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Am J Med Genet A. Author manuscript; available in PMC May 5, 2008.
Published in final edited form as:
PMCID: PMC2366099
NIHMSID: NIHMS44550
Folate and One-Carbon Metabolism Gene Polymorphisms and Their Associations With Oral Facial Clefts
Abee L. Boyles,1* Allen J. Wilcox,1 Jack A. Taylor,1 Klaus Meyer,2 Åse Fredriksen,2 Per Magne Ueland,2 Christian A. Drevon,3 Stein Emil Vollset,4 and Rolv Terje Lie4
1Epidemiology Branch, National Institute of Environmental Health Sciences/NIH, Durham, North Carolina
2Section for Pharmacology, Institute of Medicine, University of Bergen and Haukeland University Hospital, Armauer Hansens Hus, Bergen, Norway
3Department of Nutrition, Institute of Basic Medical Sciences, Medical Faculty, University of Oslo, Oslo, Norway
4Department of Public Health and Primary Health Care, Section for Epidemiology and Medical Statistics, University of Bergen, Bergen, Norway
*Correspondence to: Abee L. Boyles, Ph.D., Epidemiology Branch Mail Drop A3-05, P.O. Box 12233, National Institute of Environmental Health Sciences/NIH, Durham, NC 27709. E-mail: boylesa/at/niehs.nih.gov
Folate metabolism plays a critical role in embryonic development. Prenatal folate supplementation reduces the risk of neural tube defects and probably oral facial clefts. Previous studies of related metabolic genes have associated polymorphisms in cystathionine-beta-synthase (CBS) and 5,10-methylenetetrahydrofolate reductase (MTHFR) with cleft risk. We explored associations between genes related to one-carbon metabolism and clefts in a Norwegian population-based study that included 362 families with cleft lip with or without cleft palate (CL/P) and 191 families with cleft palate only (CPO). We previously showed a 39% reduction in risk of CL/P with folic acid supplementation in this population. In the present study we genotyped 12 polymorphisms in nine genes related to one-carbon metabolism and looked for associations of clefting risk with fetal polymorphisms, maternal polymorphisms, as well as parent-of-origin effects, using combined likelihood-ratio tests (LRT). We also stratified by maternal periconceptional intake of folic acid (>400 μg) to explore gene-exposure interactions. We found a reduced risk of CL/P with mothers who carried the CBS C699T variant (rs234706); relative risk was 0.94 with one copy of the T allele (95% CI 0.63-1.4) and 0.50 (95% CI 0.26-0.96) with two copies (P = 0.008). We found no evidence of interaction of this variant with folate status. We saw no evidence of risk from the MTHFR C677T variant (rs1801133) either overall or after stratifying by maternal folate intake. No associations were found between any of the polymorphisms and CPO. Genetic variations in the nine metabolic genes examined here do not confer a substantial degree of risk for clefts. Published 2008 Wiley-Liss, Inc.
Keywords: alleles, cleft lip, cleft palate, dietary supplements, folic acid, metabolism, humans, single nucleotide polymorphisms
Folic acid clearly reduces the risk of neural tube defects [MRC Vitamin Study Research Group, 1991; Berry, 1998; Botto et al., 1999]. Whereas early work showed that folic acid could reduce the risk of facial clefts in mouse models [Munger, 2002], epidemiologic studies of facial clefts have provided inconsistent results [Shaw et al., 1995, 2006; Hayes et al., 1996; Werler et al., 1999; Wong et al., 1999; Itikala et al., 2001; Munger et al., 2004; van Rooij et al., 2004]. A recent meta-analysis of 17 prospective and case-control studies supported a protective effect of folic acid supplementation on cleft lip with or without cleft palate (CL/P) and perhaps cleft palate only (CPO) [Badovinac et al., 2007]. Our own study of facial clefts in Norway provided evidence that dietary supplements with folic acid reduced the risk of isolated CL/P by 39%, but with no apparent effects on CPO [Wilcox et al., 2007]. Genetic polymorphisms in the folate and one-carbon metabolism pathway (Fig. 1) could be associated with risk of oral facial clefts (especially CL/P) possibly interacting with low maternal folic acid intake.
Fig. 1
Fig. 1
Folate and one-carbon metabolic pathway. Enzymes are enclosed in ellipses and substrates are enclosed in boxes. The cell membrane is represented by the dotted ellipse. Enzymatic steps are indicated by solid lines, whereas transport functions are indicated (more ...)
