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The Journal of Molecular Diagnostics, Vol. 18, No. 6, November 2016

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Diagnosis of Primary Ciliary Dyskinesia by a Targeted Next-Generation Sequencing Panel Molecular and Clinical Findings in Italian Patients Francesca Boaretto,* Deborah Snijders,y Cecilia Salvoro,* Ambra Spalletta,* Maria Luisa Mostacciuolo,* Mirella Collura,z Salvatore Cazzato,x Donatella Girosi,{ Michela Silvestri,{ Giovanni Arturo Rossi,{ Angelo Barbato,y and Giovanni Vazza* From the Departments of Biology* and Women’s and Children’s Health,y University of Padova, Padova; the Cystic Fibrosis and Respiratory Pediatric Center,z Arnas Children Hospital, Palermo; the Department of Mother and Child Health,x Salesi Children’s Hospital, Ancona; and the Pediatric Pulmonology Unit,{ Istitute Giannina Gaslini, Genova, Italy Accepted for publication July 15, 2016. Address correspondence to Giovanni Vazza, Ph.D., Department of Biology, University of Padova, Via U. Bassi 58/b, 35131 Padova, Italy. E-mail: [email protected].

Primary ciliary dyskinesia (PCD) is a rare genetic disorder that alters mucociliary clearance, with consequent chronic disease of upper and lower airways. Diagnosis of PCD is challenging, and genetic testing is hampered by the high heterogeneity of the disease, because autosomal recessive causative mutations were found in 34 different genes. In this study, we clinically and molecularly characterized a cohort of 51 Italian patients with clinical signs of PCD. A custom next-generation sequencing panel that enables the affordable and simultaneous screening of 24 PCD genes was developed for genetic analysis. After variant filtering and prioritization, the molecular diagnosis of PCD was achieved in 43% of the patients. Overall, 5 homozygous and 27 compound heterozygous mutations, 21 of which were never reported before, were identified in 11 PCD genes. The DNAH5 and DNAH11 genes were the most common cause of PCD in Italy, but some population specificities were identified. In addition, the number of unsolved cases and the identification of only a single mutation in six patients suggest further genetic heterogeneity and invoke the need of novel strategies to detect unconventional pathogenic DNA variants. Finally, despite the availability of mutation databases and in silico prediction tools helping the interpretation of variants in next-generation sequencing screenings, a comprehensive segregation analysis is required to establish the in trans inheritance and support the pathogenic role of mutations. (J Mol Diagn 2016, 18: 912e922; http://dx.doi.org/10.1016/j.jmoldx.2016.07.002)

Primary ciliary dyskinesia (PCD) (MIM#244400) is a rare genetic disorder characterized by morphologic and functional alterations of the motile cilia of ciliated epithelial cells. Motile cilia are present in the embryonic node, respiratory tract, cerebral ventricles, and oviduct cells, and defects in these structures cause a variety of signs and symptoms. In the respiratory system, PCD causes altered mucociliary clearance that promotes the development of chronic rhinosinusitis, medium otitis, rhinitis, and bronchiectasis.1 These chronic conditions may lead to severe consequences in adulthood such as permanent hearing loss due to recurrent ear infections and irreversible pulmonary function failure.2,3 Furthermore, many other organs in the cardiovascular and reproductive systems can be affected, leading to congenital heart defects and infertility.4

Defects in ciliary motility in the embryonic node lead to situs inversus totalis in about 40% to 50% of PCD patients or to variable forms of heterotaxy in about 12% of the cases.4,5 PCD usually presents early in life and affects males and females equally. Disease prevalence in different populations is largely unknown, and the available estimates range from 1 in 20,000 to 1 in 40,000.6,7 However, these numbers are likely underestimated because the diagnosis is often missed early in life.7 PCD is inherited as a recessive trait, and mutations in 34 genes, coding for structural proteins as well as proteins Supported by the Italian Ministry of Health project RF-VEN-20081201767. Disclosures: None declared.

Copyright ª 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2016.07.002

PCD Diagnosis by Targeted Gene Panel involved in axoneme assembly, have been implicated in the disease to date.8,9 With the exception of two rare X-linked forms, characterized by syndromic PCD with retinitis pigmentosa (RPGR) and mental retardation (OFD1),10,11 all of the other forms are due to biallelic (homozygous or compound heterozygous) mutations in autosomal genes. Further genetic heterogeneity is however expected because about half of patients do not carry mutations in any of these known genes.12 The diagnosis of PCD is challenging, considering the absence of specific symptoms; nevertheless, the early identification of the disease is important to prevent irreversible complications. At present, PCD diagnosis is mainly based on the identification of ciliary ultrastructural defects by transmission electron microscopy (TEM) and/ or abnormalities in ciliary waveform and beat pattern by high-speed video microscopy (HSVM).13 In addition, nasal nitric oxide measurement and immunofluorescent assays have become available in the past years in support of PCD diagnosis.14 Given the technical and practical limitations of each of these techniques, different tools are usually combined in the diagnostic process. In this context, the emerging genetic knowledge of PCD and the availability of next-generation sequencing (NGS) technologies have great potential to improve accuracy and robustness of PCD diagnosis as well as early patient assessment.15 In this study, a custom NGS panel for the parallel screening of 24 PCD genes was designed and used to molecularly characterize a cohort of Italian PCD patients.

