The human 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a major enzyme in the folate-dependent regulation of methionine and homocysteine concentrations. Different MTHFR mutations lead either to severe homocystinuria as a multisystem disorder or to moderate hyperhomocysteinaemia, which is a common risk factor for disorders ranging from cardiovasculopathy to spina bifida. The N-terminal part of the human MTHFR gene is incompletely characterised. We report the completed genomic structure of this gene including three novel exonic sequences on the basis of a 5'-RACE and a 4.2 kb cloned fragment of human genomic DNA. We demonstrate the existence of four MTHFR transcripts differing in their first exons. The diversity of transcripts is due to alternative transcription initiation and alternative splicing. Three putative polypeptides of 657, 698, and 680 amino acids are encoded. The novel genomic sequence described here includes putative promoter regions as suggested by the presence of regions homologue to binding sites for SP1, AP1, AP2, CAAT or GC boxes. Furthermore, we provide evidence that there are no TATA-box elements to regulate the human MTHFR gene. The results of our study render the full-length characterisation of affected alleles in severe homocystinuria and moderate hyperhomocysteinaemia due to MTHFR deficiency and provide a basis for investigating the regulation of the human MTHFR gene.

Controlled data on the association of MTHFR genotypes, hyperhomocysteinaemia and their interaction with factor V G1691A with childhood thrombosis are not yet available. Therefore we conducted a case-control study comparing 141 childhood patients with venous thrombosis with 345 healthy controls. The MTHFR C677T genotypes, FV G1691A and prothrombin G20210A were evaluated; in addition, fasting homocysteine concentrations were measured in a subgroup of 60 children and 80 healthy controls. 10.4% of the healthy control population showed the MTHFR TT genotype, 34.2% the CT genotype and 55.4% the CC variant. MTHFR genotypes account for fasting homocysteine concentrations in healthy controls (CC: 5.5 micromol/l (4-7.2); CT: 7 micromol/l (3.9-9.8); TT: 12.1 micromol/l (7.7-13.3)) with an upper age-specific 95th percentile of 8.3 micromol/l. The following frequencies (patients versus controls), odds ratios (OR) and 95% confidence intervals (CI) were found for single defects: MTHFR 677TT genotype (10.6% vs. 10.4%; OR/CI: 1.02/0.54-1.93; P = 0.99) and CT genotype (43.8% vs. 34.2%; OR/CI: 2.12/1.42-3.16; P = 0.0000). A combination of FV G1691A mutation and MTHFR 677CT genotype was found in 9.9% of patients and in 2.9% of the controls (OR/CI: 3.8/1.64-8.75; P = 0.027). Fasting homocysteine median (range) concentrations in the patient group were significantly higher than in the controls (7 micromol/l (3-23) vs. 5.5 micromol/l (3-8.4): P = 0.0004), and homocysteine concentrations >8.3 micromol/l were found in 40% of patients vs. 2.5% of the controls (OR/CI: 22/2.64-183; P = 0.0003). Conclusion Data of this childhood case-control study suggest that mildly elevated fasting homocysteine concentrations >8.3 micromol/l and the CT genotype of the MTHFR C677T variant are significant risk factors for venous vascular occlusion in children.

SummaryClassical homocystinuria due to cystathionine beta-synthase deficiency is one of the disorders revealing a high risk of thromboembolic events and vascular disease. This autosomal-recessively inherited metabolic disorder is considered to be rare with an estimated prevalence of 1:130,000 in the German population. In this study, we developed a novel multiplex PCR generating allele specific fragment lengths to analyse individual genotypes of the two most frequent cystathionine beta-synthase alterations, the I278T mutation, which is worldwide found on up to the half of homocystinuric alleles, and the adjacent polymorphism 844ins68. Screening of 200 unrelated German control subjects revealed a frequency of heterozygosity of 1.5% for I278T corresponding to a calculated frequency of homozygosity of 1:17.800. Our data indicate that homocystinuria due to cystathionine β-synthase deficiency is a frequently unrecognized disorder resulting in a high risk of thromboembolic events.

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