Treatment of Inherited Homocystinurias

Review Article

Treatment of Inherited Homocystinurias Manuel Schiff1

Henk J. Blom2

1 Reference Center for Inherited Metabolic Diseases, APHP and Inserm

U676, Hôpital Robert Debré, Paris, France 2 Metabolic Unit, Department of Clinical Chemistry and Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center Amsterdam, The Netherlands

Address for correspondence and reprint requests Manuel Schiff, MD, PhD, Centre de Référence Maladies Métaboliques, Hôpital Robert Debré, 48, boulevard Sérurier, 75019 Paris, France (e-mail: [email protected]).

Abstract

Keywords

► homocystinurias ► cystathionine βsynthase deficiency ► remethylation disorders

Inherited homocystinurias, have in common, accumulation of homocysteine with subsequent neurotoxicity; they also encompass two distinctive clinical entities: classical homocystinuria due to cystathionine β-synthase (CBS) deficiency and the rare inborn errors of cobalamin and folate metabolism. In the latter group, remethylation disorders of homocysteine to methionine (chiefly CblC defect and 5,10-methylenetetrahydrofolate reductase [MTHFR] deficiency) are by far the most frequently encountered situations. The natural history of CBS deficiency is relatively well known and described. Similarly, clinical presentations of remethylation defects are becoming better recognized and reported. Conversely, few data are available regarding treatment of these disorders, especially for remethylation defects. In this review, after an overview of the metabolic pathophysiology and the clinical features of inherited homocystinurias due to CBS deficiency, CblC defect, and MTHFR deficiency, we focus on present and prospective therapeutic approaches.

Introduction The many conditions associated with high homocysteine levels encompass a wide range of clinical manifestations.1 The normal level of plasma total homocysteine (tHcy) is below 15 µM. However, the threshold of tHcy above which a metabolic disorder of homocysteine metabolism should be suspected and a specific therapy initiated is around 50 µM.2,3 Indeed, these metabolic conditions mainly include inherited disorders of homocysteine metabolism and acquired nutritional cobalamin (Cbl, vitamin B12) or folate deficiencies. Inherited disorders of homocysteine metabolism comprise disorders of the transsulfuration pathway with cystathionine β-synthase (CBS) deficiency (or classical homocystinuria) and disorders of remethylation of homocysteine to methionine. The latter includes 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency and inherited disorders of Cbl absorption, transport, and intracellular metabolism4,5 and the rare congenital folate malabsorption disorder.6 Intracellular remethylation defects include disorders that have defective methionine synthesis in common: MTHFR

deficiency impairs methyl-tetrahydrofolate synthesis, defective lysosmal release of cobalamin (CblF and CblJ) and defects in cytosolic reduction and transport of hydroxocobalamin (CblC and CblD) impair the synthesis of both methyl- and adenosylcobalamin, and isolated deficiencies of methionine synthase (CblE and CblG) or CblD variant with isolated homocystinuria are associated with defective methyl cobalamin synthesis. In CBS deficiency, in addition to severe elevations of tHcy, plasma methionine level is also increased. In remethylation disorders, in addition to increased tHcy there is a low (or lownormal) level of methionine in plasma due to the ineffective conversion (remethylation) of homocysteine to methionine. Regarding the prevalence of the remethylation disorders, CblC defect (the most frequent inherited disorder of intracellular Cbl metabolism) and MTHFR deficiency are, albeit rare, by far the most frequent. In this article we review therapeutic aspects of CBS deficiency and remethylation disorders limited to MTHFR deficiency and CblC defects, although our conclusions on CblC defect and MTHFR deficiency may be extrapolated to some of the other remethylation defects.

received June 19, 2012 accepted after revision September 11, 2012

Copyright © by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.

DOI http://dx.doi.org/ 10.1055/s-0032-1329883. ISSN 0174-304X.

Downloaded by: Vrije Universiteit. Copyrighted material.