Materials and Methods Patients In total, 44 unrelated index patients were collected from four different Italian tertiary hospitals. In addition, seven affected proband relatives and 83 healthy family members (parents, brothers, and sisters) were recruited for this study. The study conforms to the Helsinki Declaration regarding ethical principles for medical research, and written informed consent for DNA analysis was obtained for all participating subjects. Collected medical and anamnestic data included family history, physical examination, presenting symptoms, complications, and follow-up information.

PCD Diagnosis PCD was defined by typical clinical features and PCDspecific abnormal findings in at least two of the following methods: nasal nitric oxide measurement, HSVM analysis, or ultrastructural analysis by TEM.16 The combination of bronchiectasis, chronic sinusitis, and situs inversus (Kartagener syndrome) was considered PCD even if the above criteria were not completely fulfilled.

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Nasal nitric oxide measurements were defined low and PCD-specific when at least three readings per patient were 20) and balance (strand ratio between 0.4 and 0.6 and heterozygous allele ratio between 0.4 and 0.6). Variants were checked for previously reported causative mutations; both PCD published works and mutation databases [Human Gene Mutation Database (HGMD): www.hgmd.cf.ac.uk, and Leiden Open Variation Database (LOVD): www.lovd.nl, last accessed April 2016] were consulted. Unreported variants were then searched in dbSNP (b146, GRCh37p13), 1000 Genomes (phase3, v5b.20130502), and ExAC (r0.3) repositories, to filter out common single nucleotide polymorphisms. Here, the in-house scripts were useful to distinguish already reported single nucleotide polymorphisms from new variants matching a single nucleotide polymorphism position but with different alleles. Both rare (minor allele frequency 20% of patients were removed as ascribable to sequencing errors, mostly occurring in homopolymeric regions. First, under the hypothesis of an autosomal recessive model, homozygous or compound heterozygous variants were considered; subsequently, monoalleic variants, either known or unknown, were also evaluated.

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Variants were then classified based on their expected pathogenicity according to the American College of Medical Genetics and Genomics (ACMG) guidelines, as recently published.19 For a schematic flowchart of variant classification see Supplemental Figure S1. The considered criteria accounted for i) type of variants (eg, nonsense, missense), ii) presence of well-established functional studies, iii) variant frequency (rare or novel, mutational hotspot), iv) inheritance (trans heredity, de novo events), v) segregation in affected brothers, vi) phenotype coherence, and vii) in silico predictions. Null variants (nonsense, frameshift, canonical splice-site, multiexon deletions) were assumed to have a very strong effect. Instead, in silico analyses were performed for missense and splice-site variants; each candidate mutation was evaluated with three independent algorithms, two for variant effect predictions and one for site conservation. CONDEL version 2.0 (http://bg.upf.edu/fannsdb) and MutationTaster2 (http://www.mutationtaster.org) were used to test missense substitutions, and splice-site changes were assessed using NNSplice version 0.9 (http://www.fruitfly. org/seq_tools/splice.html) and HumanSplicingFinder version 3.0 (http://www.umd.be/HSF3/). A nucleotide position was considered highly conserved whenever the relative PhyloP score provided by ION Reporter was >2. Full concordance of all of the three computational predictions was considered as supportive evidence for pathogenicity (PP3 criteria of ACMG guidelines) (Supplemental Table S2). Finally, novel synonymous variants were analyzed to evaluate possible alterations of splicing consensus motifs. According to the ACMG recommendations, only variants classified as pathogenic, likely pathogenic, and variants of uncertain significance (VUSs) were retained; all these candidate causative mutations were confirmed by Sanger sequencing (Table 2). Inheritance and segregation were also checked whenever additional family members were available. For de novo mutations, both paternity and maternity were confirmed for the proband.

Results Clinical Phenotype A total of 51 patients from 44 families with probable or definite PCD were enrolled in the study. The proportion of male versus female sex was balanced (53% versus 47%), with an age range of 6 months to 69 years. A single affected individual was present in 37 families, and seven pedigrees included two patients. Parental consanguinity was documented in one family. Situs solitus was observed in 29 individuals (57%), whereas 20 (39%) had situs inversus, and 2 (4%) had situs ambiguous. The majority of patients experienced neonatal respiratory distress (33%), recurrent pneumonia (72%), bronchiectasis (64%), and sinusitis (55%). All patients underwent a ciliary biopsy and were tested for ultrastructural defects by TEM using a qualitative and

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PCD Diagnosis by Targeted Gene Panel quantitative approach. Clear ultrastructural defects were evident in 35 patients (69%), and 14 (27%) showed normal ultrastructure on TEM analysis. Two patients showed IDA defects. Of the 35 individuals with confirmed ciliary defects, 12 had isolated ODA defects, three had central pair defects, one had microtubular disorganization defects, and 19 had combined ODA þ IDA defects.

