A number of studies have demonstrated that the common polymorphism 677C-->T in the gene encoding 5, 10-methylenetetrahydrofolate reductase (MTHFR) leads to a thermolabile variant with decreased enzyme activity and to mildly elevated plasma homocysteine. 677TT homozygosity was shown to be more frequent in NTD probands compared with controls in some studies. Recently, another polymorphism, 1298A-->C, in the MTHFR gene was described and combined heterozygosity 677CT/1298AC was suggested to be an additional risk factor for NTD. The present study examines the genotype and haplotype distribution of the two polymorphisms in the German population and evaluates the impact on NTD individuals and their relatives. To determine the haplotype of all individuals tested, we developed an easy-to-perform ARMS-RFLP test. Our data show that the two polymorphisms are in linkage disequilibrium in the general population and in NTD individuals. There was no statistically significant difference in allele and genotype frequency between probands (patients, fetuses) and controls (P > 0.10) and between observed and expected values for mother-child pairs (P > 0.80). Taking into account gender, an increased rate of 677CT heterozygotes was found in affected and unaffected males compared to affected and unaffected females. A family-based association study using a multiallelic transmission disequilibrium test (TDT) also shows that transmission rates do not deviate significantly from equilibrium (P > 0.50). Thus, our data provide no evidence for an association between NTD phenotype and MTHFR 677C/T-1298A/C genotypes and haplotypes.

Our data show no evidence for an association between the C677T mutation and the occurrence of the SB phenotype. Therefore we cannot support the hypothesis that the MTHFR variant does account for a significant genetic predisposition to the SB phenotype in the studied German patients.

A number of studies have demonstrated that the common polymorphism 677C-->T in the gene encoding 5, 10-methylenetetrahydrofolate reductase (MTHFR) leads to a thermolabile variant with decreased enzyme activity and to mildly elevated plasma homocysteine. 677TT homozygosity was shown to be more frequent in NTD probands compared with controls in some studies. Recently, another polymorphism, 1298A-->C, in the MTHFR gene was described and combined heterozygosity 677CT/1298AC was suggested to be an additional risk factor for NTD. The present study examines the genotype and haplotype distribution of the two polymorphisms in the German population and evaluates the impact on NTD individuals and their relatives. To determine the haplotype of all individuals tested, we developed an easy-to-perform ARMS-RFLP test. Our data show that the two polymorphisms are in linkage disequilibrium in the general population and in NTD individuals. There was no statistically significant difference in allele and genotype frequency between probands (patients, fetuses) and controls (P > 0.10) and between observed and expected values for mother-child pairs (P > 0.80). Taking into account gender, an increased rate of 677CT heterozygotes was found in affected and unaffected males compared to affected and unaffected females. A family-based association study using a multiallelic transmission disequilibrium test (TDT) also shows that transmission rates do not deviate significantly from equilibrium (P > 0.50). Thus, our data provide no evidence for an association between NTD phenotype and MTHFR 677C/T-1298A/C genotypes and haplotypes.

Neural tube defects (NTD) are among the most common congenital malformations in humans. The current view is that there are no major genes causing NTDs, but combinations of sequence variants in different genes have additive effects on determining the malformation. Therefore it is important to identify such sequence variants to get a better understanding of NTD pathogenesis. Studies on animal models have shown that BMP4 and NOG are involved in the patterning of the neural tube. We therefore performed a single-strand conformation analysis (SSCA) mutation screen for both genes in 179 spina bifida aperta (SBA) patients. Our SSCA screen revealed four missense mutations in BMP4 and one in NOG. It is likely that these mutations have acted together with other gene variants in independently segregating loci as susceptibility factors in these SBA cases. In addition, a case-control association study provides evidence for a genotype disequilibrium of BMP4 polymorphism 455T?C (V152A) in exon 5. The frequency of the heterozygous 455TC genotype is lower in cases than in controls (nominal P=0.017), although allele frequencies are similar in both groups. A possible explanation for this finding might be that BMP4 455TC heterozygosity at this site is a protective factor in the normal population, although this hypothesis cannot be proven to date.

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