Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome 1 3

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1 Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome 1 3 S Jill James, Marta Pogribna, Igor P Pogribny, Stepan Melnyk, R Jean Hine, James B Gibson, Ping Yi, Dixie L Tafoya, David H Swenson, Vincent L Wilson, and David W Gaylor See corresponding editorial on page 429. ABSTRACT Background: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. Objective: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. Design: The frequency of the MTHFR 677C T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. Results: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). Conclusion: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene. Am J Clin Nutr 1999;70: KEY WORDS Methylenetetrahydrofolate reductase, Down syndrome, folate, homocysteine, mutation, DNA methylation, MTHFR 677C T mutation, trisomy 21 INTRODUCTION Down syndrome is a complex genetic disease resulting from the presence and expression of 3 copies of the genes located on chromosome 21 (trisomy 21). In most cases, the extra chromosome stems from the failure of normal chromosomal segregation during meiosis (meiotic nondisjunction) (1). The nondisjunction event is maternal in 95% of cases, occurring primarily during meiosis I in the maturing oocyte, before conception (2). Down syndrome occurs with an estimated frequency of 1 in 600 live births and 1 in 150 conceptions (3). Despite the prevalence of this common genetic disease, the cellular and molecular mechanisms underlying meiotic nondisjunction and trisomy 21 are not yet understood. Both clinical and experimental studies have shown that genomic DNA hypomethylation is associated with chromosomal instability and abnormal segregation. For example, a rare autosomal disorder, ICF syndrome (immune deficiency, centromeric instability, and facial anomalies), is characterized by pericentromeric hypomethylation (4) and impaired chromosome segregation (5). In cultured plant and animal cells, chemically induced DNA hypomethylation with 5-azacytidine treatment induces chromosomal instability and aneuploidy (6, 7). Several investigators have suggested that the chromosomal instability and aneuploidy exhibited in human tumors is related to genome-wide DNA hypomethylation (8, 9). We and others have shown that dietary folate and methyl deficiency in vivo results in DNA hypomethylation (10, 11), DNA strand breaks (12), and abnormal gene expression (13, 14). MTHFR acts at a critical metabolic juncture in the regulation of cellular methylation reactions (15), catalyzing the conversion 1 From the Food and Drug Administration National Center for Toxicological Research, the Division of Biochemical Toxicology, Jefferson, AR; the University of Arkansas for Medical Sciences, the Department of Biochemistry and Molecular Biology and the Department of Dietetics and Nutrition, Little Rock; the Arkansas Children s Hospital, the Division of Pediatric Genetics, Little Rock; Trisomy-21 Research, Inc, San Jose, CA; the Saginaw Valley State University, the Department of Chemistry, University Center, MI; and the Institute for Environmental Studies and Institute for Mutagenesis, Louisiana State University, Baton Rouge. 2 Supported by a grant from the FRIENDS of Trisomy-21 Research, Inc, and the FDA Office of Women s Health. 3 Address reprint requests to SJ James, National Center for Toxicological Research, Division of Biochemical Toxicology, HFT 140, 3900 NCTR Road, Jefferson, AR jjames@nctr.fda.gov. Am J Clin Nutr 1999;70: Printed in USA American Society for Clinical Nutrition 495

2 496 JAMES ET AL of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for the remethylation of homocysteine to methionine (Figure 1). The C to T transition mutation at position 677 within the MTHFR gene (677C T) causes an alanine to valine substitution in the MTHFR protein and reduced enzyme activity. Relative to the normal C/C genotype, the specific activity of MTHFR is reduced 35% with the heterozygous C/T genotype and 70% with the homozygous T/T genotype. This reaction is important for the synthesis of S-adenosylmethionine (SAM), the major intracellular methyl donor for DNA, protein, and lipid methylation reactions. Reduced MTHFR activity results in an increased requirement for folic acid to maintain normal homocysteine remethylation to methionine. In the absence of sufficient folic acid, intracellular homocysteine accumulates, methionine resynthesis is reduced, and essential methylation reactions are compromised. An increase in homocysteine and a decrease in methionine results in a decreased ratio of SAM to S-adenosylhomocysteine (SAH), which