Mutational Screening An overall analysis of NGS data showed consistent and uniform results for multiple runs, with an average coverage depth of 161 and 97.1% of the targeted bases covered >20. Six exons failed to be amplified in all cases and were covered by Sanger sequencing. Technically, the designed PCD panel turned out to be robust in terms of both coverage and read depth for all of the 24 genes and patient samples. NGS sequencing of the 24 PCD genes revealed a total of 995 variants across the 44 index patients, with an average of about 350 variants per sample. After quality and falsepositive filtering, 169 rare (minor allele frequency G and c.3484C>T in DNAH5). In agreement with the autosomal recessive inheritance of the disease, the majority of them (n Z 26) were disrupting mutations (nonsense, coding indels, and splice acceptor/donor site) that likely cause a loss of protein function. Following the ACMG guidelines for mutation interpretation, 33 of 37 mutant alleles were classified as pathogenic or likely pathogenic, and four were categorized as VUS. Remarkably, all of the VUSs were missense changes, a type of substitutions that require several supporting elements to sustain a pathogenic effect according to the ACMG classification scheme (ie, segregation analysis, in silico predictions, functional studies) (Supplemental Table S4). Despite this, several lines of evidence support the presumed causality of these variants: i) all of the VUSs are not reported as polymorphisms, ii) HSVM and TEM phenotypes are consistent with the involved gene, and iii) three of them are in compound heterozygosity with a clear pathogenic mutation and the fourth is inherited in homozygosity by the proband (11177) and by his affected brother (11354). Considering the biallelic mutation carriers, a confirmative molecular diagnosis of PCD was established for 43% of PCD cases (Figure 1). More in detail, the causative gene was identified in about one-third of the patients categorized as probable PCD and in one-half of those with a definite diagnosis. Among these latter, the success rate was about 50% in patients with ODA or ODA þ IDA defects, and a molecular diagnosis was achieved for all cases exhibiting central pair and doublet disorganization (3 of 3). Despite the small sample size, these findings suggest that the identification of hallmark ultrastructural changes of PCD remains the mainstay of current clinical and molecular diagnosis. The genes with the highest incidence of mutations were DNAH5 (26.3%), followed by DNAH11 (15.8%), LRRC6 (10.5%), and ARMC4 (10.5%). Further, seven genes (CCDC114, SPAG1, CCDC103, CCDC39, RSPH9, RSPH4A, and HYDIN) were each mutated once, accounting all together for 36.8% of the investigated PCD series (Supplemental Figure S2). Of note, no biallelic mutations were identified in the remaining 14 genes. All of the probands showed a coherent correlation between the mutated gene and the functional and ultrastructural findings. No laterality defects were observed in patients with RSPH9, RSPH4A, and HYDIN mutations, and no defects in the dynein arms were seen in patients carrying mutations in DNAH11, RSPH9, RSPH4A, and HYDIN. Only in one case, IDA defects were observed in a DNAH11mutated patient (11057); however, IDA defects by themselves cannot be considered as pathogenic, because they are frequently associated with recurrent infections.29

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Boaretto et al Table 1

Clinical Characteristics, Ciliary Ultrastructural, and Functional Abnormalities of Patients in the Study Clinical symptoms/history Neonatal Recurrent URTI

Recurrent LRTI

Fam. ID Ind. ID Sex

Age (years) Situs inversus

RDS

Bronchitis Bronchitis without with Rhinitis Sinusitis Otitis wheezing wheezing Bronchopneumonia Bronchiectasis

1 2 3 4 4 5 6 7 8 9 10 11 12 13 14 15 15 16 16 17 17 18 19 20 21 21 22 23 23 24 25 26 27 28 29 30 31 32 33 33 34 35 36 37 38 39 40 41 42 43 44

9 3 3 6 7 51 24 17 13 3 20 9 8 50 36 18 11 27 22 23 32 3 13 10 20 10 3 18 18 17 19 14 19 12 3 10 18 18 7 4 8 69 21 16 18 8 16 13 7 1 3

Y N Y Y Y NA Y N N N N N Y N NA N N N N Y Y N Y Y N Y Y N N N Y N Y N Y N Y N N N N N N N N N NA N N N N

Y Y N Y Y Y N Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N

11047 11048 11049 11051 * 11052 11053 11054 11055 11056 11069 11070 11071 11072 11073 11074 11075 11076 11077 11078 11111 11079 11144 11145 11163 11164 11165 11166 11167 11169 11170 11171 11173 11050 11057 11174 11175 11176 11177 11354 11179 11180 11181 11182 11183 11184 11198 11212 11216 11228 11236

F F M M M F F M M F F M M M F M F M M F F M M F F F F M M M M M M M M M F M M M F F F F F F F F M M F

N N Y Y Y þ CHD Y Y Y Y Y Y Y Y Y Y N Y N N N N Dextrocardia/CHD N N N N N N N N N Y Y N Y N N N Y Dextrocardia N N N N N N N N Y Y N

