HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by James, S J. Articles by Gaylor, D. W Search for Related Content PubMed PubMed Citation Articles by James, S J. Articles by Gaylor, D. W Agricola Articles by James, S J. Articles by Gaylor, D. W

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome^1,2,3

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.

See corresponding editorial on page 429.

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 72079. E-mail: jjames{at}nctr.fda.gov.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 (677CT) 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 677CT 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 677CT 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.

Key Words: Methylenetetrahydrofolate reductase • Down syndrome • folate • homocysteine • mutation • DNA methylation • MTHFR 677CT mutation • trisomy 21

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 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 (677CT) 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 677CT 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.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. The C-to-T substitution at nucleotide 677 (677CT) 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.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

MTHFR 677CT 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 677CT 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.

Methotrexate cytotoxicity
Peripheral blood lymphocytes were isolated by using heparin-containing Vacutainer CPT tubes (Becton Dickinson and Co, Orangeburg, NY) according to the manufacturer's protocol. Washed cells were seeded into 6-well plates at 1 x 10⁹ 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% CO₂ incubator before addition of 0.22 or 0.44 µmol 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 µmol/L) were calculated from peak areas and standard calibration curves by using GOLD NOUVEAU software (Beckman Instruments).

Briefly, 200 µL plasma was mixed with 50 µL of 1.5 µmol EDTA/L, 50 µL of 1.4 mol NaBH₄/L (prepared fresh each day), and 10 µL 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 µL ice-cold 10% metaphosphoric acid was added in 20-µL increments to precipitate proteins. After being centrifuged for 15 min at 20000 x 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 C₁₈ column (4.6 x 150 mm; MC Medical, Inc, Tokyo) was used with a mobile phase consisting of 50 mmol NaH₂PO₄/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 SIGMASTAT 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 x (square root of the variance). The antilogarithms of these limits give the approximate 95% CI for the OR.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 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 677CT MTHFR mutation

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 677CT 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 677CT 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 677CT 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 with or without the 677CT 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 ß-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 µg 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 677CT 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. The data presented in this initial report indicate that abnormal folate metabolism and a 677CT mutation in the MTHFR gene are maternal risk factors for Down syndrome; nonetheless, these observations should be considered preliminary until confirmed in subsequent studies.

	ACKNOWLEDGMENTS

 
We thank the mothers who contributed their time and effort to our study; Melanie Ehrlich, Stephanie Sherman, and Charles A Thomas for critical review of the manuscript and helpful discussions; and Karalee Wetzel and Tamera Ragan, DS-FIRST, Inc, for their help in recruiting the subjects.