Participants
Of 300,000 babies delivered in Norway from 1996 to 2001, 676 were referred for oral facial cleft corrective surgery. Twenty-four babies who died after birth or whose mothers did not speak Norwegian were excluded. From the 652 remaining, 573 (377 CL/P cases and 196 CPO cases) agreed to participate in the study (88%). The number of cases dropped slightly at each subsequent analytic step (Table I). Control families were collected using the same exclusions (763 of 1,022 randomly sampled live births). We used these controls only to estimate Hardy-Weinberg equilibrium (HWE) and population minor allele frequencies (MAFs). Future references to the “full” data set include case families only. All parents provided informed consent. Further details of the overall study design and implementation have been previously published [Wilcox et al., 2007].
TABLE I
TABLE I
Case Family Datasets
Genotyping
Genotyping of cases, controls, and their available first-degree relatives was performed using a matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) multiplex method [Meyer et al., 2004]. We examined 13 frequently-studied polymorphisms in nine genes related to one-carbon metabolism (Table II): solute carrier family 19, member 1 (SLC19A1); transcobalamin II (TCN2); methylenetetrahydrofolate dehydrogenase 1 (MTHFD1); 5,10 methylenetetrahydrofolate reductase (MTHFR); 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR); 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR); betaine-homocysteine methyltransferase (BHMT); cystathionine beta synthase (CBS); and paraoxonase 1 (PON1).
TABLE II
TABLE II
MALDI-TOF MS Genotyping of Folate and Homocysteine Pathway Genes
PON1 rs854560 had a lower genotyping call rate (92% vs. 98% for the other polymorphisms). Furthermore, it generated a high level of Mendelian inconsistencies and was not in HWE. For these reasons we excluded PON1 rs854560 from subsequent analyses. Of 194 genotyped CPO cases, genetic information was unavailable for 2 mothers (1%) and 12 fathers (6%). For 367 CL/P cases, genetic information was unavailable for 13 mothers (4%) and 26 fathers (7%).
Folic Acid Supplementation
Soon after delivery, mothers were mailed questionnaires regarding their vitamin intake during each of the 6 months prior to and the first 3 months of pregnancy (questionnaire available online http://www.niehs.nih.gov/research/atniehs/labs/epi/studies/ncl/question.cfm). Mothers who reported taking a folic acid supplement or multivitamin during this time were asked for the product name. Women were also asked to send an empty pill bottle or product label to the study office for verification of the amount of folic acid. Women were considered supplemented if they took folic acid for at least 1 month during a 3-month window beginning 1 month before the last menstrual period and continuing through the first 2 months of pregnancy. This covers the crucial time period when the facial structures of the embryonic lip and palate are fusing.
Further details on assignment of folic acid dose are provided elsewhere [Wilcox et al., 2007]. Daily intake of 400 μg or more of folic acid was associated in these data with a 39% reduction in CL/P. No protective effect was seen with lower doses. Also, no protective effect was seen in CPO. We explored interactions of variations in one-carbon metabolism genes with folic acid intake by stratifying women into less than 400 μg/day and more than 400 μg/day.
Statistical Analyses
Familial genotypes inconsistent with Mendelian inheritance were identified with MEGA2 [Mukhopadhyay et al., 2005]. Of the 561 successfully genotyped families, eight had four or more inconsistencies. These were considered higher than expected due to random assay error alone, suggesting that samples may have been mislabeled. The whole families were excluded in subsequent analyses. All markers were tested for HWE using a Pearson’s chi-squared test with one degree of freedom. Disequilibrium can indicate poor assay performance (as in PON1 rs854560) in addition to other possibilities, such as recent admixture.
The remaining 12 polymorphisms were analyzed for child genotype effects, maternal genotype effects, and parent-of-origin (POO) effects using a combined likelihood ratio test (LRT) that utilizes unaffected siblings when parental genotypes are missing [Rampersaud et al., 2007]. The Combined LRT for Candidate Gene Studies, Version 1.0 (www.chg.duke.edu/research/lrt.html) was implemented with SAS (version 9.1.2, Cary, NC) software. Based on the maximum-likelihood-based method for analysis of triad data originally described by Weinberg et al. [1998], this implementation allows unaffected siblings from incompletely genotyped families to contribute to the LRT of association. POO effects are fitted by an iterative method relying on the expectation-maximization (EM) algorithm. When one or both parents are unavailable, sibling genotypes are utilized by the expectation step of the algorithm. No phenotypic information from these individuals is used. The LRTs for child’s genotype risk and maternal genotype risk are 2-df tests, whereas the POO test involves a single parameter and is a 1-df test. Wald 95% confidence intervals were calculated from the model coefficient and standard error estimates and exponentiated to determine the relative risk with 95% CIs. We did not undertake a formal test of gene-environment interaction in this dataset as it is not currently implemented with this software. All polymorphisms were tested for child genotype, maternal genotype, and POO in the full dataset as well as in the two subsets stratified by total daily folate supplementation (less than 400 or >400 μg).
