopje, The F.Y.R of Macedonia

Background: Lysosomal storage diseases (LSDs) encompass >50 different inherited metabolic diseases due to deficiency of lysosomal catabolic enzymes, each characterized by the accumulation of specific substrates. Many LSDs might affect the liver usually manifesting with elevated transaminases and hepatosplenomegaly. Case Report: 3 patients with LSDs diagnosed at Gastroenterohepatology Department initially referred for hepatosplenomegaly will be presented.

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Clinical observation of 53 treatment naïve Fabry patients Tsuboi K 1, Yamamoto H 1, Goto H 1 1

LSD Center, Nagoya Central Hospital, Nagoya, Japan

S274 Background: Fabry disease is a rare metabolic storage disease resulting from deficient activity of the lysosomal α-galactosidase A, which leads to disordered glycosphingolipid metabolism and accumulation of the lipid substrate globotriaosylceramide (Gb3). Two enzyme replacement preparations are available, agalsidase alfa (Replagal) and agalsidase beta (Fabrazyme), which are produced using different methods. Agalsidase beta is produced in CHO cells and agalsidase alfa is produced in human cells. Methods: In this study, we investigated the efficacy and safety of the ERT in 53 treatment naïve Fabry patients. 36 naïve Fabry patients, who received the ERT with agalsidase alfa were enrolled into the clinical trial (Naïve study alfa) and 17 naïve Fabry patients, who received the ERT with agalsidase beta were enrolled into the clinical trial (Naïve study beta). Results: We regularly measured plasma Gb3, Lyso-Gb3, LVMI, BNP, eGFR, and also investigated the incidence of adverse events, appearance of infusion reactions in all patients. For the efficacy, plasma Gb3/Lyso-Gb3 decreased during the ERT, and the cardiac functions and renal functions were mostly maintained to be stable. However, the disease continuously progressed in the patients who were observed to be already compromised at the baseline. This shows that an early start of the ERT is most likely to be effective. For the safety, the incidences of administration-related reactions was low, suggesting good tolerability. Discusssion: As the number of patients was limited in this study, it is necessary to analyze the results carefully. However, these results suggested the efficacy and safety of the ERT, agalsidase alfa 0.2 mg /kg and agalsidase beta 1.0 mg /kg were equivalent. Agalsidase beta and agalsidase alfa are different drugs with different origins, therefore agalsidase beta 1.0 mg /kg is not a high dose and agalsidase alfa 0.2 mg /kg, a low dose.

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Treatment for Anderson-Fabry Disease Peripheral Neuropathy with Adeno-Associated Virus Vector Encoded human α-Galactosidase A Gene in Murine Fabry Disease Model Higuchi T 1, Shimada Y 1, Kobayashi H 1 2, Ida H 1 2, Ohashi T 1 2 1 Div Gene Thera, The Jikei Univ, Tokyo, Japan, 2Dep Pedi, The Jikei Univ, Tokyo, Japan

Background: Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutation of the α-galactosidase A (GLA) gene resulting in deficient activity of GLA. This causes storage of various glycolipids including globotriaosylceramide (Gb3) in many tissues. An enzyme replacement therapy (ERT) using recombinant human GLA (rhGLA) has been developed and improves various clinical, pathological and biochemical parameters of FD. Most of the neuropathies in FD patients present in a “glove and stocking type” distribution with sensory abnormalities. Fabry neuropathy is a small fiber neuropathy (SFN) showing acute severe burning pain sensation, called Fabry pain crisis or acroparaesthesia and/or chronic pain. ERT efficacy is not sufficient for treatment of Fabry pain. Methods: We studied the therapeutic effect of adeno-associated virus mediated gene transfer for Fabry acroparaesthesia. We performed treatment of murine FD model (6 w.o.) with adeno-associated virus serotype 10 (AAV rh10) vector encoded human GLA gene injected i.v. (5 × 1011 v.g.c./kg). Results: Three weeks after injection of the AAV vector, GLA activity was dramatically increased in heart, liver, and spleen, and moderately increased in central nervous system (cerebrum and cerebellum), and peripheral nervous system (DRG in the lumbar region) (2 ~ 3 fold). The Murine Fabry disease model showed dysesthesia compared to Wild-Type C57BL/6 J mouse. By von Frey up-down test, dysesthesia were marginally improved in AAV treated mice compare to non-treated FD mice. Discusssion: Our data indicates that gene therapy using AAV rh10 encoding hGLA gene could be a new therapeutic approach for Fabry peripheral neuropathy. Conflict of Interest declared.

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 A-104

Successful unrelated cord blood transplantation in newborn sibling with infantile Krabbe disease Ishige M 1, Yagasaki H 1, Ogawa E 1, Takano C 1, Sakai N 2, Yamamoto T 3, Terui K 3, Chin M 1, Mugishima H 1, Takahashi S 1 Dept Pediatr, Nihon Univ, Tokyo, Japan, 2Dept Pediatr, Osaka Univ, Suita, Japan, 3Dept Pediatr, Hirosaki Univ, Hirosaki, Japan 1

Background: Krabbe disease is an autosomal recessive disorder characterized by demyelination in the central and peripheral nervous system due to deficiency of galactosylceramidase. In the infantile form, the symptoms appear before 6 months and the patients usually die within the first 2 years of life. Early hematopoietic stem cell transplantation in asymptomatic newborns is recommended for good neurologic improvement. Here, we report sibling cases with infantile Krabbe disease who underwent successful unrelated cord blood transplantation (CBT) on days 42 and 40. Case Report: Case 1 was an elder brother and Case 2 was a younger sister. Their brother died of Krabbe disease at 1.9 years of age. Their enzyme activity level in peripheral blood lymphocytes at birth was 0.09 nmol/mg/h and 0.05 nmol/mg/h (normal range, 1.0–2.0) respectively. They presented no signs of neurodevelopmental delay and a cranial magnetic resonance image study was also normal. The protein levels in central spinal fluid (CSF) increased to 330 mg/dl and 482 mg/dl (normal range, 10–40) respectively. Results: They received 7/8 and 5/8 allele-matched cord blood units, which included the total nucleated cells of 1.44 × 108/kg and 4.79 × 108/kg. The conditioning regimen consisted of fludarabine, citarabine, busulfan and melphalan. Neutrophil engraftment with full-donor chimerism was achieved on day 18 (Case 1) and day16 (Case 2) and no acute and chronic GVHD was observed. Neither life-threatening infection nor transplant-related organ damage occurred. The enzyme activity increased to 0.6 nmol/mg/h and 1.1 nmol/ mg/h, and the protein levels in CSF improved to 199 mg/dl and 298 mg/dl at 3 months of age respectively. At present, Case 1 is confined to a wheelchair, but without mental retardation at 7 years of age. Case 2 is well doing at 11 months of age. Discusssion: Early CBT with this conditioning regimen was feasible and possibly improved neurological outcomes as well as survival in infantile Krabbe disease.

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Effect of 12 years of enzyme replacement therapy on plasma and urine glycosaminoglycans in attenuated mucopolysaccharidosis I patients Zampini L 1, Padella L 1, Santoro L 1, Volpi N 2, Maccari F 2, Fiumara A 3, Giovagnoni A 1 1

Dep Odont Spec Cl Sc, UNIVPM, Ancona, Italy, 2Dep Life Sc, UNIMORE, Modena, Italy, 3Dep Ped, Univ Catania, Catania, Italy Background: To date, only few data are available on the capacity of ERT at standard doses to definitively eliminate pathological glycosaminoglycans (GAGs). We report a characterization of urine and plasma GAGs performed in order to assess the effect of ERT after 12 years in two attenuated MPS I siblings. Case Report: The brother (sibling1) commenced weekly laronidase infusions at a dose of 0.5 mg/kg at the age of 5 months and the sister (sibling 2) at the age of 5 years, shortly after the diagnosis. Urine samples were analyzed by DMB and electrophoretic methods. Plasma GAG disaccharides were evaluated by HPLC. RESULTS Sibling1 Total urinary GAGs excretion was slightly above the upper normal limits for age during the last 6 years. Urinary GAGs electrophoresis showed

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 the presence of DS and CS with a ratio of about 50/50. Plasma GAGs before ERT were 18.2 μg/ml (nv 5.27 ± 3.01) with a CS/DS ratio of 25/75 (nv 100/0). After 12 years of ERT, plasma GAGs have remained within the normal range for age, with traces of DS. Sibling2 Total urinary GAGs excretion was elevated before ERT and normalized after 4 months of therapy until 8.5 years of age. At the age of 10.5 years GAGs levels increased to 89 μg/creat (nv 37 ± 18) and subsequently normalized. Urinary GAGs were characterized by the presence of DS and HS before ERT and normalized after 4 months of ERT up to the age of 6 years when a ratio of 50/50 of DS/CS was observed. Plasma GAGs at the age of 11.5 years were 2.8 μg/ml (nv 2.7 ± 1.12) with a CS/DS ratio of 98/2 (nv 100/0). Discusssion: Biochemical observation of these siblings reveals a moderate increase of total urinary GAGs and more notably DS later in childhood. Normal levels of plasmatic GAGs were observed with only traces of DS. No abnormal levels of HS present before ERT were detected in urine and plasma. Based on the present results, we can suppose that the amount of supplied enzyme could contribute to the insufficient GAGs degradation.

A-106

The bone status in children with Gaucher’s disease.