has been associated with DNA hypomethylation (14, 16, 17). On the basis of these metabolic considerations, we hypothesized that the 677C T mutation may predispose to aberrant DNA methylation and increased risk of meiotic nondisjunction. Supporting this possibility, metabolic data are presented suggesting that abnormal folate and methyl metabolism are associated with the risk of Down syndrome. SUBJECTS AND METHODS Study population and specimen collection Mothers of children with Down syndrome were recruited by advertisements placed in support-group newsletters and on the Internet. From the group of 108 responders, 57 mothers were selected for participation. Selection criteria were birth of a child with karyotypically confirmed full trisomy 21 and <40 y of age at the time of conception. The participating mothers resided in 16 different states and Canada. Each participant was asked to recruit a control mother who resided in the same geographic area, was approximately the same age and of the same social class, and had experienced no miscarriages and no abnormal pregnancies. Written, informed consent approved by the Food and Drug Administration s Research Involving Human Subjects Committee and the University of Arkansas for Medical Sciences Human Research Advisory Committee was obtained from all participants. A family health history and ethnicity questionnaire was completed by most participants and a food-frequency questionnaire (18) was completed by most mothers of children with Down syndrome. Participants residing in different states were sent kits containing 2 heparin-containing evacuated tubes for collection of fasting blood samples at a local laboratory. Samples were returned to the National Center for Toxicological Research the next day. Lymphocytes were isolated from whole blood by centrifugation through ficoll-hypaque and stored at 20 C until analyzed for genotype. Lymphocytes from local participants were isolated immediately and used to evaluate in vitro methotrexate sensitivity in cell culture. Separated plasma, obtained within 0.5 h of blood collection, was sent frozen on dry ice in separate tubes for subsequent HPLC analysis of homocysteine and methionine. MTHFR 677C T mutation identification Genomic DNA was extracted from lymphocytes by using standard procedures (19). For genotype analysis, the MTHFR gene was amplified by polymerase chain reaction followed by restriction enzyme digestion with Hinf I (New England Biolabs, Beverly, MA) by using primers and conditions described previously (20). The presence of the 677C T mutation within the MTHFR gene creates an Hinf I restriction site that is detected by the appearance of a 175 base pair fragment on a 3% agarose gel. Genotyping was conducted in a blind fashion without prior knowledge of the case or control status of the subjects. FIGURE 1. The C-to-T substitution at nucleotide 677 (677C T) mutation in the methylenetetrahydrofolate reductase (MTHFR) gene decreases the activity of MTHFR and the synthesis of 5-methyltetrahydrofolate (5-methyl-THF), which is used for the remethylation of homocysteine to methionine. Insufficient synthesis of 5-methyl-THF and methionine results in a decrease in methionine and in S-adenosylmethionine (SAM) and an accumulation of homocysteine and S-adenosylhomocysteine (SAH). A reduction in SAM:SAH reduces the efficiency of DNA (cytosine-5-)-methyltransferase and is associated with DNA hypomethylation. dtmp, deoxythymidine monophosphate; dump, deoxyuridine monophosphate; B-12, vitamin B-12.

3 ABNORMAL FOLATE METABOLISM AND DOWN SYNDROME 497 TABLE 1 Characteristics of responding age-matched control mothers and mothers of children with Down syndrome (DS mothers) 1 Control mothers DS mothers C/C C/T + T/T C/C C/T + T/T (n = 15) (n = 17) (n = 10) (n = 32) Mean age at conception (y) (all pregnancies) (all pregnancies) (DS pregnancy) (DS pregnancy) Mean number of live birth pregnancies 2.3 (35/15) 2.4 (41/17) 3.0 (30/10) 2.9 (92/32) Percentage of miscarriages (%) Ethnicity White (mixed European) 13/15 [88] 14/17 [82] 9/10 [90] 30/32 [94] Hispanic 2/15 [13] 2/17 [12] 0/10 [0] 2/32 [6] Asian 0/15 [0] 1/17 [6] 1/10 [11] 0/32 [0] Black 0/15 [0] 0/17 [0] 0/10 [0] 0/32 [0] At time of conception Taking vitamin supplement containing 400 g folic acid 4/15 [27] 4/17 [24] 2/10 [20] 9/32 [28] Following a weight-loss diet 0/15 [0] 0/17 [0] 4/10 [40] 7/32 [22] Heavy alcohol consumption (3 4 mixed drinks/d) 0/15 [0] 0/17 [0] 1/9 [11] 2/32 [6] Maternal family history Twins in extended family 2/14 [14] 3/17 [14] 2/9 [22] 9/30 [30] Heart disease 5/14 [36] 6/17 [35] 2/9 [22] 17/30 [57] Cancer 8/14 [57] 11/17 [65] 5/9 [56] 19/30 [62] 1 Percentage