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Epstein CJ. Down syndrome (trisomy 21). In: Stansbury JB, Wyngarden JB, Fredrickson DS, eds. The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill, 1995:749–95.
2. Antofnarakis SE, Petersen MB, McInnis MG, et al. The meiotic stage of nondisjunction in trisomy 21: determination by using DNA polymorphisms. Am J Hum Genet 1992;50:544–50.[Medline]
3. Hernandez D, Fisher EMC. Down syndrome genetics: unravelling a multifactorial disorder. Hum Mol Genet 1996;5:1411–6.[Abstract]
4. Ji WZ, Hernandez R, Zhang XY, et al. DNA demethylation and pericentromeric rearrangements of chromosome 1. Mutat Res 1997; 379:33–41.[Medline]
5. Jeanpierre M, Turleau C, Aurius A, et al. An embryonic-like methylation pattern of classical satellite DNA is observed in ICF syndrome. Hum Mol Genet 1993;2:731–5.[Abstract/Free Full Text]
6. Harrison JJ, Anisowicz A, Gadi IG, Raffeld M, Sager R. Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes. Proc Natl Acad Sci U S A 1983;80:6606–10.[Abstract/Free Full Text]
7. Leyton C, Mergudich D, de la Torre C, Sans J. Impaired chromosome segregation in plant anaphase after moderate hypomethylation of DNA. Cell Prolif 1995;28:481–96.[Medline]
8. Lengauer C, Kinzler KW, Vogelstein B. DNA methylation and genetic instability in colorectal cancer cells. Proc Natl Acad Sci U S A 1997;94:2545–50.[Abstract/Free Full Text]
9. Qu GZ, Grundy P, Narayan A, Ehrlich M. Frequent hypomethylation in Wilms tumors of pericentromeric DNA in chromosomes 1 and 16. Cancer Genet Cytogenet 1998;76:196–201.
10. Pogribny IP, Miller BJ, James SJ. Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat. Cancer Lett 1997;115:31–8.[Medline]
11. Christman JK, Sheikhnejad G, Dizik M, Abileah S, Wainfan E. Reversibility of changes in nucleic acid methylation and gene expression induced in rat liver by severe dietary methyl deficiency. Carcinogenesis 1993;14:551–7.[Abstract/Free Full Text]
12. Pogribny IP, Basnakian AG, Miller BJ, Lopatina NG, Poirier LA, James SJ. Breaks in genomic DNA and within the p53 gene are associated with hypomethylation in livers of folate/methyl deficient rats. Cancer Res 1995;55:1894–901.[Abstract/Free Full Text]
13. Pogribny IP, Muskhelishvili L, Miller BJ, James SJ. Presence and consequence of uracil in preneoplastic DNA from folate/methyl deficient rats. Carcinogenesis 1997;8:2071–6.
14. Wainfan E, Poirier LA. Methyl groups in carcinogenesis: effects on DNA methylation and gene expression. Cancer Res 1992;52:2071s–7s.[Medline]
15. Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111–3.[Medline]
16. De Cabo SF, Hazen MJ, Molero ML, Fernández-Piqueras J. S-adenosyl-L-homocysteine: a non-cytotoxic hypomethylating agent. Experientia 1994;50:658–9.[Medline]
17. Balaghi M, Wagner C. DNA methylation in folate deficiency—use of CpG methylase. Biochem Biophys Res Commun 1993;193:1184–90.[Medline]
18. Block G, Hartman AM, Dresser CM, Carroll MD, Gannon J, Gardener L. A data-based approach to diet questionnaire design and testing. Am J Epidemiol 1986;139:1190–6.[Abstract/Free Full Text]
19. Ausebel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA. In: Current protocols in molecular biology. New York: Wiley-Interscience, 1989:2.3.1–2.3.3.
20. Chen J, Giovannucci E, Kelsey K, et al. A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer. Cancer Res 1996;56:4862–4.[Abstract/Free Full Text]
21. Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James SJ. A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection. Nutr Biochem 1999 (in press).
22. Broxson EH Jr, Stork LC, Allen RH, Stabler SP, Kolhouse JF. Changes in plasma methionine and total homocysteine levels in patients receiving methotrexate infusions. Cancer Res 1989;49:5879–83.[Abstract/Free Full Text]
23. Freeman S, Grantham M, Hassold T, et al. Cytogenetic and molecular studies of spontaneous human abortions. Am J Hum Genet 1996;49(suppl):916A (abstr).
24. Lamb NE, Feingold E, Savage A, et al. Characterization of susceptible chiasma configurations that increase the risk for maternal nondisjunction of chromosome 21. Hum Mol Genet 1997;6:1391–9.[Abstract/Free Full Text]
25. Almeida A, Kokalj-Vokac N, Lefrancois D, et al. Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines. Hum Genet 1993;91:538–46.[Medline]
26. Hernandez R, Frady A, Zhang XY, Varela M, Ehrlich M. Preferential induction of chromosome 1 multibranched figures and whole-arm deletions in a human pro-B cell line treated with 5-azacytidine or 5-azadeoxycytidine. Cytogenet Cell Genet 1997;76:196–201.[Medline]
27. Karpen GH, Allshire RC. The case for epigenetic effects on centromere identity and function. Trends Genet 1997;13:489–96.[Medline]
28. De Cabo SF, Santos J, Fernández-Piqueras J. Molecular and cytological evidence of S-adenosyl-L-homocysteine as an innocuous undermethylating agent in vivo. Cytogenet Cell Genet 1995;71:187–92.[Medline]
29. van der Put NMJ, Steegers-Theunissen RPM, Frosst P, et al. Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. Lancet 1995;346:1070–1.[Medline]
30. van der Put NM, Eskes TK, Blom HJ. Is the common 677CT mutation in the methylenetetrahydrofolate reductase gene a risk factor for neural tube defects? A meta-analysis. Q J Med 1997;90:111–5.[Abstract/Free Full Text]
31. van der Put NM, Thomas CM, Eskes TK, et al. Altered folate and vitamin B₁₂ metabolism in families with spina bifida offspring. Q J Med 1998;62:1022–51.
32. Rozen R. Molecular genetics of methylenetetrahydrofolate reductase deficiency. J Inherit Metab Dis 1996;19:589–94.[Medline]
33. Molloy A, Daly S, Mills JM, et al. Methylenetetrahydrofolate reductase associated with low red cell folates: implications for folate intake recommendations. Lancet 1997;349:1591–3.[Medline]
34. Czeizel AE, Dudas I. Prevention of the first occurrence of neural tube defects by periconceptional vitamin supplementation. N Engl J Med 1992;327:1832–5.[Abstract]
35. MRC Vitamin Study Research Group. Folic acid and the prevention of neural tube defects: results of the Medical Council Vitamin Study. Lancet 1991;338:131–7.[Medline]
36. Motulsky AG. Nutritional ecogenetics: homocysteine-related arteriosclerotic vascular disease, neural tube defects, and folic acid. Am J Hum Genet 1996;58:17–20.[Medline]
37. Rosen R. Genetic predisposition to hyperhomocysteinemia: deficiency of methylene tetrahydrofolate reductase (MTHFR). Thromb Haemost 1997;78:523–6.[Medline]
38. Canfield MA, Annegers JF, Brender JD, Cooper SP, Greenberg F. Hispanic origin and neural tube defects in Houston/Harris County, Texas. Am J Epidemiol 1996;43:12–24.
39. Strassburg MA, Greenland S, Portigal LD, Sever LE. A population-based case-control study of anencephalus and spina bifida in a low-risk area. Dev Med Child Neurol 1983;25:632–41.[Medline]
40. Down syndrome prevalence at birth—United States, 1983–1990. Teratology 1997;56:31–6.