The relative risks of CL/P and CPO associated with one or two copies of all polymorphisms in the full data set are shown in Figure 2. Among CL/P, the only LRT reaching statistical significance was for the maternal effect of CBS rs234706 variant (LRT P value = 0.008). Figure 3 includes the relative risks of CL/P and P values for the three LRTs (child, mother, POO) in each of the full dataset and two subsets for the four SNPs that had a P value less than 0.05 on any LRT: CBS rs234706 (Fig. 3A), MTHFR rs1801133 (Fig. 3B), BHMT rs3733890 (Fig. 3C), and TCN2 rs1801198 (Fig. 3D). All three groups showed a protective association with two copies of the CBS rs234706 T variant. Whereas one copy of the T allele in the full dataset was not protective (0.94, 95% CI 0.63-1.4), two copies did show a protective effect (0.5, 95% CI 0.26-0.96). For the <400 μg group, one copy had a relative risk of 0.96 (95% CI 0.62-1.5) and two copies had a risk of 0.61 (95% CI 0.3-1.2). The folic acid-supplemented group showed the same trend (one copy 0.9, 95% CI 0.32-2.5; two copies 0.18, 95% CI 0.03-1.04). Thus, while offspring of mothers homozygous for this CBS variant may have some decreased risk of CL/P, its association was not appreciably modified by maternal folate supplementation.
Fig. 2
Fig. 2
Fig. 2
Relative risks of CL/P (A) and CPO (B) and their 95% confidence intervals of zero (ref), one, and two copies of each polymorphism for child’s genotype and mother’s genotype in the full dataset. Estimates are graphed vertically for each (more ...)
Fig. 3
Fig. 3
Fig. 3
Relative risks of CL/P with 95% CIs and LRT P values in the full, <400, and >400 μg folate datasets. The four SNPs where any LRT P-value was less than 0.05 are shown: (A) CBS rs234706, (B) MTHFR rs1801133, (C) BHMT rs3733890, and (more ...)
We did not observe an increased risk of CL/P with the maternal C677T MTHFR T allele of rs1801133 in the full data set (one maternal copy 0.62, 95% CI 0.40-0.93; two copies 0.51, 95% CI 0.26-1.01; Fig. 3B). In the subset of families where mothers took less than 400 μg of folic acid, the MTHFR T allele was protective for the maternal genotype (LRT P = 0.037, one copy 0.60, 95% CI 0.39-0.92; two copies 0.44, 95% CI 0.21-0.95). We saw no evidence for a protective effect in the subset who took more than 400 μg of folate supplementation (one copy 0.84, 95% CI 0.26-2.6; two copies 0.90, 95% CI 0.16-4.9).
In the subset of 50 families with CL/P and folic acid supplementation, LRT’s with P values less than 0.05 were found for three SNPs. For CBS rs234706, the LRT of child’s genotype was 0.02, but the relative risks showed no dose dependent trend for this variant (one copy 0.33, 95% CI 0.11-1.1; two copies 1.3, 95% CI 0.39-4.2). The test of BHMT rs3733890 produced a P-value of 0.04 for maternal genotype, again with no trend according to the number of variant alleles (one copy 1.3, 95% CI 0.44-3.8; two copies 0.78, 95% CI 0.09-6.4; Fig. 3C). A significant POO test was found for TCN2 rs18011989 (0.11, 95% CI 0.027-0.48; Fig. 3D), but it was based on even fewer families: 10 cases received a G allele from their father, while only six received it from their mother.
These data from a population-based case-control study in Norway provide the largest sample size to date for the analysis of genes related to folate and homocysteine metabolism and oral facial clefts. In this population, folic acid supplements in early pregnancy reduced the risk of CL/P but not CPO [Wilcox et al., 2007], irrespective of genotype. We provide data here on associations of 12 polymorphisms in nine of the one-carbon pathway genes including stratification by maternal periconceptional folate supplementation. Previous studies of these genes in clefts are summarized in Table III.