B a c k g r o u n d : We p r e s e n t a 9 - y e a r f o l l o w - u p o f a b o y w i t h mucopolysaccharidosis type II (MPS II) who had idursulfase therapy initiated at the age of 3 months and compare his clinical course to his healthy twin brother as well as to his affected, elder sister who began enzyme replacement therapy at the age of 6 years. Methods: Detailed anthropometric features, ultrasound studies of liver and spleen volumes, echocardiography and psychological tests, joint range of motion (ROM) and skeletal radiographs were carried out in two siblings. In healthy twin brother anthropometric parameters and psychological tests were performed at the same time. Results: After 9 years of treatment, the patient has not developed any evident clinical manifestations of MPS II. He did not develop coarse facial features or organomegaly, his cardiac function remained normal. At the age of 9 years, the patient does not present body disproportion associated with MPS II, has normal height (75percentile) with the only disproportion appearing in head proportion—head circumference). There is a minor restriction of movement in the hip joints. The only difference when compared with his healthy twin brother is lower IQ (Termann-Merrill 104 vs. 121) . Treatment started at 6 years of age in his older sister did not result in clinical improvement. Discusssion: Our study suggests that early initiation of enzyme replacement therapy may significantly slow or even prevent the development of disease manifestations and therefore completely change the natural history of MPS II.

Nikitina A 3, Bergaliev A 3, Bogdanova S 3, Vissarionov S 3, Kostik M 1, Larionova V 2 3 A-108 1

State Pediatric Medical University, Saint-Petersburg, Russian Federation, 2 Center For Rare Dis and Mol Medicine, Saint-Petersburg, Russian Federation, 3The Turner Sci Res Inst for Child Orth, Saint-Petersburg, Russian Federation Background: Gaucher’s disease (GD) type I is caused by mutations in the gene encoding the enzyme glucocerebrosidase which leads to decreased function. The main current treatment strategy is an enzyme-replacement therapy (ERT). The bone involvement in GD is frequent. It can appear in bone fractures and secondary bone deformities, so early identification of GD is crucial. The degree of bone involvement determines the therapeutic dosage of ERT. Case Report: We examined 3 sibs with GD (boys 11 and 5 years old and girl 10 years old) confirmed by genetic and biochemical tests. We used the bone MRI, dual-energy-X-ray absorptiometry (DEXA) of total body (TB) and lumbar spine (LS), biochemical markers of bone and mineral metabolism: P1NP-the marker of bone synthesis and β-Cross Laps – the marker of bone resorption. Results: We have not found any specific MR-changes in bone in all cases. All children had linear growth failure. All children had received ERT in standard doses with imiglucerase. The data of DEXA in 11 yo boy: TB and LS was normal, in 10 yo girl TB DEXA was normal, LS DEXA was decreased, bone mineral density = −1.3 SD, in 5 yo boy TB and LS were both decreased and were −1.3 SD and −1.5 SD, accordingly. All children despite the data of DEXA had similar bone metabolic changes and showed a decreased rate of bone remodeling. 11 yo boy had P1NP – 632,4 ng/ml (651,8 – 919,6) and βCross Laps 1,18 ng/ml (1,63 – 1,94), 10 yo girl had P1NP – 490,4 ng/ml (670,2 – 1042,5) and β-Cross Laps 1,34 ng/ml and 5 yo boy had P1NP – 507,7 ng/ml (667,1 – 975,3) and β-Cross Laps 1,17 ng/ml (1,55 – 1,77). Discusssion: All children had stereotypical changes in bone metabolism— decreased bone synthesis and bone resorption despite the data of bone mineral density. We suggest that decreased bone metabolism is a reason for linear growth failure and the increased doses of ERT are possibly needed.

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Enzyme replacement therapy for mucopolysaccharidosis type II from 3 months of age: 9-year follow up Tylki-Szymanska A 2, Rozdzynska-Swiatkowska A 1, Marucha J 2, Lugowska A 3 1 Anthrop Lab, CMHI, Wasaw, Poland, 2Dep of Ped, Nutrit and Metab Dis, CMHI, Warsaw, Poland, 3Dep of Gen, Inst of Psych and Neuro, Warsaw, Poland

Infantile Pompe disease (IPD) presenting with severe fetal-neonatal arrhythmia: rapid cardiac response to weekly high dose enzyme replacement therapy Ceglie T 1, Pagliardini V 1, Biamino E 1, Ricci F 1, Banaudi E 2, Porta F 1, Spada M 1 1

Depart of Pediatrics, University, Turin, Italy, 2Div Cardiology, Reg Margh Child Hosp, Turin, Italy Background: Pompe disease is an inherited disorder characterized by lysosomal acid alpha-glucosidase (GAA) deficiency, resulting in progressive glycogen storage in myocardium and skeletal muscle. The natural history of IPD without treatment is fatal cardiorespiratory failure usually within 1 year. Enzyme replacement therapy (ERT) with recombinant human GAA (rh-GAA) is now available and the identification of patients at an early stage of the disease becomes mandatory. Current ERT regimen is based on the administration of 20 mg/kg/eow, but in a subset of IPD patients this dosage might be not optimal to halt the progression of the disease. Case Report: A boy was born at 38 weeks of gestation complicated by a bradycardia revealed at 29 weeks by US examination. At birth he presented with respiratory distress, hypotonia, and bradycardia associated with diffuse high QRS voltage complexes, ST-T changes and short PR interval at ECG. As echocardiogram showed hypertrophic cardiomyopathy (interventricular septum diastolic wall thickness: 10.2 mm), screening for IPD was performed, revealing no residual GAA activity in dried blood spot. CRIM status resulted positive. Repeated episodes of paroxysmal supraventricular tachycardia (PSVT) occurred in the following days unresponsive to adenosine and vagal nerve stimulation and requiring frequent cardioversion procedures. ERT therapy with a high dose regimen of 40 mg/kg administered every week was started at the age of 14 days. Results: A progressive cardiac improvement characterized by the reversal of the cardiomyopathy within the age of 3 months and the disappearance of PSVT at the age of 4 months was observed, allowing the withdrawal of antiarrhythmic drugs. At the last follow-up (14 months), the patient walks independently and shows normal echocardiographic and ECG findings. Discusssion: Higher dosage regimen associated with very early initiation of ERT may have a dramatic positive impact on the short term outcome in IPD patients.

S276 22. Glycosylation disorders/CDG, protein modification disorders

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Neonatal gastrointestinal involvement associated with a significant decrease of coagulation factors revealing MPI-CDG (CDG-Ib) Abily-Donval L 1, Guemann A S 2, Torre S 1, Auger M 1, Mouterde O 3, Pinquier D 1, Dupre T 5, Seta N 5, Pinto-Cardoso G 1, Marret S 1, Bekri S 4, Girard M 6, De Lonlay P 2 1 Dept Neonatal Medicine, Rouen Univ Hosp, Rouen, France, 2Ped Metab Dis Dept, Necker Univ Hosp, Paris, France, 3Dept of Med Ped, Rouen Univ Hosp, Rouen, France, 4Dept of Metab Bioch, Rouen Univ Hosp, Rouen, France, 5 Dept of Bioch, Bichat Univ Hosp, Paris, France, 6Ped Hepatology Unit, Necker Univ Hosp, Paris, France

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 diagnosed at birth via ABR testing. He developed port-A-cath thrombosis, and at 13 months of age edema secondary to hypoalbuminemia in the setting of mild hepatic enlargement. He suffered from frequent status epilepticus and recurrent infections, which led to his death at 15 months of age. Results: LC/MS of serum transferrin showed a CDG type 1 pattern, and serum Nglycan analysis by MALDI-TOF/TOF revealed the presence of tetrasaccharide NeuAc-Gal-GlcNAc2, a biomarker of ALG1-CDG. ALG1 analysis showed one pathogenic mutation c.773C > T and the VUS c.208 + 13G > A. In silico characterization of the VUS predicted creation of new splice donor site. Discusssion: We describe an ALG1-CDG patient with compound heterozygosity for mutation S258L in ALG1 and a novel variant c.208 + 13G > A. In addition to typical clinical features, he exhibited hearing loss due to auditory neuropathy, and thrombosis, previously unrecognized clinical features of ALG1-CDG. Hypoalbuminemia due to synthetic liver dysfunction, recurring status epilepticus and infections may be poor prognostic features and have been previously associated with the S258L mutation.

A-111 Background: MPI-CDG (CDG-Ib) is a glycosylation disorder characterized by a homogeneous clinical presentation: liver and gastrointestinal damage, moderate hyperinsulinemia and thrombotic risk. We report a case of MPI-CDG with an unusual presentation. Case Report: This newborn was hospitalized at 3 weeks of life for diarrhea and edema. He had many liquid stools per day with weight stagnation. Dietary measures were ineffective. Physical examination revealed pallor, splenomegaly without hepatomegaly and edema. Laboratory tests showed hypoproteinemia (23 g/L) with hypoalbuminemia, moderate hepatic cytolysis (ASAT: 83 UI/L and ALAT: 74 UI/L). Serum GGT, bilirubin, blood ammonia and renal function were normal. The hypoalbuminemia was found to be related to protein-losing enteropathy. On the fortieth day, a decrease was noted of the prothrombin time (TP: 28 %), factor V (16 %), factor II (18 %), factor VI (19 %) and factor X (29 %) without any increase of cytolysis or cholestasis. The patient presented with uncontrolled hypoglycemia related to hyperinsulinism and episodes of central catheter-related thrombosis. Persistence and progressive worsening of symptoms led us to raise the hypothesis of mannosephosphate isomerase deficiency (MPI-CDG). Mannose treatment was started promptly at a dose of 0.17 g/kg 5 times per day and resulted in a quick clinical and biological improvement. Results: The diagnosis of MPI-CDG was confirmed by finding serum glycoprotein hypoglycosylation and a deficient activity of mannosephosphate isomerase. Discusssion: Liver disease in MPI-CDG is characterized by bile duct dystrophy and portal fibrosis mimicking Congenital Hepatic Fibrosis. In the long term, liver damage appears to persist with a risk of developing portal hypertension. In the literature, the prothrombin time and other coagulation factors may be reduced moderately at the diagnosis of MPI-CDG. In the present patient, the original mode of presentation with a significant reduction of coagulation factors is unusual.