N NA N Y N Y N N Y N N N N Y Y Y Y N Y Y Y N N N Y Y Y Y Y Y Y Y Y N Y Y Y Y N N Y Y Y Y N N N N Y N N

N Y N NA N N N N N N N Y N N N Y Y N N N Y Y N N N N N N Y N N N N N Y Y Y N N N Y N N Y N N N N Y N N

N NA N Y Y Y Y N Y Y Y N Y Y N Y Y Y Y N Y Y Y Y Y Y Y Y N Y N Y N Y Y N Y Y N Y Y Y Y Y N N N Y Y Y N

Y NA Y N Y Y N N Y N Y N N Y N N N N N Y N Y Y N N N N N Y N N N N Y N Y N N N N N N N N Y Y Y Y Y N Y

Y NA Y NA Y Y Y N Y N Y Y N Y Y N Y Y Y Y Y Y Y Y Y Y Y N Y Y N Y Y Y N N N Y N N Y Y Y Y Y Y Y Y Y N Y

Y NA NA Y NA Y Y N Y N Y N N N Y N Y Y Y Y Y N Y N Y Y Y Y Y Y Y Y Y Y N N Y N N N Y Y Y Y N N Y Y Y N N (table continues)

*DNA not available. y Diagnosis made on clinical symptoms (situs inversus þ recurrent sinusitis þ bronchiectasis). F, female; M, male; CHD, congenital heart defect; CP, central pair defects; HSMV, high-speed video microscopy; IDA, complete absence of inner dynein arm; LRTI, lower respiratory tract infection; MTd, microtubular disorganization; N, No; NA, data not available; NO, nitric oxide; NU, normal ultrastructure; ODA, complete absence of outer dynein arms; PCD, primary ciliary dyskinesia; RDS, respiratory distress syndrome; TEM, transmission electron microscopy; URTI, upper respiratory tract infections; Y, Yes.

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PCD Diagnosis by Targeted Gene Panel Table 1 (continued) Diagnostic findings

Clinical symptoms/history

Cough

Allergy

Asthma

NO nas ppb

TEM

HSVM

Diagnosis

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N Y Y N Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y

N NA N N N N N Y Y N N N Y N N N N N N Y Y N N N N N N N N N N Y N N N N Y N N N N N N N N N Y N N N N

N NA N N N N N Y Y N N N N Y N N N N N N N N N N Y N N N N N N N N N N Y N N N N N N N N Y N Y Y N N N

170 NA NA NA NA NA NA 208 42 NA 46 NA NA NA NA 7 7 NA NA 8 NA NA NA 493 52 29 80 13 39 99 42 45 18 NA NA 27 566 400 NA NA 317 654 42 115 116 162 NA NA 74 NA NA

ODA þ IDA ODA þ IDA ODA þ IDA ODA þ IDA ODA þ IDA IDA ODA þ IDA ODA þ IDA ODA þ IDA ODA ODA þ IDA ODA ODA þIDA ODA ODA þ IDA ODA ODA ODA ODA CP CP ODA þ IDA CP ODA þ IDA ODA ODA ODA þ IDA ODA ODA ODA ODA þ IDA ODA þ IDA ODA þ IDA Partial ODA þ IDA IDA NU NU NU NU NU NU NU NU NU NU NU NU Partial ODA þ IDA MTd NU NU

Immotile Immotile Immotile Immotile Immotile Dyskinetic Immotile Immotile Immotile Immotile Immotile NA NA NA NA NA NA NA NA Slow beating, stiff Slow beating, stiff Immotile Reduced amplitude Low frequency, stiff Immotile Immotile Immotile Immotile Immotile Immotile Immotile Immotile Immotile Dykinetic Immotile Immotile, stiff High frequency, immotile Stiff Dyskinetic Immotile Regular, immotile, stiff, high frequency High frequency, stiff Immotile, stiff Regular Stiff Immotile Low frequency Normal, low frequency NA Atypical pattern Low frequency

PCD PCD PCD PCD PCD PCDy PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD PCD Probable Probable Probable Probable Probable Probable Probable Probable Probable Probable Probable Probable Probable Probable Probable PCD Probable Probable

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Boaretto et al Table 2

Primers Used for Screening the DNAAF1 Gene and for Validating the Detected Mutations

Gene

Primer name

Amplicon position

Forward sequence

Reverse sequence

DNAAF1 DNAAF1 DNAAF1 DNAAF1 DNAAF1 DNAAF1 DNAAF1 DNAAF1 DNAAF1 DNAAF1 DNAAF1 DNAAF1 ARMC4 ARMC4 ARMC4 CCDC103 CCDC114 CCDC114 CCDC39 DNAH11 DNAH11 DNAH11 DNAH11 DNAH11 DNAH11 DNAH5 DNAH5 DNAH5 DNAH5 DNAH5 DNAH5 DNAH5 DNAH5 DNAH5 DNAH5 DNAI1 HYDIN HYDIN LRRC6 LRRC6 LRRC6 SPAG1