TABLE III
TABLE III
Previous Studies of Folate Gene Associations With Oral Facial Clefts
Folic acid derivatives provide essential single-carbon units for nucleic acid synthesis and methylation reactions—both of which are necessary for cell division, proper gene expression, and maintenance of chromosome structure during fetal development [Morrison et al., 1998]. Polymorphisms in SLC19A1 or TCN2 could impair one-carbon metabolism by limiting the cellular availability of folate or vitamin B12. Reactions within the folate metabolic cycle are complex, with multiple enzymes converting folate into several chemical forms. Genes encoding proteins that perform or support these conversions include MTHFD1, MTHFR, MTR, and MTRR. MTHFR polymorphisms are widely studied in coronary artery disease, neural tube defects, and facial clefts [selected recent publications: Bennouar et al., 2007; Chevrier et al., 2007; Munoz et al., 2007]. The folate cycle is coupled to one-carbon metabolism by the MTR-mediated conversion of homocysteine to methionine. This conversion is also performed by BHMT in a non-folate dependent reaction [van der Linden et al., 2006]. Homocysteine can also be converted to cystathionine by CBS [Morrison et al., 1998], or generated from homocysteine thiolactone by PON1 [Jakubowski et al., 2000].
In this study, mothers with a synonymous SNP, rs234706, in CBS had slight protection from CL/P that was not influenced by periconceptional folate supplementation (Fig. 3A). These results were modest and not concordant with a previous study of this gene in clefts [Rubini et al., 2005]. CBS performs the rate-limiting step in the transsulfuration pathway that degrades homocysteine [Finkelstein, 1990], and this SNP was associated with a decrease in the total homocysteine/cystathionine ratio [Fredriksen et al., 2007]. While the SNP is a silent transition at codon position 233, it may be in linkage disequilibrium with transcriptional regulatory elements [Aras et al., 2000].
Our study provides modest evidence for a protective effect of the C677T T allele of MTHFR in CL/P risk (Fig. 3B). This SNP causes an alanine at codon 222 in the catalytic region of the enzyme to be changed to a valine [Frosst et al., 1995]. Enzymatic function is lower in the thermolabile MTHFR protein encoded by this polymorphism, and it has been associated with higher levels of plasma homocysteine and lower levels of folate and betaine [Frosst et al., 1995; Fredriksen et al., 2007]. It is not readily apparent how this variant would provide protection from CL/P in the offspring of mothers who carry it. Some studies have suggested protection from CL/P by the MTHFR T allele in mothers [van Rooij et al., 2003; Gaspar et al., 2004; Nurk et al., 2004] and children [Shaw et al., 1998; Grunert et al., 2002; Shotelersuk et al., 2003; Gaspar et al., 2004], although none reached statistical significance. A meta-analysis including these studies found no effect on CPO (OR 1.0, 95% CI 0.9-1.2) and a slightly increased risk of CL/P (OR 1.2, 95% CI 0.9-1.5).
A similar protective effect of the T allele has been consistently seen in colorectal cancer [Chen et al., 1996; Ma et al., 1999; Slattery et al., 1999; Le Marchand et al., 2002, 2005; Ulvik et al., 2004; Jiang et al., 2005]. One possible explanation is that when folate is low, the wild-type enzyme depletes 5,10-CH2THF for methylation at the expense of pyrimidine synthesis leading to uracil misincorporation and DNA strand breaks. Thus the TT genotype would slow the conversion of 5,10-CH2THF to 5-CH3THF and close this “window of opportunity” for DNA damage [Brockton, 2006].
In this study the protective effect of the mother’s or child’s T allele was strongest when mothers did not take supplemental folate, as seen in a previous analysis of a subset of these data [Jugessur et al., 2003]. The T allele was also found to be protective against spina bifida occulta and anencephaly [Relton et al., 2003], while it is considered a risk factor for myelomeningocele in some populations [van der Put et al., 1995; de Franchis et al., 1998; O’Leary et al., 2005]. Studies of simulated data suggest that reversals in allelic association could also be due to multi-locus effects or inter-locus correlations [Lin et al., 2007].