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Schiff, Blom

Biochemical and Pathophysiologic Aspects Homocysteine is a sulfur amino acid not used for protein synthesis but synthesized from the essential amino acid methionine via transmethylation. It is located at a branchpoint of metabolic pathways and is either irreversibly degraded via the transsulfuration pathway to cysteine or remethylated back to methionine (►Fig. 1). In the transmethylation pathway (►Fig. 1) methionine adenosyltransferase activates the methyl group of methionine by ATP via synthesis of S-adenosylmethionine (AdoMet).7 AdoMet donates methyl groups to, for example, DNA, RNA, proteins, guanidinoacetate, and phosphatidylethanolamine. By doing so, S-adenosylhomocysteine (AdoHcy), which is a potent inhibitor of most methylation reactions, is formed. AdoHcy hydrolase hydrolyzes AdoHcy to adenosine and homocysteine. The methylation reactions are ubiquitously distributed and have crucial cellular functions, especially in the central nervous system.

lysis of cystathionine to cysteine and α-ketobutyrate. CBS is mainly expressed in the liver and kidneys. Besides protein synthesis, cysteine is used in the synthesis of taurine and glutathione, a strong antioxidant, and involved in transport processes and detoxification of many xenobiotics.

Remethylation Homocysteine remethylation to methionine is catalyzed by the methionine synthase enzyme,8 which transfers a methyl group from 5-methyltetrahydrofolate (5-methylTHF) via cobalamin to homocysteine. This reaction links the folate cycle with homocysteine metabolism (►Fig. 1). Methionine synthase is ubiquitously expressed, probably in all cells except the red blood cells. Another homocysteine remethylation system, betaine-homocysteine methyltransferase (►Fig. 1), is mainly expressed in the liver and kidneys.9 Betaine-homocysteine methyltransferase remethylates homocysteine using a methyl group derived from betaine and is presumably responsible for up to 50% of homocysteine remethylation. Betaine is formed via oxidation of choline.

Transsulfuration In the transsulfuration pathway (►Fig. 1) homocysteine is irreversibly degraded to cysteine by the action of two pyridoxal phosphate (the active form of vitamin B6)–dependent enzymes: CBS and cystathionine γ-lyase. CBS catalyzes the condensation of homocysteine and serine to cystathionine, and cystathionine γ-lyase subsequently catalyzes the hydro-

Folate Folate is a B vitamin whose role is to transfer and transport one-carbon groups; it can be formyl in its most oxidized form or methyl in its most reduced form. The circulation form is 5-methylTHF, which is formed from 5,10-methyleneTHF by MTHFR, a vitamin B2 requiring enzyme. In addition,

Fig. 1 Homocysteine metabolism and the folate cycle. CBS and MTHFR deficiencies (surrounded by a black dotted circle) and MTR and MTRR deficiencies can all lead to accumulation of homocysteine. AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; AICAR, 5aminoimidazole-4-carboxamide ribonucleotode; ATP, adenosine triphosphate; BHMT, betaine-homocysteine methyltransferase; CBS, cystathionine β-synthase; CTH, cystathionine γ-lyase; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; FAICAR, formyl-AICAR; MAT, methionine adenosyltransferase; MTHFD, methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; SAHH, S-adenosylhomocysteine hydrolase; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; TYMS, thymidylate synthase. (Adapted from: Blom HJ, Smulders Y. Overview of homocysteine and folate metabolism. With special references to cardiovascular disease and neural tube defects. J Inherit Metab Dis 2011;34(1):75–81.) Neuropediatrics


Treatment of Inherited Homocystinurias


Schiff, Blom

5,10-methyleneTHF donates a one-carbon unit in the conversion of dUMP into dTMP, and 10-formylTHF can donate onecarbon groups for purine biosynthesis (►Fig. 1).

