European Journal of Human Genetics (1998) 6, 257–265 © 1998 Stockton Press All rights reserved 1018–4813/98 $12.00 http://www.stockton-press.co.uk/ejhg ORIGINAL PAPER Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency Leo AJ Kluijtmans1, Udo Wendel1,2, Erik MB Stevens1, Lambert PWJ van den Heuvel1, Frans JM Trijbels1 and Henk J Blom1 1Department of Pediatrics, University Hospital Nijmegen, The Netherlands 2University Children's Hospital, University of D¨usseldorf, Germany Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is an inborn error of folate metabolism, and is inherited as an autosomal recessive trait. MTHFR is a key enzyme in folate-dependent remethylation of homocysteine, and reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Patients with this severe enzymatic deficiency are biochemically characterised by homocystinuria and hypomethioninaemia, and may suffer from neurological abnormalities, mental retardation and premature vascular disease. Here we report the molecular basis of severe MTHFR deficiency in four unrelated families from Turkish/Greek ancestry. By use of reverse-transcriptase (RT)-PCR, subsequently followed by direct sequencing analysis, we were able to identify four novel mutations in the MTHFR gene: two missense (983AG; 1027TG) and two nonsense (1084CT; 1711CT) mutations. Furthermore, a splice variant containing a premature termination codon, was observed in one patient, probably as a secondary effect of the 1027TG missense mutation. The ongoing identification and characterisation of mutations in the MTHFR gene will provide further insight into the heterogeneity of the clinical phenotype in severe MTHFR deficiency. Keywords: methylenetetrahydrofolate reductase; mutations; inborn error of metabolism; folate; splice variant Introduction donor of the methyl group in the remethylation of homocysteine to methionine. In addition to its involve- Methylenetetrahydrofolate reductase (MTHFR) [EC ment in protein synthesis, methionine is converted to 1.5.1.20] is a key enzyme in the folate-dependent S-adenosylmethionine (AdoMet), the most predomi- remethylation of homocysteine. The enzyme reduces nant methyl donor in the human body. MTHFR, a 5,10-methylenetetrahydrofolate to 5-methyltetrahydro- flavoprotein, is allosterically regulated by AdoMet; folate, the predominant circulating form of folate, and high levels of AdoMet inhibit MTHFR activity and thereby the remethylation of homocysteine to Correspondence: Dr HJ Blom, Department of Pediatrics, methionine.1,2 University Hospital Nijmegen, PO Box 9101, 6500 HB MTHFR deficiency, the most common inborn error Nijmegen, The Netherlands. Tel: + 31 24 3613469; Fax: + 31 24 3,4 3618900; E-mail: [email protected] of folate metabolism, is inherited as an autosomal Received 16 July 1997; revised 25 November 1997; accepted 2 recessive trait and may be subdivided into severe and December 1997 thermolabile MTHFR deficiency. Patients with the Journal: European Journal of Human Genetics Article: 5200182 Molecular basis of MTHFR deficiency L A J Kluijtmans et al 258 severe form have severely elevated homocysteine levels case report by Hyland et al23 (case 2) and by Wendel in blood and urine, a decreased concentration of and Bremer.24 Both parents originate from the same methionine in plasma, and display a wide range of village, but without known consanguinity. The girl was clinical features, such as neurological abnormalities, microcephalic, hypotonic, restless, and was unable to sit mental retardation, and premature vascular disease. without support. Plasma homocystine concentrations Residual MTHFR activity in cultured fibroblasts is were in the range of 15–25 µm/L, homocysteine- usually < 20% compared with controls, whereas vir- cysteine mixed disulphide 25–35 µm/L, and methionine tually no residual activity can be detected in isolated concentrations 4–5 µm/L. Diagnosis of severe MTHFR lymphocytes. The variety in clinical presentation, age of deficiency was established by absence of MTHFR onset, and residual MTHFR activity in cultured fibro- activity in extracts of cultured fibroblasts. Therapy was blasts, suggest the existence of a strong heterogeneity in initiated, and she received 20 g of betaine and 15 mg of mutations causing severe MTHFR deficiency. folinic acid daily. Plasma methionine levels increased to The recent localisation of the MTHFR gene on >25µm/L, and plasma homocystine concentration chromosome 1 and the elucidation of the MTHFR decreased to trace amounts. Her clinical condition 5 cDNA permitted the molecular characterisation of improved substantially; however, now at 17 years of patients with severe MTHFR deficiency. Goyette et al age, she is still severely mentally retarded. reported the identification of mutations