Although based on a small sample size, there was a significant LRT of BHMT rs3733890 in the >400 μg folate supplemented subset (Fig. 3C). This polymorphism was also associated with risk in NTD cases whose mothers took supplements with folic acid around conception [Boyles et al., 2006]. The suggestion of a POO effect for TCN2 in >400 μg folate supplemented CL/P cases was based only on a few families (Fig. 3D).
Protection from CPO by folate supplementation is weak, if present at all [Badovinac et al., 2007; Wilcox et al., 2007]. We found no evidence of association between CPO and the polymorphisms studied here. Segregation analyses of CL/P have shown a significant genetic component to clefts but are inconclusive as to the mode of inheritance [Lidral and Moreno, 2005]. An extensive Danish study found evidence of genetic heterogeneity in CL/P [Marazita et al., 1984] making it difficult to identify single gene or environment risk factors associated with risk in all affected people.
If we had corrected for the multiple tests performed, even with a method less stringent than a Bonferroni correction, it is unlikely that even our lowest P-value of 0.008 would remain significant. We also did not perform haplotype-based analysis in the three genes with two polymorphisms. Based on the single polymorphism results there was little reason to believe that such tests would strengthen our results, and this would have further increased the overall number of tests.
Our study fell well short of capturing all the genetic variation in these genes. Expanded genotyping with haplotype tagging SNPs could provide more comprehensive results. Also this set of nine genes does not cover the entire metabolic pathway. For example, mouse knockouts of folate binding protein 1 have major craniofacial defects [Tang and Finnell, 2003]; therefore the human homolog, folate receptor 1, could harbor risk-conferring polymorphisms. Furthermore, the protective effect of folic acid supplementation could act via other developmental pathways. Several WNT signaling pathway genes are regulated by folate and must function properly for normal fetal growth and differentiation [Krapels et al., 2006].
Future research must capture more of the genetic variation in the folate and homocysteine pathway with haplotype tagging SNPs and additional genes. More direct analytical methods for evaluating gene-gene and gene-gene-environment interactions could be more powerful than SNP-by-SNP approaches. Haplotype-based association testing of gene-gene and gene-environment interactions may provide insight into the complex relationship between folate supplementation and the risk of oral facial clefts.
ACKNOWLEDGMENTS
The authors would like to thank Robert McConnaughey for his data management and graphical assistance. This research was supported, in part, by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences and the Thematic Program on Perinatal Nutrition, Faculty of Medicine, University of Oslo.
Grant sponsor: Intramural Research Program of the NIH; Grant sponsor: National Institute of Environmental Health Sciences; Grant sponsor: Thematic Program on Perinatal Nutrition, Faculty of Medicine, University of Oslo.
Footnotes
This article is a US Government work and, as such, is in the public domain in the United States of America.
  • Aras O, Hanson NQ, Yang F, Tsai MY. Influence of 699C- >T and 1080C- >T polymorphisms of the cystathionine beta-synthase gene on plasma homocysteine levels. Clin Genet. 2000;58:455–459. [PubMed]
  • Badovinac RL, Werler MM, Williams PL, Kelsey KT, Hayes C. Folic acid-containing supplement consumption during pregnancy and risk for oral clefts: A meta-analysis. Birth Defects Res A Clin Mol Teratol. 2007;79:8–15. [PubMed]
  • Beaty TH, Hetmanski JB, Zeiger JS, Fan YT, Liang KY, VanderKolk CA, McIntosh I. Testing candidate genes for non-syndromic oral clefts using a case-parent trio design. Genet Epidemiol. 2002;22:1–11. [PubMed]
  • Bennouar N, Allami A, Azeddoug H, Bendris A, Laraqui A, El Jaffali A, El Kadiri N, Benzidia R, Benomar A, Fellat S, Benomar M. Thermolabile methylenetetrahydrofolate reductase C677T polymorphism and homocysteine are risk factors for coronary artery disease in moroccan population. J Biomed Biotechnol. 2007;1:80687. [PMC free article] [PubMed]
  • Berry A. Focusing on folic acid. Task force organizing to promote consumption of folic acid. AWHONN Lifelines. 1998;2:19–20. 19. [PubMed]
  • Blanton SH, Kolle BS, Hecht JT, Mulliken JB, Martin ER. No evidence supporting MTHFR as a risk factor in the development of familial NSCLP. Am J Med Genet. 2000;92:370–371. [PubMed]
  • Botto LD, Moore CA, Khoury MJ, Erickson JD. Neural-tube defects. N Engl J Med. 1999;341:1509–1519. [PubMed]
  • Boyles AL, Billups AV, Deak KL, Siegel DG, Mehltretter L, Slifer SH, Bassuk AG, Kessler JA, Reed MC, Nijhout HF, George TM, Enterline DS, Gilbert JR, Speer MC. Neural tube defects and folate pathway genes: Family-based association tests of gene-gene and gene-environment interactions. Environ Health Perspect. 2006;114:1547–1552. [PMC free article] [PubMed]
  • Brockton NT. Localized depletion: The key to colorectal cancer risk mediated by MTHFR genotype and folate? Cancer Causes Control. 2006;17:1005–1016. [PubMed]
  • Chen J, Giovannucci E, Kelsey K, Rimm EB, Stampfer MJ, Colditz GA, Spiegelman D, Willett WC, Hunter DJ. A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer. Cancer Res. 1996;56:4862–4864. [PubMed]
  • Chevrier C, Perret C, Bahuau M, Zhu H, Nelva A, Herman C, Francannet C, Robert-Gnansia E, Finnell RH, Cordier S. Fetal and maternal MTHFR C677T genotype, maternal folate intake and the risk of nonsyndromic oral clefts. Am J Med Genet Part A. 2007;143A:248–257.