The accumulating homocysteine (in all three conditions) is a well-known multisystem toxic agent,1 either directly or indirectly via conversion to AdoHcy (►Fig. 1), that potentially inhibits many essential methyltransferases.10 Direct homocysteine toxicity involves endothelial toxicity1 and neuronal cell death.11 Remethylation defects (CblC and MTHFR) not only result in severe elevations of homocysteine but also in a shortage of methionine, required for protein synthesis and AdoMet synthesis. The latter causes via this route an additional reduction of cellular methylation capacity, especially in the central nervous system.12 CblC defect13 is an inborn error of intracellular cobalamin metabolism due to a genetic defect in MMACHC. After normal dietary intake, intestinal absorption, and blood transport, cobalamin is delivered in the cytoplasm where it becomes bound to MMACHC. This protein has been shown to catalyze dealkylation of alkylcobalamins such as adenosylcobalamin and methylcobalamin14 and the decyanation of cyanocobalamin.15 In the absence of normal MMACHC, neither methylcobalamin (the cofactor of methionine synthase) nor adenosylcobalamin (the cofactor of methylmalonyl-CoA mutase) is functional, with subsequent defects of methionine synthase (remethylation defect) and methylmalonyl-CoA mutase (methylmalonic acid [MMA] accumulation), respectively (►Fig. 2). In MTHFR deficiency, the methyl donor 5-methylTHF cannot be produced (►Fig. 1), which secondarily impairs methionine synthase function and subsequent remethylation (►Fig. 2). If homocysteine toxicity associated with methionine cellular depletion likely explains the pathogenesis of MTHFR deficiency, the pathophysiology of CblC defect remains incompletely understood.16,17 One potential additional mechanism in CblC defect is the trapping of 5-methylTHF due to methionine synthase dysfunction and irreversible activity of MTHFR. This causes folates to accumulate as 5-methylTHF, resulting in cellular deficiency of the different folates. Toxic accumulation of MMA in CblC defect might play an additional role.17 Besides MTHFR deficiency and CblC defect, remethylation of homocysteine to methionine can also be impaired in abnormalities of any of the steps of folate or Cbl metabolism (dietary intake, intestinal absorption, blood transport of Cbl by transcobalamin, cellular uptake, and intracellular metabolism). Remethylation disorders related to intracellular Cbl metabolism dysfunction include CblF, CblJ, CblC, CblD, CblDHCy, CblE, and CblG defects (►Fig. 2).

Biochemical Features of CBS Deficiency, CblC Defect, and MTHFR Deficiency The characteristic biochemical pattern of CBS deficiency is, in addition to severely elevated plasma homocysteine (preferably measured as tHcy), high-normal to elevated serum methionine and low-normal to reduced serum cysteine. Remethylation disorders combine homocysteine accumulation with increased cystathionine and low-normal serum

Fig. 2 Intracellular cobalamin metabolism pathway and its defect. To date, 10 complementation-group defects of the cobalamin pathway have been described. After binding to transcobalamin receptor (TC-R), cobalamin bound to TC enters the cell via lysosome-mediated endocytosis and is released through proteolysis. Export from the lysosome into the cytoplasm is defective in patients with the cblF and recently described 68 CblJ defects. The steps in the cytosol after lysosomal release are defined by the complementation groups cblC and cblD. The exact form of cobalamin at this stage is unclear (as indicated by “Cblx”). In the cytosol, cobalamin is reductively methylated by methionine synthase reductase (cblE) to methylcobalamin, the cofactor for methionine synthase (cblG). After its transport into the mitochondrion, cobalamin is converted to adenosylcobalamin, the cofactor for methylmalonyl–coenzyme A mutase (mut), by cobalamin adenosyltransferase (cblB). CblD defect can cause isolated methylmalonic aciduria (CblD-MMA complementation group), isolated homocystinuria (CblD-HCy), or both (CblD). In all the earlier mentioned conditions with defective remethylation (TC and TC-R deficiency, CblF, CblJ, CblC, CblD, CblD-HCy, CblE and CblG defects), there is homocysteine accumulation due to dysfunction in methionine synthesis. (Adapted from: Coelho D, Suormala T, Stucki M, et al. Gene identification for the cblD defect of vitamin B12 metabolism. N Engl J Med 2008;358(14):1454–1464).

methionine. In CblC defect, MMA accumulates both in plasma and urine.

CBS Deficiency, CblC Defect, and MTHFR Deficiency: Clinical Overview CBS Deficiency CBS deficiency is characterized by developmental delay/intellectual disability, ectopia lentis and/or severe myopia, skeletal abnormalities, and thromboembolism. The expression of all clinical signs is extremely variable.18 Two phenotypic variants are recognized, B6-responsive homocystinuria and B6-nonresponsive homocystinuria. B6-responsive homocystinuria is usually milder than the nonresponsive variant.19 In most untreated individuals ectopia lentis may occur around 8 years of age. Individuals are often tall and slender with a “marfanoid” habitus and are prone to osteoporosis. Thromboembolism is the major cause of early death and morbidity. IQ in individuals with untreated homocystinuria ranges widely, from 10 to 138.18 In B6-responsive untreated individuals the mean IQ is 79 versus 57 for those who are B6nonresponsive. Neuropediatrics