in brackets. C/C, homozygous normal genotype with C at position 677 on both alleles; C/T, heterozygous genotype with T substitution on one allele; T/T, homozygous genotype with T substitution on both alleles. Methotrexate cytotoxicity Peripheral blood lymphocytes were isolated by using heparincontaining Vacutainer CPT tubes (Becton Dickinson and Co, Orangeburg, NY) according to the manufacturer s protocol. Washed cells were seeded into 6-well plates at cells/l in RPMI 1640 medium containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and 2 mg phytohemagglutinin/l (Sigma, St Louis). Cells were incubated for 2 d in a humidified 5% CO 2 incubator before addition of 0.22 or 0.44 mol methotrexate/l. Cells were harvested after an additional 24 h and the number of remaining live cells was counted by trypan blue exclusion. Results are expressed as the mean percentage of viable cells after methotrexate exposure. Plasma homocysteine and methionine Total homocysteine and methionine concentrations in plasma were determined by using HPLC (Beckman Instruments, Fullerton, CA) with a Coulochem II detector and model 580 solvent delivery system (ESA, Inc, Chelmsford, MA). The detector was equipped with a model 5010 analytic cell and model 5020 guard cell (ESA, Inc). The guard cell was set at 970 mv, electrode 1 at 400 mv, and electrode 2 at 870 mv. Concentrations of homocysteine and methionine (in mol/l) were calculated from peak areas and standard calibration curves by using GOLD NOUVEAU software (Beckman Instruments). Briefly, 200 L plasma was mixed with 50 L of 1.5 mol EDTA/L, 50 L of 1.4 mol NaBH 4 /L (prepared fresh each day), and 10 L isoamyl alcohol. After a 30-min incubation with gentle shaking at room temperature, samples were placed on ice for 5 min. To the samples on ice, 100 L ice-cold 10% metaphosphoric acid was added in 20- L increments to precipitate proteins. After being centrifuged for 15 min at g at room temperature, samples were neutralized by dropwise addition of 2 mol TRIZMA/L (Sigma). Twenty microliters of filtered supernate was injected into the HPLC system and thiol separation was accomplished according to the method described by Lakritz et al (21) with modifications. An MCM C 18 column ( mm; MC Medical, Inc, Tokyo) was used with a mobile phase consisting of 50 mmol NaH 2 PO 4 /L, 0.2 mmol octane sulfonic acid/l, and 2% acetonitrile, adjusted to ph 2.7 with metaphosphoric acid. Statistics Results for continuous data (eg, homocysteine and methionine) are expressed as means ± SEMs. Comparisons between groups were evaluated with a paired Student s t test with SIG- MASTAT software (Jandel Scientific, San Rafael, CA). For enumeration data (eg, the number of individuals with various genotypes), comparisons of percentages between groups were evaluated with a one-sided chi-square test corrected for continuity. The variance of the logarithm of the odds ratio (OR) is approximately the sum of the reciprocals of the number of individuals in each group. The 95% CIs for the log OR is ± 1.96 (square root of the variance). The antilogarithms of these limits give the approximate 95% CI for the OR. RESULTS Characteristics of the study population In Table 1, the data collected from the structured questionnaires are stratified by genotype among the responding mothers of children with Down syndrome and control mothers. There were no significant differences between groups in terms of mean age at conception, mean number of pregnancies, or maternal family history of cancer. The participating mothers were white (of mixed European descent) and there were no significant differences in ethnicity between groups. Compared with the control mothers, mothers of children with Down syndrome reported a

4 498 JAMES ET AL TABLE 2 Frequency of the MTHFR C-to-T substitution at nucleotide 677 in age-matched control mothers and in mothers of children with Down syndrome (DS mothers) Control mothers DS mothers Odds ratio Genotype (n = 50) (n = 57) (95% CI) P 1 n[%] n [%] Homozygous normal (C/C) 24 [48] 15 [26.3] 2 Heterozygous mutant (C/T) 22 [44] 34 [59.6] 2.5 (1.0, 5.7) <0.04 Homozygous mutant (T/T) 4 [8] 8 [14.0] 3.2 (0.8, 12.5) <0.10 Combined mutant (C/T + T/T) 26 [52] 42 [73.6] 2.6 (1.2, 5.8) <0.03 Mutant allele frequency Chi-square analysis. 