  • de Franchis R, Buoninconti A, Mandato C, Pepe A, Sperandeo MP, Del Gado R, Capra V, Salvaggio E, Andria G, Mastroiacovo P. The C677T mutation of the 5,10-methylenetetrahydrofolate reductase gene is a moderate risk factor for spina bifida in Italy. J Med Genet. 1998;35:1009–1013. [PMC free article] [PubMed]
  • Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem. 1990;1:228–237. [PubMed]
  • Fredriksen A, Meyer K, Ueland PM, Vollset SE, Grotmol T, Schneede J. Large-scale population-based metabolic phenotyping of thirteen genetic polymorphisms related to one-carbon metabolism. Hum Mutat. 2007;28:856–865. [PubMed]
  • Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, et al. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10:111–113. [PubMed]
  • Gaspar DA, Matioli SR, de Cassia Pavanello R, Araujo BC, Alonso N, Wyszynski D, Passos-Bueno MR. Maternal MTHFR interacts with the offspring’s BCL3 genotypes, but not with TGFA, in increasing risk to nonsyndromic cleft lip with or without cleft palate. Eur J Hum Genet. 2004;12:521–526. [PubMed]
  • Grunert RR, Braune A, Schnackenberg E, Schloot W, Krause HR. Genetic differences in enzymes of folic acid metabolism in patients with lip-jaw-palate clefts and their relatives. Mund Kiefer Gesichtschir. 2002;6:131–133. [PubMed]
  • Hayes C, Werler MM, Willett WC, Mitchell AA. Case-control study of periconceptional folic acid supplementation and oral clefts. Am J Epidemiol. 1996;143:1229–1234. [PubMed]
  • Itikala PR, Watkins ML, Mulinare J, Moore CA, Liu Y. Maternal multivitamin use and orofacial clefts in offspring. Teratology. 2001;63:79–86. [PubMed]
  • Jakubowski H, Zhang L, Bardeguez A, Aviv A. Homocysteine thiolactone and protein homocysteinylation in human endothelial cells: Implications for atherosclerosis. Circ Res. 2000;87:45–51. [PubMed]
  • Jiang Q, Chen K, Ma X, Li Q, Yu W, Shu G, Yao K. Diets, polymorphisms of methylenetetrahydrofolate reductase, and the susceptibility of colon cancer and rectal cancer. Cancer Detect Prev. 2005;29:146–154. [PubMed]
  • Jugessur A, Wilcox AJ, Lie RT, Murray JC, Taylor JA, Ulvik A, Drevon CA, Vindenes HA, Abyholm FE. Exploring the effects of methylenetetrahydrofolate reductase gene variants C677T and A1298C on the risk of orofacial clefts in 261 Norwegian case-parent triads. Am J Epidemiol. 2003;157:1083–1091. [PubMed]
  • Krapels IP, Vermeij-Keers C, Muller M, de Klein A, Steegers-Theunissen RP. Nutrition and genes in the development of orofacial clefting. Nutr Rev. 2006;64:280–288. [PubMed]
  • Le Marchand L, Donlon T, Hankin JH, Kolonel LN, Wilkens LR, Seifried A. B-vitamin intake, metabolic genes, and colorectal cancer risk (United States) Cancer Causes Control. 2002;13:239–248. [PubMed]
  • Le Marchand L, Wilkens LR, Kolonel LN, Henderson BE. The MTHFR C677T polymorphism and colorectal cancer: The multiethnic cohort study. Cancer Epidemiol Biomarkers Prev. 2005;14:1198–1203. [PubMed]
  • Lidral AC, Moreno LM. Progress toward discerning the genetics of cleft lip. Curr Opin Pediatr. 2005;17:731–739. [PMC free article] [PubMed]
  • Lin PI, Vance JM, Pericak-Vance MA, Martin ER. No gene is an island: The flip-flop phenomenon. Am J Hum Genet. 2007;80:531–538. [PubMed]