Pathophysiology

Schiff, Blom

CblC Defect and MTHFR Deficiency Clinical signs of remethylation defects are mainly neurologic. Neonatal and early-onset patients exhibit acute neurologic distress. In childhood, patients exhibit nonspecific mental retardation often associated with acquired microcephaly. Without appropriate therapy remethylation-defective patients (CblC and MTHFR) may develop acute or rapidly progressive neurologic deterioration, sometimes leading to death. Adolescents and adults exhibit, after a period of normal development or mild developmental delay, a rapid mental and/or psychiatric deterioration. These patients typically have signs of subacute combined degeneration of the cord. In addition, adults can be asymptomatic or present with isolated stroke. In CblC defect, 5-methylTHF accumulates due to the block at methionine synthase (the 5-methylTHF trap), causing a functional cellular folate deficiency. This explains the hematologic signs (megaloblastic bone marrow leading to macrocytic anemia and/or pancytopenia) that are not present in MTHFR deficiency.20 In CblC defect severe (occasionally fatal) multisystem deterioration may occur. This includes hemolytic and uremic syndrome, cardiomyopathy, and interstitial pneumonia, which all share an identical pathologic hallmark (i.e., thrombotic microangiopathy).16 In addition, a peculiar and poorly understood retinopathy with nystagmus is often present. In both groups of inherited homocystinurias isolated arterial and venous occlusive disease may present at any age. Due to the low incidence of arteriosclerosis and thrombosis in children or adolescents, homocystinurias should be excluded in any case presenting with these clinical manifestations.

Newborn Screening Expanded newborn screening programs with tandem mass spectrometry on dried blood spot allow the screening of CBS deficiency, which is detected by an elevated methionine level and a high methionine-to-phenylalanine ratio confirmed by a second-tier molecular and biochemical assays. However, these current screening approaches fail in detecting large groups of CBS-deficient patients, in particular those who are pyridoxine responsive. For remethylation defects an elevation of propionylcarnitine (C3) may indicate CblC defect. However, elevation of C3 is a common finding with poor specificity, unless a second-tier test is used for the determination of MMA and tHcy in the same blood spot. MTHFR deficiency is not currently detected by expanded newborn screening programs; however, as recently proposed21 and efficiently demonstrated, the implementation of a low methionine cutoff point, followed by tHcy determination in the same blood spot, appears to be a sensitive and specific test for the screening of remethylation disorders in the newborn period.22

Focusing on Neurologic Signs: When Should the Child Neurologist Be Searching for Inherited Homocystinurias? Because newborn screening does not allow unequivocal diagnosis of any of the inherited homocystinurias (see earlier), early identification and prompt management are crucial to ensure Neuropediatrics

the best possible outcome, especially in CBS and MTHFR deficiencies (treatable disorders). Neurologic symptoms are the most common symptoms in these disorders with some agerelated specific aspects (►Table 1). Neonates and young infants affected with remethylation defects exhibit acute neurologic deterioration, which may be fatal or leave severe neurologic impairments, often with hydrocephalus.23 Neonatal seizures, sometimes with a burst suppression pattern, may be seen in MTHFR deficiency.24 Older children typically exhibit progressive triphasic deterioration. After an initial period of normal development, older children acquire microcephaly and nonspecific psychomotor retardation (second phase). They may exhibit abrupt deterioration (third phase) with respiratory failure, which may be fatal. Adolescents show normal development or mild developmental delay initially and then may experience rapid neurologic and/or behavioral/psychiatric deterioration.25 A few remethylation defective patients may have symptoms of subacute combined degeneration of the spinal cord. Adults may be asymptomatic or present with isolated stroke or other arterial and/or venous occlusive disease. These thromboembolic symptoms can also manifest at any age. CBS-deficient patients may exhibit nonspecific developmental delay in infancy, sometimes with autistic features.26 Lens dislocation gives a peculiar visual behavior. Marfanoid features may be present. A stroke or stroke-like episode may be seen in CBS-defective patients at any age. Similarly, these patients may exhibit unexplained psychiatric acute symptoms (schizophrenia-like). These neuropsychiatric symptoms may remain apparently isolated, but a thorough clinical history and examination will often reveal associated features such as marfanoid habitus, skeletal abnormalities, lens dislocation in CBS deficiency, megaloblastic bone marrow in CblC defect, and, for all the disorders (CBS, CblC, MTHFR), arterial or venous occlusive disease.

