Benedicte Christensen

Genetic polymorphisms in methylenetetrahydrofolate reductase and methionine synthase, folate levels in red blood cells, and risk of neural tube defects

Folic acid administration to women in the periconceptional period reduces the occurrence of neural tube defects (NTDs) in their offspring. A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR), 677C-->T, is the first genetic risk factor for NTDs in man identified at the molecular level. The gene encoding another folate-dependent enzyme, methionine synthase (MTR), has recently been cloned and a common variant, 2756A-->G, has been identified. We assessed genotypes and folate status in 56 patients with spina bifida, 62 mothers of patients, 97 children without NTDs (controls), and 90 mothers of controls, to determine the impact of these factors on NTD risk. Twenty percent of cases and 18% of case mothers were homozygous for the MTHFR polymorphism, compared to 11% of controls and 11% of control mothers, indicating that the mutant genotype conferred an increased risk for NTDs. The risk was further increased if both mother and child had this genotype. The MTR polymorphism was associated with a decreased O.R. (O.R.); none of the cases and only 10% of controls were homozygous for this variant. Red blood cell (RBC) folate was lower in cases and in case mothers, compared to their respective controls. Having a RBC folate in the lowest quartile of the control distribution was associated with an O.R. of 2.56 (95% CI 1.28-5.13) for being a case and of 3.05 (95% CI 1.54-6.03) for being a case mother. The combination of homozygous mutant MTHFR genotype and RBC folate in the lowest quartile conferred an O.R. for being a NTD case of 13.43 (CI 2.49-72.33) and an O.R. for having a child with NTD of 3.28 (CI 0.84-12.85). We propose that the genetic-nutrient interaction--MTHFR polymorphism and low folate status--is associated with a greater risk for NTDs than either variable alone.

Correlation of a Common Mutation in the Methylenetetrahydrofolate Reductase Gene With Plasma Homocysteine in Patients With Premature Coronary Artery Disease

Mild hyperhomocysteinemia, a risk factor for occlusive arterial disease, can be caused by disruptions of homocysteine metabolism. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate, the methyl donor for homocysteine remethylation to methionine. A common mutation in MTHFR, an alanine-to-valine substitution, may contribute to mild hyperhomocysteinemia in coronary artery disease (CAD). To test this hypothesis, we studied 152 patients with CAD by mutation analysis, MTHFR enzymatic assays, and measurements of plasma homocysteine and several vitamins. The MTHFR mutation was associated with reduced enzymatic activity and increased enzyme thermo-lability in these patients. The difference in the prevalence of the homozygous mutant genotype between the CAD patients (14%) and an unmatched group of healthy subjects (10%) was not significant. However, individuals with the homozygous mutant genotype had higher plasma homocysteine, particularly when plasma folate was below the median value. This genetic-environmental interaction is proposed to be a risk factor for CAD.

Characterization of six novel mutations in the methylenetetrahydrofolate reductase (MTHFR) gene in patients with homocystinuria

Severe deficiency of methylenetetrahydrofolate reductase (MTHFR) is the most common inborn error of folate metabolism. Patients are characterized by severe hyperhomocysteinemia, homocystinuria and a variety of neurological and vascular problems. Eighteen rare mutations have been reported in this group of patients. Two polymorphisms which cause mild enzyme deficiencies have been described (677C→T and 1298A→C). The first sequence change encodes a thermolabile enzyme and is associated with mild hyperhomocysteinemia. Six novel point mutations are described in patients with severe deficiency of MTHFR, along with their associated polymorphisms and clinical phenotypes. Of the two nonsense mutations (1762A→T, 1134C→G) and four missense mutations (1727C→T, 1172G→A, 1768G→A, and 358G→A), one was identified in the N‐terminal catalytic domain, while the others were located in the regulatory C‐terminal region. All four residues affected by missense mutations are conserved in one or more MTHFRs of other species. This report brings the total to 24 mutations identified in severe MTHFR deficiency, with two mutations identified in each of 22 patients. Hum Mutat 15:280–287, 2000.

9 Effects of folate deficiency on embryonic development

While there is strong evidence that folate deficiency including the use of anti-folate drugs in early pregnancy is teratogenic and may lead to a range of serious abnormalities of the developing fetus including intrauterine death, the mechanism(s) for these effects have not yet been delineated. In neural tube defects, there is increasing evidence that marginal folate status exacerbates the effect of an underlying genetic defect in the mother, the fetus, or both. An abnormal relationship between the ingestion of folate and the folate levels in red blood cells has been found in women who have given birth to infants with neural tube defects. Periconceptional folate supplementation has been shown to give effective protection against the development of neural tube defects. The mechanism of the prevention is as yet unknown. However, folic acid will not prevent all cases of neural tube defect. Moreover, neither determinations of periconceptional vitamin profiles (Mooij et al, 1993) nor determinations of methylmalonic acid and homocysteine levels will detect all women at risk. Anencephaly and spina bifida can be identified prenatally by detection of excessive levels of alpha-fetoprotein in amniotic fluid and maternal serum and by ultrasonographic scanning (Wilson and Rudd, 1993). Both genetic counselling and prenatal diagnosis should be offered to women who are recognized to be at high risk. Research into the underlying biology of neural tube defects is of major importance. The identification of underlying genetic defects would allow for genetic testing and better counselling of families at risk for the occurrence of a neural tube defect.

Sibs with anencephaly, anophthalmia, clefts, omphalocele, and polydactyly : Hydrolethalus or acrocallosal syndrome?