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Expanding the clinical and molecular phenotype of ALG1-CDG: a case study Gavrilova R H 1, Patterson M C 1, Raymond K 1 1

Deciphering Immunological aspects of Congenital Disorders of Glycosylation (CDG): a model for common diseases Monticelli M 1 5, Ferro T 1, Ferreira V 2, Jaeken J 3, Videira P A 1 4 1 CEDOC, NOVA Medical School, Lisbon, Portugal, 2Portoguese Association for CDG, Lisbon, Portugal, 3Center for Metabolic Disease, Leuven, Belgium, 4 Dep Life Sciences, Univ NOVA, Lisbon, Portugal, 5Dep Biology, Univ Federico II, Naples, Italy

Background: Glycosylation of cell surface proteins has a key role in all interactions between cells and between cells and their environment. Since the immune response is based upon contacts between cells and molecules, there is a great probability that glycans on glycoproteins may have a significant role in a variety of common diseases like cancer and inflammation. Congenital Disorders of Glycosylation (CDG) are a group of serious life-altering disorders caused by an abnormal or absent synthesis of the glycans, that cause multi-organ failure with a special involvement of the nervous system. The immune deficiency aspects of CDG remain unknown. CDG have a childhood mortality of 15–25 % in the first 2 years of life due to severe infections, besides the organ failure, and this could be a signal of the involvement of the immune system in these diseases. Methods: A literature review was carried out using Pubmed as source of information to collect clinical evidences about the impairment of the immune response in CDG. Based on the level of involvement of the immune response, CDG were divided into two groups, with a major/minor involvement. Reiterative tables were used to display the results. Results: The immunological dysfunction can represent a minor or a major part of the phenotype in CDG, according to the CDG type. CDG with major immunological involvement are ALG12-CDG, MAGT1-CDG, MOGSCDG, SLC35C1-CDG, COG6-CDG and PGM3-CDG. Discusssion: Our results are a first step to understand and treat these pathologies and other more common diseases. Under the scope of the first worldwide established CDG Professionals and Patient Associations Working Group (CDG-PPAWG), our current research project aims at (1) boosting patient centered CDG research and (2) combining expertise from different laboratories and patient advocacy groups to increase knowledge in the field of CDG.

Mayo Clinic, Rochester, United States

Background: CDG are large group of metabolic disorders caused by defective post-translational modification of proteins and lipids. Since the original description, more than 100 CDG have been described. ALG1-CDG is the third most common N-glycosylation disorder with 57 reported cases due to 39 mutations in ALG1. Clinically it is characterized by psychomotor retardation, dysmorphism, hypotonia, epilepsy, and hypoalbuminemia. Its severity varies considerably and a lethal outcome occurs in almost half of the patients. Case Report: We present an ALG1-CDG case, heterozygous for a novel probably pathogenic variant, who exhibited previously unrecognized clinical features. As other affected patients, he showed hypotonia, intractable multifocal epilepsy, visual inattentiveness and dysmorphic features. Brain MRI showed prominent subarachnoid spaces. In addition, auditory neuropathy was

A-112

A new missense mutation in two ALG2-CDG Argentinean siblings Papazoglu M 3, Pereyra M 4, Ng B 5, Gamboni B 4, Amorosi C 2, Dodelson de Kremer R 3, Freeze H 5, Asteggiano C G 1 1 CONICET-CEMECO-UNC-UCC, Cordoba, Argentina, 2 CONICETINIMEC-UNC, Cordoba, Argentina, 3CONICET-CEMECO-UNC, Cordoba, Argentina, 4Hospital Notti, Mendoza, Argentina, 5Sanford-Burnham Research Institute, La Jolla, San Diego, United States

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Disruption of the glycan synthesis frequently results in a multisystem disease with neurological involvement. Congenital Disorders of Glycosylation (CDG) are a group of genetic diseases due to defects in the synthesis or remodeling of glycoconjugate glycans. Assembly of N-linked glycans starts at the outside of the ER membrane and involves 14 steps resulting in a lipid-linked oligosaccharide. ALG2 encodes the human mannosyltransferase that transfers a mannose residue from GDP-Man to Man1GlcNAc2-PP-dolichol at the cytosolic side of the ER. Nine patients with ALG2-CDG have been reported: one patient presenting bilateral iris coloboma, unilateral cataract, infantile spasms, severe developmental disability and abnormal coagulation factors [Thiel et al. 2003], and on the other hand, 8 patients with congenital myasthenic syndrome-14, with tubular aggregates (Cossins et al. 2013; Monies et al. 2014). Case Study: Multisystem manifestations were observed in two siblings (aged 8 and 10 years) born from non-consanguineous parents. They presented refractory epilepsy with flexion spasms, psychomotor disability, facial dysmorphism, and weak tendon reflexes. Eye fundus was normal, EEG showed hypsarrhythmia and brain MRI frontotemporal atrophy. Methods: Consisted of isoelectrofocusing (IEF) and capillary zone electrophoresis (CZE) of serum transferrin, and whole exome sequencing. Results: A CDG type 1 pattern was detected by IEF and CE. Exomic sequencing revealed a homozygous missense mutation (c.752G > T; p.R251H) in exon 2 of ALG2. Only DNA of the mother was available showing heterozygosity for this mutation. Discusssion: We have detected ALG2-CDG siblings carrying a new mutation. Screening for CDG is important also in Latin America. Supported by CONICET/FONCYT/UCC/Sanford-Burnham Inst. [email protected]

23. Neurotransmitter and creatine related disorders

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Aromatic L-Amino Acid Decarboxylase (AADC) deficiency in a child with an early myopathic onset Battini R 1, Lenzi S 2 5, Carducci C 3, Astrea G 5, Artiola C 3, Carducci C A 3, Frosini S 5, Leuzzi V 4 IRCCS FSM and Ped Neurol Unit UCSC, Pisa and Rome, Italy, 2University of Pisa, Pisa, Italy, 3Dpt Experim Med, Sapienza Univ, Rome, Italy, 4Dpt Ped Child NeuroPsy Sapienza Univ, Rome, Italy, 5Dpt Develop Neurosci IRCCS FSM, Pisa, Italy 1

Background: L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive inborn error in neurotransmitter metabolism that leads to combined serotonin and catecholamine deficiency; clinically it is characterized by vegetative symptoms, oculogyric crises, dystonia, and severe neurologic dysfunction, usually beginning in infancy or childhood. Case Report: We report on a 3-year-old boy who presented with fluctuating palpebral ptosis since birth. Other early clinical characterizations were hypotonia with fatigability and fluctuation of alertness, sleep and eating disorders and nasal stuffiness. Psychomotor and language developments were delayed. Oculogyric crises and dystonic movements of the upper limbs were observed at the age of 2.6 years and suggested a neurotransmitter disorder. Results: The decrease of both homovanillic acid and 5-hydroxyindoleacetic acid combined with the increase of 3-O-methyldopa and 5-hydroxytryptophan in CSF examination suggested aromatic AADC deficiency. Sequencing of DDC detected two predictably pathogenic mutations (c.272C > T/p.Ala91Val and c.1228 T > G/p.Cys410Gly), the second being novel. Dopamine and transdermal rotigotine treatments resulted in an initial improvement of the clinical picture with reduced fatigability and less fluctuating palpebral ptosis and alertness, but after 2 weeks the child experienced insomnia, hyperactivity alternating with somnolence, clumsiness and oculogyric crises reappearance. By reducing gradually rotigotine doses the child has shown the beneficial effects previous obtained.

Discusssion: The present case suggests that even in a presentation mimicking myopathy oculogyric crises are valuable “red flag” of AADC neurotransmitter disorder and the dopamine treatment alone can represent a useful therapy strategy.

24. Disorders of vitamins, cofactors and trace elements

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Identification of a novel mutation in the ATP7A gene in a Turkish patient with menkes disease Kose M 1, Kagnici M 1, Gurbuz G 2, Edizer S 2, Akinci G 2, Yilmaz U 2, Eraslan C 2, Diniz G 3, Ozkul T 2, Unalp A 2 1 Div Metab Dis, Behcet Uz Child Hosp, IZMIR, Turkey, 2Div Ped Neurology, Behcet Uz Child Hosp, IZMIR, Turkey, 3Div Pathology, Tepecik Hosp, IZMIR, Turkey

Background: Menkes disease, also known as kinky hair disease, is an infantileonset X-linked recessive neurodegenerative disorder caused by diverse mutations in a copper-transport gene, ATP7A. Affected patients are characterized by progressive hypotonia, seizures, failure to thrive and death in early childhood. Here, we report a case of Menkes disease presenting with intractable seizures and infantile spasms. Case Report: A 11-month-old male infant had visited our pediatric clinic for lethargy, floppy muscle tone, poor oral intake and partial seizures. His hair was kinky, yellow colored and fragile. Symptoms of seizure were characterized by left facial twitching and left arm clonic movements. EEG showed hypsarrhythmia. Vascular tortuosity and diffuse brain atrophy with callosal thinning were detected in an MRI scan. Light microscopy of hair showed pili torti and trichorrexis nodosa. Biochemical markers showed low levels of serum copper (9.0 μg/dL, reference range: 70–130 μg/dL) and ceruloplasmin (5.6 mg/dL, reference range: 16–31.5 mg/dL) . Results: Molecular genetic investigation detected a p.199delG (c.595_597delGGA) mutation in the ATP7A gene, and the infant was diagnosed with Menkes disease. The mutation was a novel one that has not been previously reported as a cause of MD. Discusssion: MD is a rare cause of metabolic epileptic encephalopathy. It should be kept in mind in patients with characteristic features and epilepsy resistant to anticonvulsant drugs.