Specific Issues Regarding Therapy Therapeutic Goals The general therapeutic goal is to reduce tHcy accumulation and, for remethylation disorders, to bypass the remethylation defect, thereby maintaining normal methionine and folate concentrations. This should correct the hematologic abnormalities and ensure normal neurologic development. In both conditions normalization of plasma tHcy levels is ideal, but in practice this is difficult if not impossible to achieve. However, considerable experience with CBS-defective patients has shown that treatment prevents further thromboembolic events even when the tHcy levels remain clearly above the normal range.2,3 Decreasing tHcy to 50 to 70 µM is therefore a more reasonable goal in many patients with CBS deficiency as well as remethylation disorders.

Available Treatment Options To date, all the remethylation disorders are similarly treated with the combined supplementation of vitamin B12, vitamin B9 (folate), vitamin B6, betaine, and methionine, although the dosage and route of administration may vary with the type of defect (►Table 2).




Schiff, Blom

Table 1 Most frequent neurologic symptoms encountered in inherited homocystinurias according to age CBS

CblC

MTHFR

Acute neurologic deterioration

–

þ

þ

Neonatal seizures

–

–

þ

Developmental delay

þ

þ

þ

Neonatal period and early infancy

Hydrocephalus

–

þ/

þ

Nystagmus

–

þ

–

Stroke, strokelike, isolated arterial or venous occlusive disease

þ

þ

þ

Neurologic deterioration (acute, subacute, or chronic) often triphasic (see text)

–

þ

þ

Spastic tetraparesis (myelopathy)

–

þ

þ

Peripheral neuropathy

–

þ

þ

Cerebellar ataxia

–

þ

þ

Lens dislocation (peculiar eye behavior)

þ

–

–

Autistic features, behavioral disorders

þ

–

–

Developmental delay

þ

þ

þ

Seizures

þ

þ

þ

Psychiatric symptoms

þ

þ

þ

Nystagmus

–

þ

–

Neurologic deterioration (acute, subacute, or chronic)

–

þ

þ

Combined degeneration of the spinal cord

–

þ

þ

Stroke, strokelike, isolated arterial or venous occlusive disease

þ

þ

þ

Mental retardation

þ

þ

þ

Unexplained acute psychiatric symptoms

þ

þ

þ

Seizures

þ

þ

þ

Myoclonia

–

þ

þ

Nystagmus

–

þ

–

Adolescence to adulthood

Abbreviations: Cbl, cobalamin; CBS, cystathionine β-synthase; MTHFR, 5,10-methylenetetrahydrofolate reductase.

Table 2 Main treatment options in CBS deficiency, MTHFR deficiency, and CblC defect Defect

Methionine

B6 (mg/d)

Folinic acid (mg/d)

OHCbl

Betaine 2–3 times daily (oral)

CBS

Low methionine diet

B6-R

1–5

1 mg/d to 1 mg/wka Oral

Children: 150–250 mg/kg/d Adults: 5–20 g/d

Newborn and infants: 50–250 Children and adults: 50–500 B6-NR 50–100 MTHFR

40–50 mg/db

50–100

5–30

1 mg/d to 1 mg/wk Oral


CblC

40–50 mg/db

50–100

5–15

1 mg/d Long-term frequency? Intramuscular


Abbreviations: B6-R, pyridoxine-responsive homocystinuria; B6-NR, pyridoxine-nonresponsive homocysttinuria; Cbl, cobalamin; CBS, cystathionine βsynthase; MTHFR, 5,10-methylenetetrahydrofolate reductase. a According to serum B12 levels. b Starting dosage, further adapted to methionine plasma level.