This article has been cited by other articles:

				L. M. J. W. van Driel, R. de Jonge, W. A. Helbing, B. D. van Zelst, J. Ottenkamp, E. A. P. Steegers, and R. P. M. Steegers-Theunissen Maternal Global Methylation Status and Risk of Congenital Heart Diseases Obstet. Gynecol., August 1, 2008; 112(2): 277 - 283. [Abstract] [Full Text] [PDF]

				S.S. Young, B. Eskenazi, F.M. Marchetti, G. Block, and A.J. Wyrobek The association of folate, zinc and antioxidant intake with sperm aneuploidy in healthy non-smoking men Hum. Reprod., May 1, 2008; 23(5): 1014 - 1022. [Abstract] [Full Text] [PDF]

				P. Thomas and M. Fenech Chromosome 17 and 21 aneuploidy in buccal cells is increased with ageing and in Alzheimer's disease Mutagenesis, January 1, 2008; 23(1): 57 - 65. [Abstract] [Full Text] [PDF]

				P. Thomas and M. Fenech A review of genome mutation and Alzheimer's disease Mutagenesis, January 1, 2007; 22(1): 15 - 33. [Abstract] [Full Text] [PDF]

				H.-C. Lee, Y.-M. Jeong, S. H. Lee, K. Y. Cha, S.-H. Song, N. K. Kim, K. W. Lee, and S. Lee Association study of four polymorphisms in three folate-related enzyme genes with non-obstructive male infertility Hum. Reprod., December 1, 2006; 21(12): 3162 - 3170. [Abstract] [Full Text] [PDF]

				J. M. Robbins, J. M. Tilford, T.M. Bird, M. A. Cleves, J. A. Reading, and C. A. Hobbs Hospitalizations of Newborns With Folate-Sensitive Birth Defects Before and After Fortification of Foods With Folic Acid Pediatrics, September 1, 2006; 118(3): 906 - 915. [Abstract] [Full Text] [PDF]

				T. Tamura and M. F. Picciano Folate and human reproduction Am. J. Clinical Nutrition, May 1, 2006; 83(5): 993 - 1016. [Abstract] [Full Text] [PDF]

				R.-M. Gueant-Rodriguez, J.-L. Gueant, R. Debard, S. Thirion, L. X. Hong, J.-P. Bronowicki, F. Namour, N. W Chabi, A. Sanni, G. Anello, et al. Prevalence of methylenetetrahydrofolate reductase 677T and 1298C alleles and folate status: a comparative study in Mexican, West African, and European populations Am. J. Clinical Nutrition, March 1, 2006; 83(3): 701 - 707. [Abstract] [Full Text] [PDF]

				S. Ernest, A. Hosack, W. E. O'Brien, D. S. Rosenblatt, and J. H. Nadeau Homocysteine levels in A/J and C57BL/6J mice: genetic, diet, gender, and parental effects Physiol Genomics, May 11, 2005; 21(3): 404 - 410. [Abstract] [Full Text] [PDF]

				J-L Gueant, G Anello, P Bosco, R-M Gueant-Rodriguez, A Romano, C Barone, P Gerard, and C Romano Homocysteine and related genetic polymorphisms in Down's syndrome IQ J. Neurol. Neurosurg. Psychiatry, May 1, 2005; 76(5): 706 - 709. [Abstract] [Full Text] [PDF]