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WITHDRAWN-17 June 2016

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Biotinidase: Is it an enhancing factor in binding of thyroid hormone receptor in autoimmune thyroiditis? Bulbul S F 1, Torel Ergur A 2, Odabasi Gunes S 2, Katircioglu M 1 1 Kirikkale Univ, Div Metab Dis,, Kirikkale, Turkey, 2Kirikkale Univ, Div Endocrinolgy, Kirikkale, Turkey

Background: The transcriptional effects of thyroid hormones are mediated by a group of thyroid hormone receptors (THR). Accessory nuclear factors facilitate the interaction of THRs. This can be caused by biotinylation of histones. Biotin is a water-soluble vitamin, which is covalently attached to distinct lysine residues in histones, affecting chromatin structure and mediating gene regulation. Histone is a specific acceptor of biotin transferred from biotinylated biotinidase, where, biotinylated histones increased linearly with biotinidase

S278 concentration. In this paper we aimed to inspect the biotinidase status in relation with clinical picture in children with autoimmune thyroiditis (AT). Methods: Thirty-eight children with AT and 13 healthy controls were involved in the study. Five of the patients had intensive hair loss. AT was diagnosed by clinical symptomatology, thyroid function tests, thyroid antibodies and thyroid ultrasonography. Biotinidase enzyme activity (BEA) was measured by caloric method. Results: At admission, 30 patients were euthyroid and 8 were hypothyroid. BEA of the hypothyroid, euthyroid and control groups were 7.27 ± 1.94, 6.9 ± 2.12, and 5.66 ± 1.10 U/L respectively (P = 0,925). BEA of the patients with loss of hair was lower than the whole study group (5.87 ± 0.94 U/L). Moreover, there was a weak negative correlation between anti-Tg and BEA (r = − 0.395). Discusssion: The results confirm the idea that BEA might be the enhancing factor in AT to mimic the symptoms of hypothyroid. Additionally, being lowest in patients with hair loss strengthened our hypothesis. In addition, high anti-Tg titers, which is found in AT effects BEA negatively. In due to these results; we are of opinion that oral biotin treatment may be beneficial in AT patients.

A-117

Severe Cobalamin Deficiency in Five Families Due to Different Etiologies Fu X 1, Francisco C 2, Mitchell W 3 1 Dep Pathology, Univ Southern California, Los Angeles, United States, 2Div Ped Surgery, Child Hos Los Angeles, Los Angeles, United States, 3Div of Neuro, Dep of Ped, Child Hos LA, Los Angeles, United States

Background: Cobalamin deficiency may be due to dietary restrictions, abnormal absorption, or inactivation due to nitrous oxide (N2O). It is relatively common in the elderly and less common in children. Cobalamin deficiency may lead to serious hematological and neurodevelopmental abnormalities. In some cases, the neurological damage can be permanent. Therefore, early diagnosis and adequate treatment is essential. We herein report five patients, ages 9 m to 18y, with severe cobalamin deficiency due to varying etiologies. Case Report: 1) 9 m old male presented with developmental delay, chorea, megaloblastic anemia, was entirely breast-fed. Mother has asymptomatic pernicious anemia. 2) 23 m old male who was born at home presented with failure-to-thrive, developmental delay, hand tremor, significant bowing of the legs. He was exclusively breast fed for 1 year. 3) 9y old female who is Haitian descent presented with progressive weakness, paresthesia of lower extremities, pancytopenia, and wheelchair-bound. 4) 15y old female presented with pernicious anemia, vague abdominal pain, intermittent emesis, weakness and dizziness. 5) 18y old male presented with generalized weakness, difficulty walking, numbness of fingers and feet. Results: All five patients showed elevated plasma methylmalonic acid (MMA) from 6.7 to 14.1 (0–0.29 uMol/L), elevated homocysteine (tHcy) from 27.7 to 125 ( T (p.Arg169Cys). Discusssion: We underline that transient positivity in expanded NBS may be present in SLC52A2 deficiency; a riboflavin-responsive disorder.

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Hypophosphatasia and Spinal Muscular Atrophy in one patient Pechatnikova N L 1, Kakaulina V S 1, Bruhanova N O 1, Polyakova N A 1 1 Morozov’s Moscow Children Clinical Hosp, Moscow, Russian Federation Background: Hypophosphatasia (HPP) is a rare inherited metabolic disease characterized by defective mineralization of bone and/or teeth in the presence of low activity of serum and bone alkaline phosphatase and caused by mutations in the ALPL gene. It is an extremely rare condition: prevalence is estimated at around 1: 300,000 newborns. Proximal spinal muscular atrophy (SMA) is an autosomal recessive disease caused by a genetic defect in the SMN1 gene. SMN protein is selectively necessary for survival of motor neurons, as diminished abundance of the protein results in loss of function of neuronal cells in the anterior horn of the spinal cord and causes a system-wide muscle atrophy. Prevalence is estimated at around 1:10 000 newborns. Case Report: In our clinic we had observed a boy, 9 months old, with complaints about motor development delay. During the clinical examination diffuse muscle hypotonia, motor development delay and the absence of tendon reflexes were revealed. There were no skeletal changes or any fractures. In laboratory tests the decrease level of alkaline phosphatase activity was founded 31 U/L (N 116–450 U/L). Results: DNA tests for ALPL gene and SMN1 gene were performed. In ALPL gene was detected the mutation c.889 T > A. (p.Tyr297Asn) in heterozygous state. According to international mutation database this mutation was described as pathogenic. As in HPP dominant-negative effect was described, the diagnosis of HPP was suspected with high probability. In mother’s sample the same mutation was founded in heterozygous state. The Alkaline phosphatase level in mother was decreased, she had not had any symptoms of HPP. In SMN1 gene then deletion of 7–8 exones was founded in the patient, the diagnosis of spinal muscular atrophy was confirmed. Discusssion: We report the combination of two inherited diseases in one patient, presented with muscle hypotonia an motor development delay. The decision about the treatment (enzyme replacement therapy for the HPP) is the clinical dilemma.

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 25. Miscellaneous / New Disease Group

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Novel mutations in Czech and Egyptian patients with porphyria and a population study of common wild-type allelic variant contributing to the disease. Farrag M S 1, Martasek P 1 1

Ped dept, 1st faculty Med, Charles Univ, Prague, Czech Republic

Background: The porphyrias are rare metabolic disorders resulting from a n e n z y m e d e f e c t i n t h e h a e m b i o s y n t h e s i s p a t h w a y. Hepatoerythropoietic Porphyria (HEP) is a subtype of porphyria caused by homozygous mutation in the UROD gene leading to defic i e nc y i n u ro po r phy r i no ge n de c ar bo xy l a se . E ry t h ro po i e t i c protoporphyria (EPP), another sub-type, is due to deficiency in the ferrochelatase enzyme of the heme pathway. EPP clinical expression requires coinheritance of a private FECH mutation trans- to a hypomorphic FECH*IVS3-48C allele. Methods: UROD and FECH genes Sanger sequencing. Measurement of mutated and wild type UROD enzyme activity. Results: We identified a novel mutation in the UROD gene in Egyptian patients with HEP. We show that the homozygous c. T163A missense mutation leads to a substitution of a conserved phenylalanine (amino acid 55) for isoleucine in the enzyme active site, causing a dramatic decrease in the enzyme activity (19 % of activity of wild-type enzyme). We also investigated two patients in a Czech family that manifested EPP in four generations. We found a novel mutation, c.84G > A, in the FECH gene in four individuals including the proband and his mother (G84A transition in exon 2; p.W28*). Both clinically manifested probands inherited the hypomorphic IVS3-48C allele as well, while two clinically latent individuals with FECH mutation did not. Discusssion: Novel mutations were identified that widen that heterogeneity of the disease. Low enzyme activity confirmed that the mutation was producing the disease. We also confirmed the importance of the inheritance of the low expression allele IVS3-48C in manifesting EPP. Moreover, we screened the frequency of the low expression allele in a control Czech Caucasian population. Among 312 control individuals, there were no IVS3-48C/C (c.68-23C-T) homozygotes; 35 IVS3-48C/T heterozygous individuals were detected. The frequency of IVS3-48C allele was thus found to be 5.5 % in the Czech population.

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Epigenotype, genotype, and phenotype analysis of patients in Taiwan with Beckwith–Wiedemann syndrome Lin H Y 1 2 3 4, Lin S P 1 2 3, Chuang C K 2, Tu R Y 2, Fang Y Y 2, Su Y N 5, Chen C P 6, Chang C Y 7, Liu H C 1, Niu D M 8, Chu T H 8 1 Dep Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan, 2Dep Med Res, Mackay Memorial Hospital, Taipei, Taiwan, 3Dep Medicine, Mackay Medical College, New Taipei City, Taiwan, 4Mackay Junior College Med, Nurs, Manage, Taipei, Taiwan, 5College Med, Taipei Medical University, Taipei, Taiwan, 6Dep Obs Gyn, Mackay Memorial Hospital, Taipei, Taiwan, 7Dep Pediatrics, Mackay Memorial Hospital, Hsinchu, Taiwan, 8Dep Ped, Taipei Veterans General Hosp, Taipei, Taiwan

Background: Beckwith–Wiedemann syndrome (BWS) is a congenital overgrowth disorder predisposing to tumorigenesis that results from abnormal expression or function of imprinted genes of chromosome 11p15.5.