EX1 EX2 EX3 EX4 EX5 EX6 EX7 EX8 EX9 EX10 EX11 EX12 EX20 EX19 EX11 EX4 EX7 EX4int4 EX16 EX10 EX15 EX28 EX56 EX78 EX81 EX78 EX77 EX68 EX51 EX41 EX37 int34 EX31 EX24 EXint23 EX11 EX41 EX3 EX5 EX4 EX3 int1_int2

chr16:84178694-84179267 chr16:84182532-84182888 chr16:84183762-84184044 chr16:84188119-84188460 chr16:84189025-84189517 chr16:84193145-84193538 chr16:84199348-84199642 chr16:84203426-84204063 chr16:84205787-84206264 chr16:84208173-84208375 chr16:84209374-84209997 chr16:84211066-84211565 chr10:28101329-28101781 chr10:28149489-28149834 chr10:28233707-28233943 chr17:42979705-42980004 chr19:48807111-48807331 chr19:48815194-48815441 chr3:180336876-180337304 chr7:21627597-21628096 chr7:21639300-21639599 chr7:21678484-21678881 chr7:21804964-21805321 chr7:21934190-21934504 chr7:21939503-21939834 chr5:13700646-13701123 chr5:13701232-13701610 chr5:13735175-13735481 chr5:13788760-13789093 chr5:13820456-13820630 chr5:13830064-13830428 chr5:13839262-13839755 chr5:13850703-13851002 chr5:13870729-13871228 chr5:13871481-13872007 chr9:34500632-34500928 chr16:70986199-70986983 chr16:71218670-71218894 chr8:133644917-133645280 chr8:133650115-133650492 chr8:133669051-133669383 chr8:101163517-10117389

50 -GGCAAAGTCTTCCAAACCGG-30 50 -GTCATTAACCAAGCTGAAGTTG-30 50 -TTTGGGCAGGAATGGATGTG-30 50 -GTGACCGTGACCCCTCTG-30 50 -GGTCTCACTGTGTTGCCCA-30 50 -ACCAGGACAGGATATTGGCA-30 50 -AGTTCACCTCCCCTATTTATGT-30 50 -GGACACTCCCACATTCACCT-30 50 -GGAGCCCATCTTCACCGTA-30 50 -AACTAAGGCTGGGTTGACTG-30 50 -CCCCAGGCCTAACTTTCAGA-30 50 -CTGGTATTATAGGGGCCTGCC-30 50 -GATGGTTGAAAGGGTTTGCT-30 50 -CCTAAGTTTCTTTCCAGGCTACA-30 50 -GAACAAGACTCCGCATCCAA-30 50 -GACTCCCTTTCCTTCCTTTGTG-30 50 -GGAGCGAAGTGTGGTCCCA-30 50 -CCAGTTCTTCCCATAACCGC-30 50 -AGCACTTGTTTTTGTCTCCTTT-30 50 -AAGAGGTTTGAAGGGGAACAT-30 50 -CACAACAATGCAGTCTCTTCTTTT-30 50 -TGAGTTTTGTTTTGCCGAAGT-30 50 -TCTCTCATTGTCTAAGATTGCAA-30 50 -TGACGAAAACTGAAATAGAAAGGAG-30 50 -CTATGGGACTAAAGGATGCC-30 50 -GCAAAATCTCTACTACATGATAACAAA-30 50 -TGAGAACAACTGGCTGGGAT-30 50 -AAAGGGCTTTCACAATAACTCCT-30 50 -TGTCTTCAGATTTCCCTTTAGG-30 50 -TGTCATGGCTCTCATCAAGG-30 50 -ATGACCAGGGAGATGCATGA-30 50 -ACTGAAGGCAAAGGAAGCAA-30 50 -TCCCTGTATCCTTTTGTCTCAA-30 50 -AGGGGTTCGGTATCATCAACA-30 50 -GTGTGCAAAATAGTCCAGAATCA-30 50 -GCCCAAGGAGGTACAGACAG-30 50 -CGAGAACACTGCAGGGATGA-30 50 -GGATTAGGTAGCCAAGAAACAA-30 50 -TTGGAAAGAGAATACAGTTTGCT-30 50 -GCAGGAATTATTGTAGTTGCTCA-30 50 -AGCATAATAGAAGAAACAGTC-30 50 -CGGATGGACTTGTGAGAAGC-30

50 -TTGGCTGTGAAGATCGGGTA-30 50 -GTGCCATCCTCACTCCAT-30 50 -TTCTGAGAACTAAAGGTGATCCC-30 50 -CAGGGGAAGGTGATGGACAT-30 50 -TGGCATTTCAGATACTCCAGCT-30 50 -GTAGCCATCAAGCCTATTTCCA-30 50 -TCCCAAGATTAAAGACTGGGT-30 50 -AGAAGTGAAACTCAGGCCCC-30 50 -TGGTAGACATGGTTCAAGGGT-30 50 -GGTGGAATTAAGCTGCCTGT-30 50 -TCAGGGTAAGGCTGAGTGAC-30 50 -TCACACAAACAGGAGCTAGCC-30 50 -GGAGGAAGGAGTCTAATTCAGC-30 50 -GACATGATCTTGCAGGGTCA-30 50 -TCTATGGGAATGCGGTGTTC-30 50 -AGCAGGCTTAGGTTCAGGG-30 50 -GATGGAGGCGCAGGTCCT-30 50 -TTCCCTGGGCCATTCAATCT-30 50 -GGCTACTACATGCCCATAGTG-30 50 -GGGAACATTTAGTCAGAATGGTTT-30 50 -CTCAGGAGGCAACAAGATCA-30 50 -CAGCACCAGACAGTTATTCTTCC-30 50 -GGAGATGTACTGTGGAAAGC-30 50 -CCCCTTGCATGGAGAAAAA-30 50 -GAGTGAGGAGATGCTGAGAA-30 50 -TGAGGGAATCTTGCTAATTCTT-30 50 -CATAGCTGGTTTACACTGCAGT-30 50 -CAATTGAAAAGGGCAGTGGGA-30 50 -ACTGTCAACCTGAATTCCCA-30 50 -GGGGATTCTCTTATGTTTTCAGG-30 50 -TGAATGAACTTACATCTGGCTCA-30 50 -AGTTCGGTTGTCTGTGAGTT-30 50 -TGGAGGAATGTGAGATACTGTCTT-30 50 -TGTGGGTTAGAGGGCAATAAG-30 50 -TCCAGATAATCACACAGTTCACT-30 50 -TCAAGGTGGTTAAGTGCAGAAG-30 50 -TGAGCACGTGAACCCAGC-30 50 -GCTTACACCCTCAGAGTTCCT-30 50 -AAAATCAACCTGGGGCTGAT-30 50 -ACTTCATGGTGAGCAATGCA-30 50 -GGTAAAGACATGAGCAAAAC-30 50 -TGGGCAGATAAACAGCTTAAAC-30