Major characteristics of the acrocallosal syndrome include severe mental retardation, agenesis or hypoplasia of the corpus callosum, and polydactyly of fingers and toes. In the past few years, anencephaly has also been noted, together with other midline defects. We report on a nonconsanguineous, Norwegian couple with a history of two pregnancies with a male and a female fetus, respectively, with anencephaly, median cleft lip and palate, omphalocele, and preaxial polydactyly, suggesting the diagnosis of the acrocallosal syndrome. Both fetuses also lacked eyes and nose, a finding not previously reported in the acrocallosal syndrome. Microphthalmia has been reported in the hydrolethalus syndrome, which may be caused by mutations in the same gene as the acrocallosal syndrome. The present report adds support to the hypothesis that the acrocallosal and hydrolethalus syndromes may be allelic conditions. The family history is consistent with autosomal recessive inheritance.

Effect of methionine and nitrous oxide on homocysteine export and remethylation in fibroblasts from cystathionine synthase-deficient, cb1G, and cb1E patients.

ABSTRACT: We investigated the nitrous oxide-induced inactivation of methionine synthase and the concurrent homocysteine (Hey) export in mutant fibroblasts with defects in the homocysteine catabolizing enzyme, cystathionine β-synthase, or in methionine synthase, which carries out homocysteine remethylation. The fibroblasts were incubated in various concentrations of methionine to create conditions favoring methionine conservation or catabolism. In cystathionine β-synthase-deficient cells, high medium methionine partly protected the enzyme against inactivation, as previously found in normal fibroblasts. The Hey export rate at low methionine levels was low (0.2–0.6 nmol/ h/106 cells), and increased 2–3-fold at high methionine levels. Nitrous oxide enhanced Hey export rate at low methionine, so that in the presence of nitrous oxide, the Hey export became less dependent of methionine. In cblG cells, the enzyme inactivation was moderate and independent of medium methionine. The Hey export rate was intermediate (0.5–0.8 nmol/h/106 cells) at low methionine levels, and increased moderately (<2-fold) at high methionine levels or following nitrous oxide exposure. In cblE mutants, the enzyme activity was not affected by nitrous oxide, and the Hey export was high (0.8–1.6 nmol/h/106 cells) and independent of methionine and nitrous oxide. These data suggest that Hey remethylation and cystathionine βsynthase activity are major determinants of Hey export at low and high methionine, respectively. The low susceptibility of methionine synthase to nitrous oxide in the presence of high methionine or in cblG or cblE mutants is probably related to low catalytic turnover.

Spina bifida, folate metabolism, and dietary folate intake in a Northern Canadian aboriginal population

Objectives. Inhabitants of the subarctic region of the Eastern James Bay of Northern Quebec consume a diet low in folate. This is largely secondary to poor access to plant-foods and a preferred diet high in meat, fowl, and fish as in many other northern populations. Furthermore, there is a high frequency of spina bifida in the Cree of the region. It was hypothesized that genetically altered folate metabolism as well as low folate intake contributes to the high frequency of spina bifida. Methods: A casecontrol study evaluating folate metabolism and the common 677C-T polymorphism of the gene for methylenetetrahydrofolate reductase (MTHFR) in mothers of children with spina bifida, and controls (n=23) of Cree descent from the Eastern James Bay region. These results were compared to a similar Montreal cohort (n=152) who were not of First Nations descent. Dietary intake of folate of 219 women of the Eastern James Bay region was also determined. Results: No Cree mothers of children with spina bifida were homozygous for the 677C-T polymorphism of MTHFR. Although serum cobalamin was significantly higher in Cree mothers, RBC folate was significantly lower than in the Montreal cohort. In addition, plasma homocysteine was significantly lower in the Cree. Dietary intake of folate of women in the same region was substantially lower (100 µg/day) than widely recommended daily intakes. Conclusions. In this remote Canadian aboriginal community there is no evidence of altered folate metabolism in the mothers of children with spina bifida. Nonetheless, it remains essential that culturally appropriate public health efforts be continued to increase the intake of folic acid in the hope of reducing the high frequency of spina bifida in this population.

NEW MUTATION IDENTIFIED IN FOUR UNRELATED PATIENTS WITH METHYLENETETRAHYDROFOLATE REDUCTASE DEFICIENCY. † 843

Methylenetetrahydrofolate reductase (MTHFR) deficiency is an autosomal recessive disorder typically characterized by less than 20% of normal enzymatic activity, hyperhomocysteinemia, hypomethioninemia, and a range of neurological and vascular abnormalities. Nine rare mutations in severe MTHFR deficiency have been reported. In addition, a common missense mutation, which is associated with a milder deficiency and a thermolabile enzyme, has been observed in 35% of chromosomes from the general population, and may represent a risk factor for cardiovascular disease and for neural tube defects. This polymorphism may also be present in patients with mutations for severe MTHFR deficiency. Here we report a new MTHFR mutation identified by SSCP analysis and direct sequencing. The mutation is a G to A substitution at bp 1172 which converts a glycine to an aspartic acid residue. Since this mutation eliminates a recognition site for HaeIII, restriction digestion was used to detect this mutation in 4 of 15 patients with severe MTHFR deficiency. Thus it is the most frequent severe mutation found to date. Each of the 4 patients had one copy of the mutation while 42 controls were negative. Notably, 3 of the 4 patients had relatively late onset disease presenting at ages 13, 15, and 40 years, respectively. The fourth presented with seizures at age 7 months. Of the 4, only this infant was homozygous for the polymorphism associated with a thermolabile enzyme. The mother of this early-onset patient had had a stroke at age 4 years, but no other obvious findings of MTHFR deficiency. She presumably has one mutation for severe MTHFR deficiency and at least one copy of the thermolabile polymorphism. Studies are in progress to identify other mutations in these patients and to examine possible interactions of the polymorphism with heterozygosity or homozygosity for severe MTHFR mutations.