Methods: Forty-seven patients in Taiwan with clinical suspicion of BWS were referred for diagnostic testing based on methylation profiling of H19associated imprinting center (IC) 1 and KCNQ1OT1-associated IC2 using high-resolution melting analysis, multiplex ligation-dependent probe amplification, or high-resolution quantitative methylation profiling. Results: Twenty-eight patients received a clinical diagnosis of BWS (the presence of 3 major features or 2 major features and at least 1 minor feature), 18 had suspected BWS (the presence of at least 1 major feature), and 1 had isolated Wilms’ tumor. Nineteen patients were identified with IC2 hypomethylation (including 1 with isolated Wilms’ tumor), 1 with IC1 hypermethylation, 2 with paternal uniparental disomy, and 1 with CDKN1C mutation. Several clinical features were found to be statistically different (P < 0.05) between the 2 groups—clinical diagnosis of BWS (n = 28) or suspected BWS (n = 18)—including macroglossia, pre- or postnatal gigantism, abdominal wall defect, ear creases, facial nevus flammeus, BWS score, and the molecular diagnosis rate. Molecular lesion was detected in 81 % of patients with the presence of three major features, compared with 33 % and 28 % of those with two or one major feature, respectively. The mean BWS score was 5.6 for 19 subjects with “IC2 hypomethylation”, compared with 3.8 for 2 subjects with pUPD. The BWS score of one subject with CDKN1C mutation and one with IC1 hypermethylation was 6 and 7, respectively. Discusssion: The BWS score was positively correlated with the molecular diagnosis rate (P < 0.01). The BWS database of epigenotype, genotype, and phenotype is expected to promote better genetic counseling and medical care of these patients.

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GNAL Gene Mutation and Dystonia in Two Turkish Siblings Diagnosed by Exome Sequencing Gunduz M 1, Unal O 1, Ozgul R K 2, Dursun A 2 1

Ankara Children’s Hem Oncology Div Metab, Ankara, Turkey, 2Hacettepe Univ Div Metab, Ankara, Turkey Background: Dystonia is a movement disorder characterized by sustained or intermittent muscle contractions, causing abnormal, often repetitive, movements, postures or both. Inherited dystonia can be classified by mode of inheritance and gene or chromosome locus. Hereditary neurodegenerative or metabolic disorders can be the underlying etiology. There is a group called myoclonic dystonia where some cases are hereditary and have been associated with a mutation in the dopamine-D2 receptor. Other genes associated with dystonia include CIZ1, GNAL, ATP1A3 and PRRT2, THAP1 and SLC20A2. Case Report: Here we report two siblings with GNAL-gene mutation. Results: Two girl siblings at the age of 14 years and 17 years were referred to our clinic for investigation of dystonia etiology. Parents were first cousins. Clinical findings had first occured at 1 year and 1 month of age, respectively. Craniocervical dystonia was more prominent but generalized dystonia affecting both side of the body was seen on physical examination. Both patients could not walk and short stature was an additional finding. Metabolic investigations and neurotransmitter analysis were normal. Cranial MRI findings were non-specific. Exome analysis revealed c. 1216C > T; p.R406W homozygous mutation and GNAL-gene dystonia in the patients. Discusssion: Investigating the underlying cause of dystonia is important to find out treatable causes such as dopa-responsive dystonia and other metabolic disorders. Mutations in the GNAL gene have recently been shown to cause primary torsion dystonia. The GNAL-encoded protein is important for dopamine D1 receptor function and odorant signal transduction. The predominant phenotype is dystonia, with onset in the neck and progression to the cranial region. Mutations in GNAL have been associated with adult-onset cranio-cervical dystonia. Symptoms starting in the first months of life and generalized dystonia are phenotype expanding manifestations in our patients.

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MEGCANN: A new disorder in the Irish Traveller population Fitzsimons P E 1, Walsh A 2, Mayne P D 1, Crushell E 7, Rea D 8, Mayr J A 3, Wortmann S B 4, Lynch B 5, Kelleher J 2, Green A 6 1

Dept of Clin Biochemistry, TSCUH, Dublin, Ireland, 2Dept of Neonatology, Coombe Univ Hosp, Dublin, Ireland, 3Dept of Paed Paracelsus Medica, Salzburg, Austria, 4Dept of Paed, Salzburger Landeskliniken, Salzburg, Austria, 5Dept of Neurology, TSCUH, Dublin, Ireland, 6Dept of Clin Genetics, OLCH, Crumlin, Dublin, Ireland, 7Nat Centre for Inherit Metab Dis, TSCUH, Dublin, Ireland, 8Dept of Radiology, OLCH, Crumlin, Dublin, Ireland Background: 3-Methylglutaconicaciduria (3-MGA-uria) with cataracts, neurological involvement, and neutropenia (MEGCANN) is a recently described autosomal recessive (AR) disorder characterized primarily by increased levels of 3-methylglutaconic acid (3-MGA). The phenotype is highly variable. We describe a boy born to consanguineous Irish Traveller parents with features of MEGCANN. He was small with microcephaly, had severe spasticity, little spontaneous movements and required continuous ventilator support. There were no cataracts. MRI brain showed a relatively simple gyral pattern and abnormal signal in the putaminae. He had severe neutropenia. Urine showed significant 3-MGA-uria. He was homozygous for a novel pathogenic mutation c.1424G > A p.(arg5475Gln) in the CLPB gene. He died on day 16 of life. We know of over 80 different AR conditions in the Irish Traveller population but have not knowingly seen this condition before. Methods: A retrospective review of urine organic acid analyses performed on neonates less than 4 weeks of age was undertaken over a 15 y period to identify those with 3-MGA-uria. Results: 53 samples from 49 neonates were identified with 3-MGA-uria. Those without documented neutropenia, had normal follow-up organic acid profiles or who had a genetically proven diagnosis, including Barth’s syndrome were excluded. From the 49 neonates, one newborn, possibly from the same extended Traveller family had moderate 3-MGA-uria and severe neutropenia. She had severe contractures, micrognathia and microcephaly and died without a definitive diagnosis in the neonatal period. One of her siblings born before the review period, had died at 2 weeks unable to ventilate on her own. Discusssion: Our results conclude MEGCANN is rare within the Irish population, however should be considered in any newborn with moderate 3-MGA-uria, neutropenia and neurological sequelae or older infants and children who may have a milder phenotype where intermittent neutropenia is a feature.

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Spectrum of IEMs presenting with cholestatic jaundice in Indian population Shinde D H 1, Borugale M A 1, Tawde R J 1, Jalan A B 1, Kudalkar K V 1, Jalan R A 1, Joshi M M 1, Shirke S M 1, Mahamunkar A P 1 1

NIRMAN, Div of Biochemical Genetics, Navi Mumbai, India

Background: Cholestatic jaundice is a manifestation of variety of liver diseases including IEM, viral infections, immunological disorders and variety of single gene defects like ATP8B1, ABCB4, ABCB11, PFIC1, PFIC3, NAG, SOPH, ILFS1, ILFS2, LFI1, NBAS and LARS. IEMs known to be associated are Galactosemia, Tyrosinemia Type I (HT1), CDGs, FAODs, Mitochondriopathies, CTX . Objectives: To identify the spectrum of IEM in Indian children with cholestatic liver disease, in order to help planning and rapid investigation. Methods: This is a retrospective study of 28 patients with cholestasis. Analysis was performed for galactose, GAL1-PUT, succinylacetone, sCD25, ferritin,

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 urine organic acids, amino acids, acylcarnitines, plasma sterols and transferrin isoelectric focussing. Results: We evaluated 28 patients (F = 09,M = 19) who presented with cholestatic jaundice, abdominal pain, hepatomegaly, drowsiness, acute liver failure, poor feeding and icterus. We could identify metabolic diseases in 17 patientsGalactosemia (2), HT1 (1), CPT II deficiency (1), CTX (2), CDG type I (1), CDG type II (2), suspected Mitochondriopathy (2), suspected mtDNA depletion (3), confirmed mtDNA depletion-MPV17 mutation (1). We also identified HLH(1) and HBV infection(1). In 11 patients no defect could be identified by our profile. Discusssion: It is important to establish the etiology for cholestatic jaundice early, so that appropriate therapy can be started. We could identify an etiology in 17/28 (60.7 %) patients. However the remaining 11/28 (39.3 %) will need detailed molecular studies like WES. The commonest group of IEMs was Mitochondriopathies. With prompt treatment, we could save children with Galactosemia, HLH, and CPT II. The HT1 patient died prior to treatment being initiated. Conclusion: Performing IEM screening allowed diagnosis in about 60.7 % cases within 4–5 working days thus helping in treatment and outcome.