SPAG1 EX16 RSPH4A EX3 RSPH9 EX4

chr8:101245521-101245729 50 -CCGCAGTGGTATAGCAACAG-30 chr6:116949009-116949545 50 -AGGAAGTGGAAGAGGAAGATGT-30 chr6:43624198-43624496 50 -TGACAGTGGTTGACAGGGT-30

50 -GGCTTTCACGTTCCCATCAG-30 50 -GCAAAGCTCCTACCTGAGAGA-30 50 -GACAGATCCCATGGCCTCC-30

Amplicon size (bp) 574 357 283 342 493 394 295 638 478 203 624 500 453 346 237 300 221 248 429 500 300 398 358 315 332 478 379 307 334 175 365 494 300 500 527 297 655 225 364 378 333 Approximately 500/12000 209 546 299

Despite the small sample size, the observed mutation frequencies were consistent with other data reported for European and non-European samples12; the only exception was DNAI1, a gene associated with ODA defects. DNAI1 mutations usually account for about 10% of PCD patients,21 and in the studied group only one monoalleic mutation was identified in this gene and all of the remaining patients with ODA defects carried mutations in other genes. The comparable frequencies of the involved PCD genes worldwide suggest that the distribution of the deleterious alleles is mainly driven by the rate of spontaneous mutations in each gene (mutation-selection balance) rather than by

within-population genetic dynamics. Accordingly, the majority of the mutations here identified (68% of the total) were novel and mapped along the entire coding region of a gene, including exons where mutations had never been reported before. As an example, five of six mutations identified in DNAH11 (2 nonsense, 2 missense, and 1 splice-site) were private, thus confirming previous screenings of this gene that mostly detected novel mutations.30 In line with this trend, a de novo mutational event (c.1442T>G, p.Leu481Ter) was identified in the ARMC4 gene. This mutation distribution deters the use of screening methods that focus on specific mutational hot spots and emphasizes

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PCD Diagnosis by Targeted Gene Panel Table 3

Genetic Findings of Patients Carrying Biallelic and Monoallic Mutations

Causative Proband ID gene Exon

Nucleotide change Protein change

References

Zygosity

Siblings tested

ACMG score

c.8498G>A c.5710-2A>G c.3712G>T c.11583C>A c.6132delT c.3484C>T c.3598þ1G>A c.3484C>T c.13595G>T c.5034C>A c.13183C>T c.2753G>T c.12796_ 12801delinsATA c.9304G>A c.4922C>G c.3080G>A c.2855G>A c.1442T>G

p.Arg2833His p? p.Glu1238Ter p.Ser3861Arg p.Ala2045Glnfs p.Gln1162Ter p? p.Gln1162Ter p.Gly4532Val p.Cys1678Ter p.Arg4395Ter p.Gly918Val p.Phe4266_ Asn4267delinsIle p.Gly3102Ser p.Ser1641Ter p.Cys1027Tyr p.Trp952Ter p.Leu481Ter

New Failly et al20 New Djakow et al21 New New New New New New New New Boon et al22

Comp het

Only child

Comp het

Only child

Comp het

Aff brother

Likely ODA þ IDA Pathog/Pathog Pathog/Likely ODA Pathog Pathog/Pathog ODA

Comp het

Aff brother

Pathog/Pathog

Comp het

Aff brother

Homo Comp het

Only child Only child

Likely ODA Pathog/Pathog Pathog/Pathog IDA VUS/Likely NU Pathog

Comp het

Only child

11144

Ex.4 Ex.3 LRRC6 Ex.5 Ex.4 SPAG1 Ex.16 Ex. 1-2 CCDC103 Ex.4 Ex.4 CCDC114 Int.4 Ex.7 CCDC39 Ex.16 HYDIN Ex.41 Ex.3 RSPH4A Ex.3

c.426A>C c.220G>C c.598_599delAA c.299T>C c.2014C>T 11,971 bp dely c.331C>T c.461A>C c.372þ1G>A c.742G>A c.2190delA c.6369delT c.211C>T c.1391G>A

p.Leu142Phe p.Ala74Pro p.Lys200Glufs p.Ile100Thr p.Gln672Ter p? p.Arg111Ter p.His154Pro p? p.Ala248Serfs p.Glu731Asnfs p.Asp2124Thrfs p.Arg71Ter p.Gly464Glu