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Late infancy and childhood

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Regarding CBS deficiency, therapy is more standardized with a longer experience available.19 It is based on (1) increasing residual CBS activity with the use of vitamin B6 (in B6-responsive patients); (2) decreasing the load on the affected pathway and replacing deficient products with a low methionine diet, limitation of natural proteins, amino acid mixture, special low-protein foods, and supplementation of cysteine (200 to 500 mg/d); and (3) increasing remethylation to methionine with folate, vitamin B12, and betaine to reduce tHcy accumulation. Vitamin B12 is the cofactor of methionine synthase. Its natural form, hydroxocobalamin (OHCbl),27 is more effective than the synthetic form, cyanocobalamin (CNCbl).28–31 In addition, in control fibroblasts, intracellular uptake and retention are significantly higher for OHCbl than for CNCbl.32 In CBS deficiency OHCbl may be given orally (1 mg/d to 1 mg/wk) according to the serum B12 level3 to prevent Cbl deficiency, which may be related to MTHFR inhibition due to high AdoMet. In remethylation disorders initial treatment includes daily parenteral administration of OHCbl (1 mg/d). If MTHFR deficiency is confirmed, switching to oral OHCbl (1 mg/d to 1 mg/wk) or even stopping OHCbl supplementation may deserve discussion. Conversely, in CblC defect lifelong highdose intramuscular OHCbl injections are needed. The optimal interval between intramuscular injections remains to be determined,20 but recent data support the need for longterm daily doses.13 Moreover, as indicated by a report of a patient with CblC defect, higher OHCbl doses may sometimes be required to reduce the biochemical abnormalities and, possibly, to achieve clinical control.33 Specifically in CblC defect, the use of OHCbl might be favored becasue the MMACHC gene product also catalyzes the in vitro decyanation of cyanocobalamin,15 potentially explaining why CNCbl is relatively ineffective compared with OHCbl as demonstrated in vivo.34 Folate (or vitamin B9) is available in three different forms: folic acid, a very stable synthetic form of the vitamin used, for example, in food fortification35; folinic acid (5-formyl-THF), the most stable form of the reduced and active vitamin36; and 5-methylTHF (CH3-THF), the main natural and circulating form of the vitamin.37 A folinic acid formulation for parenteral administration is available for emergency treatment, whereas the other forms are available only for oral use. Whatever the disorder, folinic acid is more appropriate because it is the most stable reduced form and folic acid may exacerbate cerebral CH3-THF deficiency, especially in MTHFR deficiency.38 In addition, because dihydrofolate reductase is a slow reductor, oral folic acid will accumulate and may even inhibit folate-dependent transports and enzymes. In CBS deficiency folinic acid should be given orally (1 to 5 mg/d) to avoid folate depletion. Moreover, folate repletion may be necessary to permit a pyridoxine response, meaning that pyridoxine responsiveness should always be tested after potential folate depletion correction.39 Furthermore, CBS deficiency may exacerbate folate deficiency from other causes. In CblC defect, long-term high-dose oral folate supplementation is added to compensate for the methylfolate Neuropediatrics

trap40 and to correct the hematologic abnormalities.31 Because of the methylfolate trap 5-methylTHF does not work satisfactorily, and folinic acid can be used. The daily dose varies from 5 to 30 mg.20,28 Conversely, MTHFR deficiency is characterized by inadequate 5-methylTHF synthesis (►Fig. 1). Moreover, as shown by the low levels of 5-methylTHF in erythrocytes and cerebrospinal fluid (CSF), severe systemic and cerebral 5-methylTHF depletion occurs in MTHFR deficiency, which provides a rationale for exogenous 5-methylTHF administration.24 However, in our experience long-term 5-methylTHF oral supplementation in a daily dose of even 45 mg failed to correct the low CSF levels of 5-methylTHF.24 Possible explanations for this lack of effectiveness include insufficient drug dosage and/or drug instability.41 However, in utero administration of 5-methylTHF, but not folic acid, increased the survival of Mthfr/ mice, confirming the key pathogenic role of 5-methylTHF deficiency.42 Folinic acid (5 to 30 mg/d) may also be given in an attempt to enhance residual MTHFR activity.28 In addition, riboflavin (vitamin B2) is a cofactor of MTHFR and therefore may stabilize the mutant protein. It may theoretically be proposed in MTHFR patients despite the lack of clinical data except for one known responsive patient reported in a review article43 and an in vitro riboflavinresponsive mutation found in an MTHFR-defective patient.44 Vitamin B6 (or pyridoxine) via its action as the cofactor for CBS is given orally in pharmacological dose in CBS-deficient patients to detect possible B6-responsive individuals. There is no consensus on the dose and duration of B6, which is usually given from 100 to 500 to 1,000 mg/d during a several-week period, after which tHcy is determined to evaluate whether B6 has been effective in lowering (or even normalizing) tHcy. As discussed earlier, B6 should always be combined with folinic acid. In B6-responsive patients long-term therapy with pyridoxine and folate prevents further deterioration. In remethylation disorders B6 might theoretically enhance homocysteine removal and may be given at a low dose (50 to 100 mg). For pyridoxine-responsive CBS-deficient patients, pyridoxine should be kept at the lowest dosage able to achieve adequate metabolic control. Because of reported high-dose pyridoxine toxicity on the nervous system,45,46 daily dosages higher than 200 to 250 mg in newborns and young infants and 400 to 500 mg/d in children and adults should probably be avoided as a long-term treatment. In pyridoxine-nonresponsive patients, a daily dosage of 50 to 100 mg of pyridoxine can be added to the treatment.45 Betaine is derived from choline and is a substrate for the enzyme betaine-homocysteine methyltransferase and therefore acts as a methyl donor47 (►Fig. 1). Oral betaine supplementation decreases homocysteine levels. Despite widespread usage there is little consensus on betaine dosage and frequency of administration. Case studies and early literature48–50 have used doses of 150 to 250 mg/ kg/d in children and 5 to 20 g/d in adults usually given two or three times daily. These early data were further confirmed by pharmacokinetic studies showing that above 200