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Mitochondrial DNA defects in Ras/MAPK disorders—does an association exist? Pelc M 1, Ciara E 1, Kowalski P 1, Iwanicka-Pronicka K 2, PiekutowskaAbramczuk D 1, Halat P 1, Jurkiewicz D 1, Trubicka J 1, Siestrzykowska D 1 , Stawinski P 3 5, Ploski R 3, Chrzanowska K 1, Pronicka E 1 4, KrajewskaWalasek M 1 1

Dept Med Genet, CMHI, Warsaw, Poland, 2Dept Audio Phon and Laryng, CMHI, Warsaw, Poland, 3Dept Med Genet, Warsaw Med Univ, Warsaw, Poland, 4Dept Paediatr Nutri and Metab Dis, CMHI, Warsaw, Poland, 5Dept Genet, Inst Phys Pathol Hearing, Nadarzyn, Poland Background: Literature data indicate a co-occurrence of RASopathies, syndromes caused by mutations in genes from the Ras/MAPK signaling pathway, and mitochondrial disorders (MD), suggesting a functional connection between this cascade and mitochondrial activity. A partial clinical overlap between both conditions is reported and decreased mitochondrial membrane potential and ATP levels combined with increased ROS occur in cells derived from mutation-positive patients. Our research aimed to verify the postulated hypothesis, that RASopathy patients may have increased susceptibility to a mitochondrial dysfunction, but a second hit mtDNA mutation is necessary for the clinical manifestation of the disease. Patients and Methods: We performed analysis of 125 molecularly confirmed Polish RASopathy patients, comprising MLPA screening for the common mtDNA mutations (n = 100) and mtDNA genome sequencing (n = 25). Results: mtDNA sequencing revealed 280 molecular variants in total (>95 % homoplasmic), including 210 in the coding region. Most of the nonsynonymous alterations were common polymorphisms, while a few identified heteroplasmic or novel variants did not seem to have a pathogenic impact. Although several variants have been associated with metabolic disorders/features in MitoMap database (LHON, DEAF, SIDS, HCM, LVNC, CPEO), the MD phenotype was excluded in the patients. In one patient with a novel RAF1 variant and his mutation-negative mother, MTND1 substitution m.3460G > A associated with LHON was identified. Discusssion: Our research suggests that the hypothesis of highly penetrant mtDNA mutations associated with a mitochondrial dysfunction in RASopathy patients may be false, as no prevalent mtDNA mutations segregating with Ras/MAPK alteration were identified. The results also indicate that some of the mtDNA “mutations” reported in literature may not correlate with MD. The study was supported by projects: NSC UMO-2011/03/N/NZ2/00516, CMHI S140/14 and 234/15.

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Different clinical presentation of LPIN1 gene mutation in the same family

Discussion: Children with HFI and heterozygous FH must follow a life-saving restricted and combined diet. The effect of Apo-E gene mutations in some cases remains unknown, yet Apo-E is involved in diet-responsive lipid profile modifications.

Aktuglu-Zeybek A C 2, Zubarioglu T 2, Kiykim E 2, Cansever M S 1, Cam H 2 A-128 1

Central lab, Cerr Med Fac, Ist Univ, Istanbul, Turkey, 2Div Metab Dis Nut, Cerr Med Fac, Ist Uni, Istanbul, Turkey Background: Autosomal recessive LPIN1 gene mutations have been identified as a cause of severe rhabdomyolysis in children. Rhabdomyolysis episodes are associated with poor prognosis and treatment options have been limited. Episodes are triggered predominantly by febrile illnesses, fasting or intense physical exercise. Here, we present two different clinical presentations in two sisters carryng the same LPIN1 mutations. Case Report: Case 1: A 4 year old female patient was admitted to hospital complaining of diffuse muscle pain and weakness. As muscle weakness was progressive, she was admitted to the pediatric intensive care unit. Diagnostic investigations including complete blood count, liver and kidney function tests, blood lactate and ammonia levels, acylcarnitine profile by tandem mass spectrometry, blood quantitative amino acid chromatography, urine organic acid analysis were all normal. Molecular analysis of LPIN1 gene revealed a novel frameshift mutation. Case 2: A 14 year old female patient was seen in the hospital after the LPIN1 mutation was detected in her younger sister. She had neither a history of rhabdomyolysis nor any complaint of muscle weakness. Her physical examination was normal with normal muscle strength. Muscle enzymes were within the normal limit. Molecular analysis of LPIN1 revealed the same homozygous frameshift mutation detected in her sister. Result and Discussion: The pathophysiology and clinical features of LPIN1 disease remains largely unknown due to limited number of patients, although severe rhabdomyolysis is the predominant presentation. Family screening is important in diagnosing asymptomatic patients and defining the clinical spectrum of the disease.

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Nutritional challenges in a patient with Hereditary Fructose Intolerance and Familial Hypercholesterolemia Capra M E 1, Pozzoli A I 1, Bensi G 1, Biasucci G 1

Retrospective review of Irish Cystinosis patients: the role of urine organic acid analysis in diagnosis. Elebert G 1, Murray C 1, Fitzsimons P E 1, O’ Shea A 1, Dolan N 2, Riordan M 2, Awan A 2, Mayne P D 1 1

Clin Bio TSCUH, Dublin, Ireland, 2Nephrology, TSCUH, Dublin, Ireland

Background: Cystinosis is an autosomal recessive disorder of lysosomal transport resulting in the intracellular accumulation of cystine. The diagnosis is often made following clinical presentation and confirmed by white cell cystine analysis. We describe a 2 year old boy who presented with waddling gait, difficulty running/climbing and polydipsia who was otherwise well. As part of a metabolic work-up a urine sample was sent for organic acid analysis which revealed increased excretion of lactate and other mitochondrial markers including tricarboxylic acid cycle intermediates and pyroglutamate. Increased pyroglutamate prompted urinary amino acid analysis which revealed a generalised aminoaciduria. The child was referred for a renal consult with a tentative diagnosis of Cystinosis. Elevated white cell cystine confirmed the diagnosis. Methods: The unusual presentation of Cystinosis in this case led to a retrospective review of the clinical and biochemical presentation of all patients diagnosed with Cystinosis in Ireland. Results: Of the 12 known patients attending TSCUH, 4 were suspected clinically and confirmed by white cell cysteine analysis, 3 had family history. Five had urine organic acids performed with increased pyroglutamate excretion. Three of these had an associated generalised aminoaciduria and two prompted urinary amino acid analysis. Discusssion The constellation of biochemical findings, in particular a generalised aminoaciduria and elevated urinary pyroglutamate would suggest either primary mitochondrial disease or Cystinosis. While this child presented with symptoms suggestive of Fanconi syndrome he did not fit the expected clinical picture of Cystinosis due to his normal growth, absence of eye signs, fair hair and complexion. This case highlights the importance of recognizing the association between urinary pyroglutamate elevation and the diagnosis of Cystinosis and the need for other further investigations such as urinary amino acid analysis.

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Dept Pediatr, Metab Dis Unit, City Hosp, Piacenza, Italy

Background: Hereditary Fructose Intolerance (HFI) is a rare disease caused by an inborn error of fructose metabolism due to a mutation of Aldolase B gene, with a prevalence of 1:20,000 newborns. Patients with HFI must follow a strict fructose-free diet to avoid symptoms such as vomiting, abdominal pain, hypoglycemia, liver dysfunction and coma. Heterozygous Familial Hypercholesterolemia (FH) has a prevalence of 1:250 individuals, but it is still underdiagnosed. FH requires a low-fat and cholesterol diet to reduce consequent cardiovascular risk. Children on special diets need to be strictly monitored to prevent impaired growth, neurodevelopmental delay and nutritional deficiencies. When two different inherited metabolic disorders occur, diet therapy is even more challenging. Case Report: A female patient was referred to our Unit at 6 month of age for vomiting and abdominal pain. A correlation between symptoms and dietary fruit introduction was elicited and the fructosuria followed by Aldolase B gene analysis confirmed the diagnosis of HFI. The patient was started on a fructosefree diet. At 2 yrs of age, total- (7.4 mM) and LDL-cholesterol (5.17 mM) blood levels were both elevated; HDL-cholesterol and triglycerides were 1.91 and 0.7 mM, respectively. Neither family history nor parents’ blood lipid profile was suggestive of FH. Nevertheless, genetic test for FH showed a de novo heterozygous mutation of the gene encoding the LDL receptor and a de novo mutation of the gene encoding the Apolipoprotein(Apo)-E. After fat and cholesterol restriction in addition to fructose free diet, clinical, biochemical and nutritional parameters were assessed every 3 months and they were steadily normal.

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A case of severe encephalopathy and movement disorder due to mutations in the TRAPPC11 gene Nascimento A 2 6, Lopez S 7, Ortez C 2, Colomer J 2, Jou C 2, Puigdelloses M 1, Corbera J 2, Rodriguez M A 1, Topf A 3, Johnson K 3, Gonzalez L 4 6, Rodriguez M J 4, Gallano P 4 6, Sacher M 5, Straub V 3, Jimenez-Mallebrera C 2 6 1 IPR-Hospital Sant Joan de Deu, Barcelona, Spain, 2Hospital Sant Joan de Deu, Barcelona, Spain, 3John Walton Muscular Dystrophy Centre, Newcastle Upon Tyne, United Kingdom, 4Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 5 Concordia University, Montreal, Canada, 6 CIBERER, Madrid, Spain, 7 Hospital Universitario, Santa Cruz de Tenerife, Spain

Background: TRAPPC11 is a component of the Transport Protein Particle (TRAPP) complex which mediates trafficking of vesicles from the endoplasmic reticulum to the Golgi system. Mutations in TRAPPC11 have been described in patients with neurological and neuromuscular diseases ranging from congenital muscular dystrophy to myopathy with ataxia and intellectual disability. Case Report: Here we report the case of a 7 year old male infant with a severe encephalopathy and mutations in TRAPPC11.