New Kott et al23 Kott et al23 New Knowles et al24 Knowles et al24 New Panizzi et al25 New Onoufriadis et al26 Merveille et al27 New New Kott et al28

11078

RSPH9

Ex.4

c.596dupA

p.Thr200Aspfs

variants CCDC114 DNAH11 DNAH5 DNAH5 DNAH5 DNAI1

Ex.4 Ex.10 Ex.77 Int.34 Ex.41 Ex.11

c.253C>T c.1848G>A* c.13486C>T c.5710-2A>G c.6763C>T c.909delG

p.Gln85Ter p.Gln616Gln p.Arg4496Ter p? p.Arg2255Ter p.Lys303Asnfs

Biallelic variants 11069 DNAH5 11072

DNAH5

11074

DNAH5

11077

DNAH5

11166

DNAH5

11057 11174

DNAH11 DNAH11

11228

DNAH11

11177 11053

ARMC4 ARMC4

11052

LRRC6

11071 11047 11051 11073 11216 11175

Monoallelic 11056 11182 11070 11163 11170 11169

Ex.51 Int.34 Ex.24 Ex.68 Ex.37 Ex.23 Int.23 Ex.23 Ex.78 Ex.31 Ex.81 Ex.15 Ex.78 Ex.56 Ex.28 Ex.20 Ex.19 Ex.11

New New New New New

Homo Aff brother Comp het Healthy brother (maternal/ de novo) Comp het Healthy sister

TEM

ODA

VUS/Pathog

NU

VUS/VUS Pathog/Pathog

NU ODA þ IDA

IDA

ODA þ IDA

Comp het

Healthy brother

Comp het

Healthy brother

VUS/Likely Pathog Pathog/Likely Pathog Pathog/Pathog

Comp het

Healthy sister

Pathog/Pathog

ODA þ IDA

Comp het

Only child

Pathog/Pathog

ODA þ IDA

Homo Comp het

Healthy sister Healthy sister

Pathog/Pathog Pathog/Pathog

MTd NU

Homo

Healthy sister

CP

New

Homo

Aff sister, healthy brother

Likely Pathog/ Likely Pathog Pathog/Pathog

CP

New New Failly et al20 Failly et al20 New New

Het Het Het Het Het Het

NA NA Only child Aff sister NA Only child

Pathog Pathog Pathog Pathog Pathog Pathog

ODA NU ODA ODA ODA þ IDA ODA

ODA

*This mutation falls in the last base of exon 10 and is predicted to affect splicing. y The complete nomenclature of this variant is c.61þ201_POLR2K:c.140þ1169SPAG1_del. ACMG, American College of Medical Genetics and Genomics; Aff, affected; Comp het, compound heterozygous; Het, heterozygous; ID, identification; IDA, inner dynein arm; NA, not applicable; NU, normal ultrastructure; ODA, outer dynein arm; Pathog, pathogenic; TEM, transmission electron microscopy; VUS, variant of uncertain significance.

the importance of a comprehensive sequencing even for the largest PCD genes. However, the generation of a global database of variants detected by NGS in PCD patients would greatly facilitate the identification of common relevant variants. In our sample, we identified eight mutations already reported in several patients with different ethnicity (ie, c.2014C>T and c.61þ201_POLR2K:c.140þ1169SPAG1_del (ID 11047,

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exons 1-2 11,971 bp del) in SPAG1, c.598_599delAA in LRRC6, c.1391G>A in RSPH4A, c.2190delA in CCDC39, c.5710-2A>G in DNAH5, c.742G>A in CCDC114, c.461A>C in CCDC103), thus confirming the existence of some recurring mutations. Also among the newly identified variants, the nonsense p.Gln1162Ter mutation in the DNAH5 gene was found in two compound heterozygous probands (11074 and 11077) in combination with other two

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A Homozygous 11%

Type of idenƟfied mutaƟons Known knonwn

12 DNAH5 27%

DNAH11 16%

8 ARMC4 11%

9

4

4 2 2

3

sit e

on

n/ rƟ o

Sp lic e-

ns en

se

se SPAG1 5%

2

1

0

RSPH4A 5%

se

CCDC39 CCDC114 CCDC103 5% 5% 5%

In

HYDIN 5%

4

le Ɵ

LRRC6 11%

de

RSPH9 5%

iss en

No mutaƟons 43%

7

6

M

Compound heterozygous 32%

New

10

No

Monoalleic mutaƟon 14%

B

Mutated PCD genes in solved cases

Figure 1

Molecular screening of 24 PCD genes in 44 unrelated index cases. A: Percentage of individuals with a molecular diagnosis versus individuals without a molecular diagnosis (left) and distribution of molecular diagnoses across 24 PCD genes (right). B: Type of identified mutations in the molecularly solved patients. PCD, primary ciliary dyskinesia.