nine was added to betaine57 and the other showed good clinical and biologic results with combined methionine– betaine therapy.58 Methionine supplementation has also been used in other MTHFR-defective patients, with variable clinical effects.59–62 As a whole, whatever the remethylation defect, methionine depletion (as attested by plasma and/or CSF low levels) is rarely corrected by betaine therapy alone, whereas in our experience the association of methionine and betaine usually corrects methionine depletion.

Additional Hypothetical Therapies: Choline and Creatine Theoretically, methionine may be spared and homocysteine accumulation prevented if less methyl groups are donated by the methyltransferase reactions using AdoMet as substrate.63 In particular, the synthesis of creatine and phosphatidylcholine consumes most methyl groups.64 As a consequence oral administration of creatine and phosphatidylcholine could lead to a reduced formation of homocysteine and so reduce accumulation in CBS deficiency. In remethylation defects methionine would be less consumed and so be spared. The use of creatine, choline, or other products of methylation in humans has not been published to our knowledge and requires further studies.

Outcome The vast experience of patients with CBS deficiency has shown that treating patients as early as possible is crucial to ensure the best possible outcome. Preferentially, patients are now diagnosed via expanded newborn screening. However, as discussed earlier, the use of methionine as biomarker unfortunately only allows the detection of B6-nonresponsive forms. All early-treated patients have a normal IQ without further complications.19 However, in adolescence, due to poor compliance, these patients may disclose complications. In late-treated patients, treatment allows stabilization and prevents further deterioration. In the few MTHFR-defective early-treated neonates (before 15 days of age; T de Koning, personal communication, 2011) the outcome is good with respect to the first years of neurologic development despite suboptimal metabolic control (tHcy 50 to 70 µM, 5-methylTHF persistently low in body fluids, but normal methionine levels, probably due to the association of betaine and methionine). If undiagnosed and/ or untreated these patients have a very poor outcome with severe impairments. On the contrary, despite some pathogenic similarity (intracellular methionine depletion due to impaired remethylation), CblC-defective patients (even the early detected and treated ones) have a particularly poor long-term outcome with multisystem involvement, thrombotic microangiopathy, and retinopathy.16,17 Of note, prenatal treatment with intramuscular OHCbl in a pregnant mother of an affected CblC fetus was reported and partially effective.65 OHCbl, 1 mg intramuscular, was administered twice Neuropediatrics