2,4 Significantly different from control mothers: 2 P < 0.02, 4 P < Both populations in Hardy-Weinberg equilibrium, P > 0.1. greater incidence of heavy alcohol consumption (equivalent of 3 4 mixed drinks/d) at the time of conception and more mothers of children with Down syndrome were following a weight-loss diet at the time of conception, regardless of genotype. Mothers of children with Down syndrome with MTHFR polymorphism reported a higher maternal family history of cardiovascular disease and twinning than did mothers of children with Down syndrome with the normal C/C genotype or control mothers. Prevalence of 677C T MTHFR mutation As shown in Table 2, the frequencies of the C/C, C/T, and T/T genotypes among the control mothers were 48%, 44%, and 8%, respectively. The corresponding frequencies among the mothers of children with Down syndrome were 26.3%, 59.6%, and 14%, respectively. These data indicated that the risk of having a child with Down syndrome was 2.6-fold higher in mothers with the 677C T substitution in one or both alleles than in mothers without the T substitution. Analyzed separately, the OR for the heterozygous C/T genotype was 2.5 and for the homozygous T/T genotype was 3.2. The frequency of the C/C genotype was significantly lower in the mothers of children with Down syndrome than in the control mothers. The overall mutant allele frequency was significantly higher in the mothers of children with Down syndrome than in the control mothers. Taken together, these data indicate that the presence of the 677C T MTHFR mutation on one or both alleles significantly increased the risk of having a child with Down syndrome. Methotrexate sensitivity Sensitivity to methotrexate cytotoxicity was evaluated in lymphocytes from mothers of children with Down syndrome and control mothers as an indicator of functional folate metabolism. Cytotoxicity with methotrexate has been associated with a reduction in intracellular 5-methyltetrahydrofolate, increased plasma homocysteine, and reduced plasma methionine (22). Lymphocytes were exposed to methotrexate in folatereplete medium to eliminate insufficient folate as an interacting variable. Under nutritionally complete conditions, cellular sensitivity to methotrexate-induced cytotoxicity should reflect the functional effect of genotype on folate metabolism. As shown in Table 3, cytotoxicity was significantly greater at the 2 different concentrations of methotrexate in lymphocytes from mothers of children with Down syndrome than in control mothers, consistent with a reduced ability to adapt to folate deprivation. A comparison of methotrexate cytotoxicity between mothers of children with Down syndrome and control mothers with C/T genotypes indicated that mothers of children with Down syndrome were significantly more sensitive to methotrexate, suggesting that the differences in methotrexate cytotoxicity were not due to the C/T genotype. These observations suggest that additional factors must be involved to functionally compromise folate metabolism in the mothers of children with Down syndrome. Plasma homocysteine and methionine concentrations Plasma concentrations of homocysteine and methionine are a reflection of current dietary patterns superimposed on genotype. Although they may not reflect dietary intake at the time a child with Down syndrome is conceived, they may indicate a genetically determined increase in folate requirement. Mean fasting plasma homocysteine concentrations in the mothers of children with Down syndrome with one or both 677T alleles was 12.0 mol/l compared with 8.3 mol/l in the corresponding control mothers (Table 4). Interestingly, the mothers of children with Down syndrome with the normal C/C genotype also had mean homocysteine concentrations that were significantly higher than those of control mothers with the C/C genotype (10.9 compared with 7.9 mol/l). The ratio of plasma homocysteine to methionine was significantly higher in mothers of children with Down syndrome than in control mothers, independent of genotype. These observations suggest that factors other than the 677C T mutation alter homocysteine and methionine concentrations in mothers of children with Down syndrome. The mean folate intake from foods as determined from the food-frequency questionnaires was 263 g/d for the mothers of children with Down syndrome with the C/C genotype and 274 g/d for the mothers of children with Down syndrome with the T substitution on one or both alleles (C/T and T/T genotypes combined). In both groups of mothers of children with Down syndrome, the folate intake from foods was below the current recommendation of 400 g/d. However, 28% of the mothers of children with Down syndrome with the MTHFR polymorphism reported taking a vitamin supplement containing 400 g folic acid at the time of conception (Table 1). Intakes of methionine and vitamin B-12 were not significantly different between genotypes. Although a reporting of intake at the time of conception would be more relevant, the current estimate is a likely reflection of adult dietary patterns.