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Results: The patient presented within the first year of life with hypotonia, spasticity, dystonic movements and hyperkinesia . He was born with microcephaly and showed cortical atrophy on brain MRI. He was never able to sit without support. His cognition was severely affected and no language was acquired. He suffered from epilepsy which was responsive to treatment. Serum CK levels were markedly elevated (2817 IU/L). Muscle biopsy showed myopathic changes, partial reduction of cytochrome c activity and normal expression of sarcolemmal proteins except for reduction of α-dystroglycan in some fibres. The patient was included in the MYOSEQ project and his DNA studied by whole-exome-sequencing. Two compound heterozygous mutations were identified in the TRAPPC11 gene. One of them has been previously described (c.1287 + 5G > A). The second mutation (c.3379_3380insT) has not been reported before and is predicted to cause a frameshift in the open reading frame and premature termination of the protein near its Cterminus. Each mutation has been inherited from either of the non-affected parents. TRAPPC11 protein levels were significantly reduced in the patient’s fibroblasts. Discusssion: This case expands the phentotypic and genetic spectrum of TRAPPC11-associated myopathies. TRAPPC11 should be considered in the differential diagnosis of patients with myopathy and/or movement disorder, elevated CK and central nervous system involvement.

even more rare form of pontocerebellar hypoplasia characterized by hypotonia, clonus, epilepsy, impaired swallowing, progressive microcephaly, spasticity, and lactic acidosis. Case Report: Here we present a female patient with neonatal seizures, global developmental delay, hypotonia, microcephaly, cardiac septal defects and hepatomegaly. Cranial magnetic resonance imaging (MRI) showed severe cerebellar and cerebral atrophy, diffuse hypomyelination, and bilateral subdural effusion. Magnetic resonance spectroscopy showed a peak of lactate. Results: Whole exome sequencing revealed a homozygous mutation (c.1037C > T; p.Thr346Ile) in RARS2 (Arginyl-tRNA synthetase 2, RARS2) gene. RARS2 encodes mitochondrial arginine-tRNA synthetase, which catalyzes an important step in translation of mitochondrialy-encoded proteins. Mutations in this gene cause Pontocerebellar hypoplasia type 6 (PCH6). Discusssion:Whole exome sequencing is an important tool for the diagnosis of this rare disease. This study was supported by TUBITAK (Project No:111S217)

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Fatty acids evaluation in biotinidase deficiency

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Borsatto T 1 2, Tucci S 3, Blom H J 3, Schwartz I V D 1 2 4 5 A case with psychomotor regression and leukoencephalopathy due to RNASEH2B gene defect Ozgul R K 1 2, Yucel-Yilmaz D 1 2, Serdaroglu E 3, Yalnizoglu D 3, Topcu M 3, Dursun A 1 1 Div Metab Dis, Hacettepe Univ Child Hos, Ankara, Turkey, 2Inst of Child Health, Hacettepe Univ, Ankara, Turkey, 3Div Pediatric Neurol, Hacettepe Univ, Ankara, Turkey

Background: Aicardi-Goutières syndrome (AGS) is a rare subacute encephalopathy characterised by leukodystrophy, basal ganglia calcification, and cerebrospinal fluid lymphocytosis. Clinical manifestations are feeding problems, irritability, psychomotor regression or delay, epilepsy, febrile episodes and chilblain skin lesions on the extremities. AGS is a genetically heterogeneous disorder and can occur due to mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1 and IFIH1 genes. Case Report: A 3-year-old girl presented with weakness, hypotonia and developmental delay. She had a normal birth and early developmental milestones, but showed psychomotor regression after a febrile episode at 13 months. Peripheral blood smear showed cell vacuolisation. Cranial magnetic resonance imaging revealed leukodystrophy, severe cortical atrophy, and mild cerebellar atrophy. Metabolic work up including lactate, lysosomal and peroxisomal screening, homocysteine, blood amino acids, urine organic acids and acylcarnitine profile was negative. Muscle biopsy resulted in nonspecific mild findings. Results: Whole exome sequencing revealed a homozygous mutation (c.412C > T; p.Leu138Phe) in RNASEH2B (Ribonuclease H2, subunit B) gene, known to be responsible for Aicardi-Goutières syndrome. Discusssion: Whole exome sequencing is an effective and rapid method to diagnose diseases with phenotypic and genetic heterogeneity. This study was supported by TUBITAK (Project No:111S217)

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1 Post Grad Prog in Gen and Mol Biol UFRGS, Porto Alegre, Brazil, 2BRAIN Lab, HCPA, Porto Alegre, Brazil, 3Univ Medical Center Freiburg, Freiburg, Germany, 4Medical Genetics Service, HCPA, Porto Alegre, Brazil, 5Dep of Genetics, UFRGS, Porto Alegre, Brazil

Background: In Biotinidase Deficiency (BD; EC 3.5.1.12), there is an inability to absorb and recycle the vitamin biotin, which results in multiple deficiencies of biotin dependent carboxylases – that are important in lipid, glucose and amino acid metabolism. After clinical suspicion or neonatal screening, the diagnosis is confirmed by biotinidase activity in plasma (total BD: < 10 % of normal activity, partial BD: 10–30 % of normal activity) but the result can be falsely reduced. Sequencing of BTD gene can be done as complementary test; genotype-phenotype association is often non informative. In experimental rats on a biotin-deficient diet, metabolic changes such as increased free fatty acids (FA) have been reported before the onset of deficiency signs. The objective of this study was to verify whether metabolites may be useful biomarkers of BD. Methods: Thirty-three Brazilian patients were included (male: 18), between 34 and 105 days of age (median: 53), who were submitted to biotinidase activity measurement in plasma after abnormal neonatal screening test for BD. The enzyme activity corresponded to partial BD (n = 11) or higher, i.e., nondeficient (n = 22). Plasma FAwere analyzed by GC-MS; relative quantification was calculated. It was possible to identify 26 different FAs and cholesterol. In addition, two indices of stearoyl-CoA desaturase (SCD, a key enzyme regulating whole body lipid composition) activity were calculated: C16:1n-7/ C16:0 and C18:1n-9/C18:0. Results: No correlation was found between the studied variables and biotinidase activity. Despite that, C16:1n-7 percentage and C18:1n-9/C18:0 ratio were significantly reduced in partial BD (p < 0.05). Discusssion: Although FA analysis by GC-MS is not easily accessible to support BD diagnosis, according to this study, partial BD seems to be associated to changes in lipid metabolism. The analyses presented here are exploratory and belong to a more comprehensive project.

Pontocerebellar hypoplasia type 6: a case with neonatal seizures, hypotonia and microcephaly diagnosed by exome sequencing

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Serdaroglu E 3, Ozgul R K 1 2, Yucel-Yilmaz D 1 2, Yalnizoglu D 3, Dursun A 1

The value of electron microscopy of axillar skin in the diagnosis of lysosomal storage or energy metabolism inherited metabolic disorders

1

2

Div Metab Dis, Hacettepe Univ Child Hos, Ankara, Turkey, Inst of Child Health, Hacettepe Univ, Ankara, Turkey, 3Div Pediatric Neurol, Hacettepe Univ, Ankara, Turkey

Karall D 1, Hofstaetter J 1, Zlamy M 1, Albrecht U 1, Haberlandt E 1, Guntersweiter D 2, Mihic-Probst D 2, Scholl-Buergi S 1

Background: Pontocerebellar hypoplasia is a heterogeneous group of rare neurodegenerative disorders. Pontocerebellar hypoplasia type 6 (PCH6) is an

1 Pediatrics, Medical University, Innsbruck, Austria, 2Clinical Pathology, University Hospital, Zuerich, Switzerland

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J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 Background: Lysosomal storage and mitochondrial disorders might show diagnostic features on axillar skin electron microscopy (EM) analysis. Thus, they can be useful to establish a diagnosis, or direct further diagnostic steps. Methods: In an era, where “old fashioned methods” are increasingly being replaced by molecular techniques (exome and whole genome sequencing), we retrospectively evaluated the value of EM of axillar skin in the diagnosis of lysosomal storage or energy metabolism inherited metabolic disorders. Results: Since 2001, 63 patients who were investigated for developmental delay with regression / neurodegeneration, underwent axillar skin biopsy both for fibroblast culture and EM. For 27/63 (=42.8 %) patients a definitive diagnosis was established. From the 48 EM biopsy results available, 6 (13 %) were diagnostic, 16 (35 %) showed abnormal results, 23 (51 %) showed normal results, and 3 were inconclusive. In 15/63 results are pending. Discusssion: From 48 axillar skin EM findings, 22 (46 %) were either diagnostic or abnormal. In view of the easy access of axillar skin, especially while undergoing other procedures needing anesthesia like i.e. magnetic resonance imaging, it is worthwhile taking the biopsy as it can be helpful / supportive in establishing a definitive diagnosis. *Mihic-Probst and Scholl-Buergi are shared senior authors.

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Pitfalls in diagnosis of Hypophosphatasia. Proshlyakova T Y 1, Rodionova S S 2, Bessonova L A 1, Belova N A 3, Mikhaylova L K 2, Bruchanova N O 4, Pechatnikova N L 4 1

FSBI Res Cent for Med Genet, Moscow, Russian Federation, 2FSI Cent Res Inst Traumat and Orthop, Moscow, Russian Federation, 3Cent Cong Path Glob Med Syst Clin, Moscow, Russian Federation, 4Child Clin Hosp named Morozov, Moscow, Russian Federation Background: Hypophosphatasia (HPP) is a rare, inherited metabolic disorder characterized by defective mineralization of bone and/or teeth in the presence of low activity of serum and bone alkaline phosphatase. HPP is caused by mutations in the gene encoding tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP, other name ALPL). Clinical features range from stillbirth without mineralized bone at the severe end to pathologic fractures of the lower extremities in adulthood at the mild end of the disease spectrum. There are six forms of hypophosphatasia, with clinical presentations varying w idely betw een the different types. HPP is often misdiagnosed. Material and Methods: We have selected 70 patients with suspected HPP based on clinical and biochemical data (ages 1 m-72y). Biochemical analysis of alkaline phosphatase activity was determined during routine biochemical analysis. Molecular-genetic analysis of ALPL gene was made by direct sequencing. Results and Discussion: We found 10 mutations in the analyzed patients and we identified 4 novel mutations which have not been described previously: c.61G > A (p.Glu21Lys), c.211C > G (p.Arg71Gly,) c . 8 8 9 T > A ( p . Ty r 2 9 7 A s n ) , c . 1 0 7 2 G > A ( p . A l a 3 5 8 T h r ) . Pathogenicity analysis in silico was performed for these mutations. Pathogenicity for all was above 95 %. These mutations are now included in the ALPL gene mutations database (www.sesep.uvsq.fr/ Database.html). Moreover, a frequent polymorphism c.787 T > C ( p . Ty r 2 6 3 H i s ) w a s f o u n d i n 1 4 p a t i e n t s a n d c . 4 5 5 G > A (p.Arg152His) was found in 2 patients. The laboratories performing analysis of HPP had difficulties in the interpretation of the identified mutations. The vast majority of patients have only one mutation in heterozygous state. Perhaps in 9 patients with one mutation there is a dominant-negative effect, or perhaps they have a mutation in combination with an extended deletion or insertion. Therefore, it is needed to develop a MLPA test system for ALPL gene and continue to search for the second mutant allele.