in the MTHFR Patient UB, a male, is the seventh child of a healthy gene in patients with a homozygous MTHFR defi- consanguineous Turkish couple. In at least three pre- 5–7 ciency, and correlated genotypes to residual MTHFR vious children of the family, MTHFR deficiency had activity in cultured fibroblasts and to clinical 6 been diagnosed. In this child, MTHFR was diagnosed phenotype. immediately after birth with plasma homocystine con- Recently, Kang et al described a thermolabile variant centrations of 15–27 µm/L, and methionine concentra- of MTHFR with a specific MTHFR activity in isolated tions of 4–6 µm/L. Therapy with betaine (60 mg/kg body lymphocytes < 50% of the control mean and with weight, daily) was initiated at the sixth day of life. increased thermolability after heat incubation for 5 min Plasma methionine levels increased to > 25 µm/L, and at 46°C.8,9 This thermolabile variant may result in plasma homocysteine decreased to trace amounts. He mildly elevated homocysteine concentrations and is was again referred to our hospital at the age of 4 years, reported to be associated with cardiovascular dis- when he was found to be severely mentally retarded, ease.10,11 In 1995 we described a 677CT transition and showed signs of dysmyelination in a cranial MRI. responsible for the thermolabile MTHFR variant.12 During the four years, betaine has only been given Recent studies have investigated this mutation as a irregularly, and even dropped for long periods of time. genetic risk factor for spina bifida13–15 and cardiovas- When regular treatment with betaine was restarted, cular disease.16–20 All studies indicate that homozygotes total plasma homocystine decreased to 80–100 µm/L, for this transition have mildly elevated plasma homo- and methionine increased to high normal values. cysteine, especially in circumstances of low folate status21,22 Patient K was a male child of a healthy consanguine- ous Turkish couple, previously presented as case 3 in In this report, we describe the identification of two 23 missense and two nonsense mutations in the MTHFR Hyland et al. Homocystinuria with low plasma gene in four unrelated families from Turkish/Greek methionine was diagnosed at the end of the first month ancestry with severe MTHFR deficiency. Furthermore, of life; however, the patient was not treated. A CAT our data contribute to the genotype/phenotype correla- scan revealed severe internal hydrocephalus, being tions in this metabolic disease. rapidly progressive during the first months. At 10 months of age, he suffered from severe psychomotoric retardation, severe muscular hypotonia, and he had no µ Patients, Materials and Methods social contact. Plasma homocystine (38–50 m/L) and homocysteine–cysteine mixed disulphide (40–60 µm/L) Patients concentrations were severely elevated, whereas plasma Patient CM was the second girl of a family of Greek- methionine concentrations were decreased and ranged Macedonian ancestry, and was admitted to our hospital from 6–10 µm/L. Specific MTHFR activity was assayed at the age of 2 because of marked psychomotor in extracts of cultured fibroblasts, but was undetectably retardation. This patient has been described before as a low. Betaine (15–20 g daily) treatment was initiated. As Molecular basis of MTHFR deficiency L A J Kluijtmans et al 259 a result, plasma methionine concentrations increased to (1:1), and 100 µl 3.0 M potassium acetate (pH 4.5). The >30µm/L, and homocystine levels decreased to incubation mixture was heated for 15 minutes at 95°C <5µm/L; homocysteine–cysteine mixed disulphide and quenched on ice for at least 10 min. After addition remained virtually unchanged. On treatment, the of 3 µl toluene, the mixture was shaken vigorously for patient showed some neurological improvement. He 15 s and centrifuged at low speed. A 2.0 µl aliquot of the became more alert and gained some social contact, but toluene phase was used for the measurement of was never able to walk. At the age of 7 he died severely radioactivity. Fibroblasts were cultured as described mentally retarded with hyperpyrrhexia. previously.27,28 Protein content of the sample was Patient U is an infant first child born from a determined by the method of Lowry et al.29 Specific consanguineous Turkish couple. At the age of 10 MTHFR activity was expressed in nmol formaldehyde/ months, she presented with severely retarded psycho- mg protein per hour. motor development, severe microcephaly, and homo- cystinuria.25 Methionine concentration in plasma was Homocysteine Determination <5µm/L and total plasma homocysteine was Determination of methionine and the non-protein > 150 µm/L. Upon betaine treatment (6–9 g daily), her bound forms of homocysteine, ie homocystine and plasma methionine increased to