mg/kg/d in two to three divided doses, there was no obvious benefit in terms of lowering tHcy (studies performed in CBS-deficient patients).51 In CBS deficiency the use of betaine is usually followed by an increase in plasma methionine and a fall in plasma tHcy. No harmful effects from raised methionine have been documented in patients on betaine therapy except one case of cerebral edema in a CBS patient with plasma methionine levels > 1,000 µM.52 In B6-nonresponsive early-treated patients a low-methionine diet alone can be highly successful with very good long-term outcomes, provided lifelong compliance is good.19,53 The clinical benefits of betaine are therefore questionable in compliant patients on a lowmethionine diet. However, in some patients (especially when compliance to the diet is poor) betaine has been of benefit and may allow an increase in natural protein intake, thus improving the nutritional status.19 In remethylation disorders, betaine increases systemic methionine levels and probably also methionine availability for the central nervous system, especially in patients with MTHFR deficiency.54 In utero, betaine therapy dramatically increased the survival of Mthfr/ mice, emphasizing a major role for methyl depletion in the pathogenesis of MTHFR deficiency.55 In CblC defect few data report a synergistic action of OHCbl with betaine in terms of lowering tHcy level.49 However, there is some uncertainty about the effectiveness of betaine therapy during the first few months of life. Some of our remethylation-defective patients exhibited persistent tHcy elevation levels during the first year of life,24 despite satisfactory intestinal absorption of betaine. This decreased effectiveness may be ascribable to liver immaturity and/or specific and yet unknown pharmacokinetic characteristics during the first few months of life. Similarly, as reported long ago,50 remethylation-defective patients exhibited a tendency toward higher betaine-to-dimethylglycine ratios in plasma (for presumably similar betaine dosages) than CBSdefective patients, as if the ability to metabolize betaine was also less effective in remethylation defects than in CBS deficiency. Oral methionine may also hold promise as an additional therapeutic measure in remethylation defects, for several reasons: cerebral methionine depletion is a key pathogenic factor, methionine might act synergistically with betaine by supplying intracellular methionine, and acute methionine loading does not lead to further homocysteine accumulation (with the attendant risk of thromboembolism), as already mentioned56 and shown in one of our MTHFR patients.24 The same is true for chronic oral methionine therapy in remethylation defects.24 With oral methionine supplementation (starting dosages of 40 to 50 mg/d) combined with betaine supplementation, stable higher-than-normal plasma methionine levels were obtained in MTHFR patients.24 In one of them the CSF methionine level normalized with methionine alone but not with betaine alone. However, the highest CSF methionine level was achieved when methionine and betaine were given in combination.24 Oral methionine therapy produced good results in two neonates with MTHFR deficiency: One had a spectacular clinical improvement when methio-

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weekly to the mother from the 24th gestational week until delivery, and both plasma tHcy concentrations and MMA in urine remained normal until delivery. Despite prenatal and postnatal treatment, adequate metabolic control, and timely achievement of developmental milestones at age 15 months, neurologic symptoms (mainly truncal hypotonia) and early-onset nystagmus developed, highlighting again the possibility of additional yet unknown pathomechanisms not overcome by available treatment. However, future studies with longer follow-up are necessary to evaluate the potential benefits of such a prenatal therapy.

Concluding Remarks and Future Prospects Early-treated CBS-defective patients have a favorable longterm outcome. One of the most important requirements for improving the outcome of MTHFR deficiency is early recognition followed by aggressive treatment. The implementation of expanded newborn screening using a low methionine cutoff should be a helpful tool in this direction. As reported over 25 years ago,66 early treatment may ensure a favorable outcome. Intracellular methionine depletion should receive considerable attention as an important pathogenic factor. Treatment aimed at normalizing methionine levels in plasma and CSF are beneficial when started early. In CblC, despite early and aggressive therapy, the outcome often remains very poor17; this may be related to a CblC-specific process (already present in fetal life65), possibly involving 5-methylTHF trapping, MMA accumulation, or yet unidentified pathogenic factors as recently highlighted, for example, by works regarding increased oxidative stress (Martinelli et al, oral communication, EMG meeting, 2011). Betaine is a useful drug, largely used in CBS deficiency in association with a low-methionine diet (which remains the key treatment), especially when compliance to the diet is poor. In remethylation defects its association with oral methionine is probably synergistic and deserves further evaluation. Despite concerns about methionine toxicity,63,67 longterm methionine supplementation is probably safe.67 Because the synthesis of choline and creatine consumes methionine and produces the majority of homocysteine via transmethylation,64 the use of choline and creatine might hold promise for treating remethylation disorders, but possibly also CBS deficiency, by decreasing the consumption of AdoMet and so of methionine and may result in reduced homocysteine production.

Acknowledgments Part of this work is the result of a workshop coordinated by MS and HJB and organized by the European Metabolic Group in 2011. We thank all participants in this workshop for their active contributions. We are also grateful to Drs. Hélène Ogier de Baulny and Jean-François Benoist for their precious help and expertise in the preparation of the manuscript.

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