different mutations. Despite being unrelated, both patients come from Southern Italy, suggesting a possible subpopulation diffusion of founder mutations. Recent testing studies identified some ancestral and recurrent mutations in the SPAG1 gene and reported an unexpected high prevalence of these in a Caucasian sample from the Czech Republic (24% of all PCD cases).24,31 If replicated, these results would have important implications for the genetic diagnostic procedure of PCD. In this cohort of Italian patients, two of the well-known ancestral SPAG1 mutations were identified, but they accounted for only 1 case of 44, thus excluding, at least in Italy, the preliminary test of SPAG1 as an effective genetic diagnostic strategy. At present, the challenge of using NGS in diagnostic screenings is to discriminate the causative mutation among the hundreds detected in each patient. The identification of a variant already reported in a mutation database, as for example HGMD and LOVD, provides support of pathogenicity, but particular attention must be paid in this circumstance. Because for example, two unrelated probands (11057 and 11078) carried two variants in the DNAH11 gene (c.8990G>A; p.Arg2997Gln and c.10739G>A; p.Arg3580His) in homozygous and heterozygous status, respectively. Both these variants are present in HGMD and LOVD databases because they were identified in a PCD patient and reported as likely pathogenic by Boon et al.22 The parental DNA analysis of both patients revealed that the variants were in cis status as inherited from the same parent. Consequently, their combination could not be considered causative of PCD. In addition, convincing mutations were found for both patients in the same gene (11057) or even in a different gene (11078). The 11057

proband had a homozygous nonsense mutation in DNAH11 (p.Arg4395Ter), and the 11078 proband was homozygous for a truncating frame-shift insertion (p.Thr200Aspfs) in RSPH9 gene. Furthermore, this latter showed slow beatings and central pair defects, a ciliary phenotype consistent with the involvement of the RSPH9 gene rather than DNAH11. These findings suggest that the causative role of p.Arg2997Gln and p.Arg3580His mutations in DNAH11 needs further support and underline how a match with a mutation database is not sufficient to define a variant as pathogenic if not corroborated by additional supportive genetic and/or functional data. In this context, it is noteworthy that the trans status of compound heterozygous variants was not assessed for a number of published PCD mutations; therefore, the availability of the proband’s relatives remains fundamental for variant interpretation. This study, to the best of our knowledge, is the first genetic screening performed systematically for 24 PCD genes in a cohort of Italian PCD patients. Despite our PCD targeted gene panel being one of the largest published to date, 25 of 44 cases (57%) did not reach a confirmative molecular diagnosis. Among them, six patients carried a single heterozygous mutation in four genes: four in DNAH5 and one in DNAH11, DNAI1, and CCDC114 each. All these monoallelic variants are disrupting mutations, all have a pathogenic ACMG score, and in all patients the observed ciliary ultrastructure defects completely agree with the involved gene. On the basis of these considerations, sequencing data of these patients were carefully evaluated, and low-covered exons were recovered by Sanger sequencing, thus excluding potential false-negative calls. In addition, 50 and 30 UTRs

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PCD Diagnosis by Targeted Gene Panel variants were considered because of a growing evidence indicating that point mutations in these regions may account for an underestimated proportion of patients with Mendelian diseases.32 Unfortunately, no candidate mutations in the UTR regions were identified. The hypothesis of a digenic inheritance was also investigated, but no combinations of relevant variants in different PCD genes were identified both in patients with a monoallelic variant and in all of the molecularly unexplained cases. These data suggest that causative mutations not detectable with typical NGS screenings of coding regions (eg, mutations in promoters, deep introns, and other regulatory sequences) may be probably undervalued in PCD. In addition, part of the missed diagnoses could possibly be explained by structural variants; a recent study has indeed proposed that deletions spanning from one to three exons may account for about 9% of PCD patients.33 However, current algorithms for the detection of large duplication/deletions from NGS data have a poor performance in terms of sensitivity and specificity, and further improvements are still required. All these observations warrant the need to implement cost-effective strategies and methods to systematically identify and interpret all kinds of genetic variation that can contribute to the disease phenotype. Nineteen patients, 11 of whom with ODA þ IDA defects, did not present any candidate change or variant in the tested genes. The genetic knowledge on PCD has grown exponentially over the past 2 years, with the identification of five new autosomal disease genes (RSPH1, RSPH3, DNAH8, CCDC151, DIRC4/GAS8). These genes are yet to be integrated into the developed NGS panel; therefore, some patients may actually carry mutations in one of them. Despite this, it should be noted that the newly identified genes seem to account for only a low causative fraction of PCD cases and that only one of them (DIRC4/GAS8) correlates with ODA þ IDA defects.34 This supports the existence of further genetic heterogeneity and predicts an ever-growing list of PCD genes in the next years. In this context, the improvement of comprehensive and accurate mutation databases, the development of new capabilities for genetic variant interpretation, as well as the establishment of national clinical and molecular registers would be crucial to grant an efficient and effective genetic diagnosis of PCD in the future. This is even more remarkable considering the forthcoming use of the whole genome sequencing in the clinical practice and the importance of the early diagnosis for timely treatments and better outcomes for PCD patients.

Acknowledgments We thank all of the families for their participation to the study. We are grateful to Drs. Nicole Enzo, Silvia Quartesan, Silvia Grancara, and Elena De Col for their organizational work.

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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.jmoldx.2016.07.002.

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