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GM3 synthase deficiency: the first Italian patient Parini R 1, Gasperini S 1, Masera N 1, Grioni D 2, Lamantea E 3, Schiliro M 1, Galimberti C 1, Selicorni A 1, Melzi M L 1, Biondi A 1, Iascone M 4 1

Dept Pediat MBBM Found S Gerardo Hosp, Monza, Italy, 2Child Neurol Dept, S Gerardo Hosp, Monza, Italy, 3Besta Neurological Institute, Milano, Italy, 4Mol Genet Lab Papa Giovanni XXIII Hosp, Bergamo, Italy Background: GM3 synthase deficiency is an autosomal recessive disease caused by mutations in the ST3GAL5 gene. It is characterized by infantile onset of refractory epilepsy, profound cognitive delay, and abnormal coreoathetotic–like movements. Complete absence of GM3 ganglioside is observed in patients’ fibroblasts and secondary deficiency of mitochondrial complexes activity has been reported. The disorder is frequent among the Amish population while it is very rare elsewhere. We diagnosed the first Italian case through clinical exome sequencing (CES) after repeated useless efforts to reach the diagnosis in a conventional way. Case Report: This is a 10-year-old girl born from consanguineous Italian healthy parents who showed developmental delay with abnormal movements at 1 year of age and was diagnosed with mitochondrial disease (partial deficiency of Complex III and pyruvate dehydrogenase). At 2 years of age, she had a diagnosis of sickle cell disease after frequent inconsolable crying. Later she developed epilepsy, non purposeful movements of limbs, profound cognitive delay and a particular facies. She never smiled nor tried to reach objects; never sat, walked or talked. Visual evoked potentials at the age of 6 years showed severe bilateral latency. Brain MRI was normal with no structural abnormalities. Results: CES showed a homozygous new missense mutation of the ST3GAL5 gene. Heterozygosity was confirmed in the parents. No mutations of genes involved in mitochondrial respiratory chain diseases were found. Discusssion: Our patient has all the features of GM3 synthase deficiency, except that her brain MRI was normal. The previous diagnosis of mitochondrial disorder prevented the search of another diagnosis, although the first one was not convincing. This disease is probably underdiagnosed at present because of its rarity and confounding biochemical data. This case emphasizes the role of CES in defining the diagnosis of complex encephalopathies.

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A novel mutation of the SLC4A4 gene in a Turkish patient with isolated proximal renal tubular acidosis and systemic involvement Seker Yilmaz B 1, Kor D 1, Bulut F D 1, Melek E 3, Ceylaner G 2, Eren H 1, Onenli Mungan N 1 1

Div Metab Dis, Univ Cukurova, Adana, Turkey, 2Div Genetics, Intergen Genetics Lab, Ankara, Turkey, 3Div Ped Nephrol, Univ Cukurova, Adana, Turkey Background: Isolated proximal renal tubular acidosis (pRTA) without a generalized defect in proximal tubule function accompanied with stunted growth, intellectual disability and ocular abnormalities may be caused by defects of the basolateral Na/HCO3 cotransporter (NBCe1). SLC4A4 gene located on chromosome 4q21 encodes for the NBCe1 protein which plays a major role in renal bicarbonate absorption via proximal tubules. Case Report: A 13 year old girl was the first child of consanguineous parents with normal stature referred to our hospital for the evaluation of short stature. The patient’s history revealed normal anion-gap metabolic acidosis together with a urinalysis indicating isolated pRTA (pH of 5, no protein, no glucose, no blood and the normal urinary excretion of amino acids), and absence of nephrocalcinosis on renal ultrasonographic examination at the age of 5. Bilateral glaucoma was diagnosed when she was 6 years old. She did not have cataract or band keratopathy. Beside intellectual disability, she had seizures which were controlled with phenobarbital from the age of 7 years.

S284 Results: We identified a novel homozygous c.1771 + 2 T > A (IVS11 + 2 T > A) mutation in SLC4A4 by whole exome sequencing. Human splicing finder and Mutation taster tools for in silico analysis predicted this variant as damaging. Discusssion:This patient emphasizes NBCe1 defects as a cause of isolated pRTA with systemic features including intellectual disability, ocular abnormalities, and epilepsy. With further functional and molecular studies, this novel mutation may increase our understanding about the role of the NBCe1 protein in pathophysiology of the multiorgan defect.

A-137 369 inborn errors of metabolism presenting with epilepsy or seizures – implications for testing Tumiene B 1 2, Maver A 3, Peterlin B 3, Utkus A 1 2 1 Dep Hum Med Gen, Vilnius Univ, Vilnius, Lithuania, 2Vilnius Univ Hosp Santariskiu Klinikos, Vilnius, Lithuania, 3Clin Institute of Med Gen, Ljubljana, Lithuania

Background: To evaluate the scope and diagnostics of inborn errors of metabolism (IEM) involving epilepsy or seizures. Methods: Searching through OMIM, Pubmed databases was used for comprising a gene catalogue of monogenic diseases involving epilepsy or seizures, genes for IEM were selected from these. Clinical characteristics including seizure severity/ frequency, availability of specific treatment and diagnostics of these IEM were evaluated. Results: A catalogue of 830 epilepsy- or seizures-associated genes was comprised by including only genes with human phenotypes, 369 (45 %) of these genes are associated with IEM. Clinical spectrum of these IEM include a vast range of associated symptoms and signs. There were 106 mitochondrial disorders, 51 congenital disorders of glycosylation, 33 lysosomal disorders. Diagnostics of these disorders include multiple biochemical genetic analyses: 106 IEM can have informative findings of biochemical genetic tests in blood and urine, 65 IEM – in CSF. Some of these IEM can be diagnosed through pathohistological, molecular genetic tests. 77 of these disorders have specific treatments. 79 diseases involve very severe, refractory seizures, 49 – symptomatic seizures due to metabolic/ electrolyte disturbances. Discussion: Almost half of all known genes for monogenic diseases involving epilepsy or seizures are those of IEM. Majority of these IEM have characteristic findings in biochemical genetic tests. However, in clinical practice combinations of various tests, repetitive investigations are frequently used and

J Inherit Metab Dis (2016) 39 (Suppl 1):S35–S284 molecular genetic testing is usual for confirmation of a diagnosis/ genetic counseling purposes. Therefore, genomic testing methods could be of value. This 369 gene catalogue can be of high importance for further studies including molecular processes involved in epileptogenesis and ictogenesis, and for clinical practice including diagnostic algorithms for epilepsy genetic testing.

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Transient severe metabolic changes delaying Sotos syndrome diagnosis in two unrelated patients Louro P 1 2, Deshpande C 1, Champion M 3 1 Dep Clin Genetics, Guy’s Hospital, GSTT, London, United Kingdom, 2Med Genetics Unit, Hosp Ped, CHUC, Coimbra, Portugal, 3Dep In Met Dis, Evelina Child Hosp, GSTT, London, United Kingdom

Background: Sotos syndrome is a rare autosomal dominant disorder characterized by a distinctive facial appearance (broad and prominent forehead with a dolichocephalic head shape, sparse frontotemporal hair, long and narrow face, long chin), mild to severe intellectual disability, and somatic overgrowth (head and/or height circumference ≥ 2 SD above the mean). Other features of Sotos syndrome include brain, cardiac and renal anomalies, seizures, behavioral problems, joint hyperlaxity, neonatal jaundice, neonatal hypotonia, and maternal preeclampsia. Transient metabolic changes are not commonly reported. Methods: We report the cases of two unrelated boys with Sotos syndrome. Results: Both patients were born with severe hypotonia and are now severely delayed. Recurrent chest infections due to unsafe swallow and aspiration lead to the placement of a feeding tube. Patient 1 was diagnosed antenatally with intra-uterine growth retardation, ventriculomegaly and single umbilical artery. He was born at 33 weeks of gestation and presented neonatal hypoglycemia and jaundice. Conjugated hyperbilirubinemia occurred from the fifth day of life to 3 months of age. At that time, abdominal US showed a homogeneous liver parenchyma. Patient 2 had investigations for hypotonia. Muscle biopsy showed complex IV deficiency and he was thought to have a mitochondrial disorder. In both patients, NSD1 testing was performed as a result of typical facial gestalt. Discussion: Nonspecific metabolic changes direct clinicians into investigating an inborn error of metabolism in a critically ill neonate. Although such course of action is appropriate in view of therapeutic implications, this might delay the establishment of the underlying genetic diagnosis.