  • Ma J, Stampfer MJ, Christensen B, Giovannucci E, Hunter DJ, Chen J, Willett WC, Selhub J, Hennekens CH, Gravel R, Rozen R. A polymorphism of the methionine synthase gene: Association with plasma folate, vitamin B12, homocyst(e)ine, and colorectal cancer risk. Cancer Epidemiol Biomarkers Prev. 1999;8:825–829. [PubMed]
  • Marazita ML, Spence MA, Melnick M. Genetic analysis of cleft lip with or without cleft palate in Danish kindreds. Am J Med Genet. 1984;19:9–18. [PubMed]
  • Martinelli M, Scapoli L, Pezzetti F, Carinci F, Carinci P, Stabellini G, Bisceglia L, Gombos F, Tognon M. C677T variant form at the MTHFR gene and CL/P: A risk factor for mothers? Am J Med Genet. 2001;98:357–360. [PubMed]
  • Martinelli M, Scapoli L, Palmieri A, Pezzetti F, Baciliero U, Padula E, Carinci P, Morselli PG, Carinci F. Study of four genes belonging to the folate pathway: Transcobalamin 2 is involved in the onset of non-syndromic cleft lip with or without cleft palate. Hum Mutat. 2006;27:294. [PubMed]
  • Meyer K, Fredriksen A, Ueland PM. High-level multiplex genotyping of polymorphisms involved in folate or homocysteine metabolism by matrix-assisted laser desorption/ionization mass spectrometry. Clin Chem. 2004;50:391–402. [PubMed]
  • Mills JL, Kirke PN, Molloy AM, Burke H, Conley MR, Lee YJ, Mayne PD, Weir DG, Scott JM. Methylenetetrahydrofolate reductase thermolabile variant and oral clefts. Am J Med Genet. 1999;86:71–74. [PubMed]
  • Morrison K, Papapetrou C, Hol FA, Mariman EC, Lynch SA, Burn J, Edwards YH. Susceptibility to spina bifida: An association study of five candidate genes. Ann Hum Genet. 1998;62:379–396. [PubMed]
  • Mostowska A, Hozyasz KK, Jagodzinski PP. Maternal MTR genotype contributes to the risk of non-syndromic cleft lip and palate in the Polish population. Clin Genet. 2006;69:512–517. [PubMed]
  • MRC Vitamin Study Research Group Prevention of neural tube defects: Results of the Medical Research Council Vitamin Study. Lancet. 1991;338:131–137. [PubMed]
  • Mukhopadhyay N, Almasy L, Schroeder M, Mulvihill WP, Weeks DE. Mega2: Data-handling for facilitating genetic linkage and association analyses. Bioinformatics. 2005;21:2556–2557. [PubMed]
  • Munger RG. Maternal nutrition and oral clefts. In: Wyszynski DF, editor. Cleft lip and palate: From origin to treatment. Oxford University Press; Oxford: 2002. pp. 170–192.
  • Munger RG, Sauberlich HE, Corcoran C, Nepomuceno B, Daack-Hirsch S, Solon FS. Maternal vitamin B-6 and folate status and risk of oral cleft birth defects in the Philippines. Birth Defects Res A Clin Mol Teratol. 2004;70:464–471. [PubMed]
  • Munoz JB, Lacasana M, Cavazos RG, Borja-Aburto VH, Galaviz-Hernandez C, Garduno CA. Methylenetetrahydrofolate reductase gene polymorphisms and the risk of anencephaly in Mexico. Mol Hum Reprod. 2007;13:419–424. [PubMed]
  • Nurk E, Tell GS, Refsum H, Ueland PM, Vollset SE. Associations between maternal methylenetetrahydrofolate reductase polymorphisms and adverse outcomes of pregnancy: The Hordaland Homocysteine Study. Am J Med. 2004;117:26–31. [PubMed]
  • O’Leary VB, Mills JL, Parle-McDermott A, Pangilinan F, Molloy AM, Cox C, Weiler A, Conley M, Kirke PN, Scott JM, Brody LC. Screening for new MTHFR polymorphisms and NTD risk. Am J Med Genet Part A. 2005;138A:99–106.