				ESHRE Capri Workshop Group Fertility and ageing Hum. Reprod. Update, May 1, 2005; 11(3): 261 - 276. [Abstract] [Full Text] [PDF]

				A. H Chen, S. M Innis, A G. F Davidson, and S J. James Phosphatidylcholine and lysophosphatidylcholine excretion is increased in children with cystic fibrosis and is associated with plasma homocysteine, S-adenosylhomocysteine, and S-adenosylmethionine Am. J. Clinical Nutrition, March 1, 2005; 81(3): 686 - 691. [Abstract] [Full Text] [PDF]

				C. A. Perry, S. A. Renna, E. Khitun, M. Ortiz, D. J. Moriarty, and M. A. Caudill Ethnicity and Race Influence the Folate Status Response to Controlled Folate Intakes in Young Women J. Nutr., July 1, 2004; 134(7): 1786 - 1792. [Abstract] [Full Text]

				M. Lucock Is folic acid the ultimate functional food component for disease prevention? BMJ, January 24, 2004; 328(7433): 211 - 214. [Full Text] [PDF]

				S. Gurbuxani, P. Vyas, and J. D. Crispino Recent insights into the mechanisms of myeloid leukemogenesis in Down syndrome Blood, January 15, 2004; 103(2): 399 - 406. [Abstract] [Full Text] [PDF]

				M. R Amorim, E. E Castilla, and I. M Orioli Is there a familial link between Down's syndrome and neural tube defects? Population and familial survey BMJ, January 10, 2004; 328(7431): 84. [Abstract] [Full Text] [PDF]

				H. Refsum, A. D. Smith, P. M. Ueland, E. Nexo, R. Clarke, J. McPartlin, C. Johnston, F. Engbaek, J. Schneede, C. McPartlin, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion Clin. Chem., January 1, 2004; 50(1): 3 - 32. [Abstract] [Full Text] [PDF]

				M. Kimura, K. Umegaki, M. Higuchi, P. Thomas, and M. Fenech Methylenetetrahydrofolate Reductase C677T Polymorphism, Folic Acid and Riboflavin Are Important Determinants of Genome Stability in Cultured Human Lymphocytes J. Nutr., January 1, 2004; 134(1): 48 - 56. [Abstract] [Full Text] [PDF]

				A. E. Czeizel and E. Medveczky Periconceptional Multivitamin Supplementation and Multimalformed Offspring Obstet. Gynecol., December 1, 2003; 102(6): 1255 - 1261. [Abstract] [Full Text] [PDF]

				B Wilcken, F Bamforth, Z Li, H Zhu, A Ritvanen, M Redlund, C Stoll, Y Alembik, B Dott, A E Czeizel, et al. Geographical and ethnic variation of the 677C>T allele of 5,10 methylenetetrahydrofolate reductase (MTHFR): findings from over 7000 newborns from 16 areas world wide J. Med. Genet., August 1, 2003; 40(8): 619 - 625. [Full Text]

				A. L. Bjorke Monsen and P. M. Ueland Homocysteine and methylmalonic acid in diagnosis and risk assessment from infancy to adolescence Am. J. Clinical Nutrition, July 1, 2003; 78(1): 7 - 21. [Abstract] [Full Text] [PDF]

				C. L. Guinotte, M. G. Burns, J. A. Axume, H. Hata, T. F. Urrutia, A. Alamilla, D. McCabe, A. Singgih, E. A. Cogger, and M. A. Caudill Methylenetetrahydrofolate Reductase 677C->T Variant Modulates Folate Status Response to Controlled Folate Intakes in Young Women J. Nutr., May 1, 2003; 133(5): 1272 - 1280. [Abstract] [Full Text] [PDF]

				R. Carmel, R. Green, D. S. Rosenblatt, and D. Watkins Update on Cobalamin, Folate, and Homocysteine Hematology, January 1, 2003; 2003(1): 62 - 81. [Abstract] [Full Text] [PDF]

				S. J. James, S. Melnyk, M. Pogribna, I. P. Pogribny, and M. A. Caudill Elevation in S-Adenosylhomocysteine and DNA Hypomethylation: Potential Epigenetic Mechanism for Homocysteine-Related Pathology J. Nutr., August 1, 2002; 132(8): 2361S - 2366. [Abstract] [Full Text] [PDF]

</

				B. N Ames, I. Elson-Schwab, and E. A Silver High-dose vitamin therapy stimulates variant enzymes with decreased coenzyme binding affinity (increased Km): relevance to genetic disease and polymorphisms Am. J. Clinical Nutrition, April 1, 2002; 75(4): 616 - 658. [Abstract] [Full Text] [PDF]