5 ABNORMAL FOLATE METABOLISM AND DOWN SYNDROME 499 TABLE 3 Sensitivity to methotrexate cytotoxicity in age-matched control mothers and in mothers of children with Down syndrome (DS mothers) 1 All genotypes combined C/T genotypes only Control mothers DS mothers Control mothers DS mothers Methotrexate concentration (n = 12) (n = 12) (n = 4) (n = 10) 0.22 mol/l 84.3 ± ± ± ± mol/l 68.9 ± ± ± ± x ± SEM. 2 Percentage of viable lymphocytes after 24-h exposure. 3 5 Significantly different from control mothers (Student s t test): 3 P < 0.001, 4 P < 0.02, 5 P < % 2 DISCUSSION Trisomy 21 is a major public health concern. It is the leading genetic cause of mental retardation and is estimated to occur in 1 of every 150 conceptions. About 80% of trisomy 21 conceptions result in pregnancy loss (23). Despite its prevalence and consequence, the biochemical and molecular mechanisms that predispose to maternal nondisjunction are not understood. Recent evidence indicates that abnormal recombination during the meiosis I prophase is associated with nondisjunction, but the mechanisms predisposing to altered recombination are unknown (24). Chemically induced chromosomal instability and aneuploidy with 5-azacytidine, an agent that irreversibly inactivates DNA (cytosine-5-)-methyltransferase, implicates DNA hypomethylation as a possible causative factor (4, 6, 7, 25, 26). Indeed, recent evidence suggests that stable centromeric DNA chromatin may depend on the epigenetic inheritance of specific centromeric methylation patterns and on the binding of specific methyl-sensitive proteins to maintain the higher order DNA architecture necessary for kinetochore assembly (27). We hypothesized that reduced MTHFR activity, secondary to the 677C T substitution, could promote DNA hypomethylation by decreasing SAM, the substrate for the DNA (cytosine-5-)-methyltransferase, or by increasing SAH, a competitive inhibitor of the DNA (cytosine-5-)- methyltransferase, or by both mechanisms (28). The data presented in Table 2 indicate that the risk of having a child with Down syndrome is strongly associated with the 677C T mutation. The marginal significance (P < 0.10) in risk in mothers with the T/T homozygous mutant genotype was most likely due to the low numbers in this group. Nonetheless, the greater frequency of the heterozygous genotype among the mothers of children with Down syndrome than in control mothers was highly significant (P < 0.03). These results contrast with the distribution of the 677C T mutation in parents of children with neural tube defects (NTDs), in whom the increased risk is more strongly associated with the homozygous T/T genotype (29). Meta-analysis of all reported studies of MTHFR mutation in mothers of children with NTDs indicated that the mean percentage incidence of the homozygous T/T genotype was 14.5% compared with an overall mean of 8.5% in unmatched control subjects (30). This difference is the basis for the identification of MTHFR polymorphism as a genetic risk factor for NTDs and is similar to the percentage difference observed in the homozygous mothers of children with Down syndrome and control mothers in our study (14% and 8%, respectively). A possible explanation for the predominance of the heterozygous genotype in the mothers of children with Down syndrome is that fetal viability may be lower in mothers with the homozygous T/T genotype and may also vary with the genotype of the fetus with Down syndrome. Because NTDs represent a postconceptional developmental failure, both the fetal and paternal genotypes can be additional interacting risk factors. By contrast, because meiotic nondisjunction with Down syndrome is preconceptional and maternal in 95% of cases, the genotype and environmental exposures of the mother are the major determinants of Down syndrome. Thus, the implication of the present study is that normal folate metabolism is important not only for postconceptional events such as neural tube closure, but also for preconceptional events such as normal chromosome segregation. Because the phenotypic expression of the MTHFR genotype varies with individual nutritional folate status, the same maternal genotype could have a variable outcome depending on the specific reproductive stage, the acute severity of folate insufficiency, or both. In mothers of children with NTDs, a significant increase relative to standard normal values in plasma homocysteine concentrations is most commonly associated with the homozygous T/T genotype (29). However, a recent reevaluation of mothers of children with NTDs with normal (C/C) and heterozygous (C/T) genotypes showed that the increase in plasma homocysteine was present TABLE 4 Plasma homocysteine and methionine concentrations in age-matched control mothers and in mothers of children with Down syndrome (DS mothers) 1 Control mothers DS mothers C/C C/T + T/T C/C C/T + T/T (n = 17) (n = 19) (n =9) (n = 32) Plasma homocysteine ( mol/l) 7.9 ± ± ± ± Plasma methionine ( mol/l) 36.7 ± ± ± ± 1.4 Homocysteine:methionine 0.24 ± ± ± ± x ± SEM. 2 Significantly different from control mothers, P < (Student s t test).

6 500 JAMES ET AL with or without the 677C T mutation (31). These results suggest that additional mutations in the MTHFR gene or in other genes involved in folate homeostasis interact to increase plasma homocysteine in mothers of children with NTDs. The significantly higher homocysteine concentrations in mothers of children with Down syndrome with the normal C/C genotype and the greater methotrexate sensitivity in the mothers of children with Down syndrome than in control subjects with the identical C/T genotype similarly suggests that other mutations may interact with MTHFR polymorphism to compromise folate homeostasis. For example, mutations in the 5-methyltetrahydrofolate homocysteine S-methyltransferase gene or in the methionine synthase reductase gene could contribute to abnormal folate metabolism in the mothers of children with Down syndrome. Both high homocysteine concentrations and low DNA methylation have been proposed as possible mechanisms leading to the failure of normal neural tube closure (32). However, a recent study showed that mutation in the cystathionine -synthase gene, resulting in the elevation of both homocysteine and methionine concentrations, is not associated with increased risk of NTDs (33). By deduction, these data suggest that a low methionine (SAM:SAH) value is the critical variable, although aberrant methylation has not yet been evaluated directly in relation to the risk of NTDs. Because supplemental folic acid normalizes homocysteine concentrations and has been shown to reduce the occurrence and recurrence of NTDs (34, 35), it is tempting to speculate that preconceptional folate supplementation could similarly reduce the incidence of Down syndrome; however, controlled prospective clinical trials will be required to validate this assumption. The Hungarian trial that established the efficacy of perinatal vitamins for prevention of NTDs also found that the incidence of Down syndrome was lower in the supplemented group (34); unfortunately, these data were not definitive because of the low number of Down syndrome cases. The recent Food and Drug Administration decision to fortify the US food supply with 1.4 g folic acid/g cereal and grain products may reduce the incidence of both Down syndrome and NTDs. Recent reports indicate that the frequency of the 677C T mutation in the MTHFR gene varies widely between different countries and ethnic groups (36). To avoid possible ethnic bias, the present study was structured such that both groups were white (of mixed European descent) and from geographically diverse areas. Although geographic controls were used, the frequency of the 677T allele in our control group of mothers (0.30) was lower than the estimate for the general US population (0.34). It is possible, however, that the mutation in the MTHFR gene occurs less frequently in mothers who have never experienced a miscarriage or an abnormal pregnancy. The mutant MTHFR allele frequency has been shown to be highest in Hispanic Americans (0.50) and lowest in African Americans (0.11) (37) and strongly correlates with the ethnic distribution of NTDs (38, 39). Of interest, the incidence of Down syndrome follows an ethnic pattern similar to that observed for NTDs, the prevalence being highest in Hispanics and lowest in African Americans (40). The similar ethnic distribution between NTDs and Down syndrome provides indirect support for the hypothesis that the risk of Down syndrome is also related to MTHFR gene mutation. The high prevalence of MTHFR polymorphism in the general population relative to the low risk and incidence of Down syndrome suggests that a mutation in the MTHFR gene alone is not sufficient for Down syndrome to occur and that a multifactorial gene-environment interaction must be involved. Interactions between diet and genotype or between genotypes may negatively affect folate metabolism and the remethylation of homocysteine to methionine. The significantly higher plasma homocysteine concentration and ratio of plasma homocysteine to methionine in the mothers of children with Down syndrome with the normal C/C genotype than in control mothers with the same genotype supports this possibility. The greater methotrexate sensitivity in the heterozygous mothers of children with Down syndrome than in the heterozygous control mothers lends further support to this possibility. 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