  • Pei L, Zhu H, Zhu J, Ren A, Finnell RH, Li Z. Genetic variation of infant reduced folate carrier (A80G) and risk of orofacial defects and congenital heart defects in China. Ann Epidemiol. 2006;16:352–356. [PubMed]
  • Pezzetti F, Martinelli M, Scapoli L, Carinci F, Palmieri A, Marchesini J, Carinci P, Caramelli E, Rullo R, Gombos F, Tognon M. Maternal MTHFR variant forms increase the risk in offspring of isolated nonsyndromic cleft lip with or without cleft palate. Hum Mutat. 2004;24:104–105. [PubMed]
  • Prescott NJ, Winter RM, Malcolm S. Maternal MTHFR genotype contributes to the risk of non-syndromic cleft lip and palate. J Med Genet. 2002;39:368–369. [PMC free article] [PubMed]
  • Rampersaud E, Morris RW, Weinberg CR, Speer MC, Martin ER. Power calculations for likelihood ratio tests for offspring genotype risks, maternal effects, and parent-of-origin (POO) effects in the presence of missing parental genotypes when unaffected siblings are available. Genet Epidemiol. 2007;31:18–30. [PMC free article] [PubMed]
  • Relton CL, Wilding CS, Jonas PA, Lynch SA, Tawn EJ, Burn J. Genetic susceptibility to neural tube defect pregnancy varies with offspring phenotype. Clin Genet. 2003;64:424–428. [PubMed]
  • Rubini M, Brusati R, Garattini G, Magnani C, Liviero F, Bianchi F, Tarantino E, Massei A, Pollastri S, Carturan S, Amadori A, Bertagnin E, Cavallaro A, Fabiano A, Franchella A, Calzolari E. Cystathionine beta-synthase c.844ins68 gene variant and non-syndromic cleft lip and palate. Am J Med Genet Part A. 2005;136A:368–372.
  • Shaw GM, Lammer EJ, Wasserman CR, O’Malley CD, Tolarova MM. Risks of orofacial clefts in children born to women using multivitamins containing folic acid periconceptionally. Lancet. 1995;346:393–396. [PubMed]
  • Shaw GM, Rozen R, Finnell RH, Todoroff K, Lammer EJ. Infant C677T mutation in MTHFR, maternal periconceptional vitamin use, and cleft lip. Am J Med Genet. 1998;80:196–198. [PubMed]
  • Shaw GM, Zhu H, Lammer EJ, Yang W, Finnell RH. Genetic variation of infant reduced folate carrier (A80G) and risk of orofacial and conotruncal heart defects. Am J Epidemiol. 2003;158:747–752. [PubMed]
  • Shaw GM, Carmichael SL, Laurent C, Rasmussen SA. Maternal nutrient intakes and risk of orofacial clefts. Epidemiology. 2006;17:285–291. [PubMed]
  • Shi M, Caprau D, Dagle J, Christiansen L, Christensen K, Murray JC. Application of kinetic polymerase chain reaction and molecular beacon assays to pooled analyses and high-throughput genotyping for candidate genes. Birth Defects Res A Clin Mol Teratol. 2004;70:65–74. [PubMed]
  • Shotelersuk V, Ittiwut C, Siriwan P, Angspatt A. Maternal 677CT/1298AC genotype of the MTHFR gene as a risk factor for cleft lip. J Med Genet. 2003;40:e64. [PMC free article] [PubMed]
  • Slattery ML, Potter JD, Samowitz W, Schaffer D, Leppert M. Methylenetetrahydrofolate reductase, diet, and risk of colon cancer. Cancer Epidemiol Biomarkers Prev. 1999;8:513–518. [PubMed]
  • Tang LS, Finnell RH. Neural and orofacial defects in Folp1 knockout mice [corrected] Birth Defects Res A Clin Mol Teratol. 2003;67:209–218. [PubMed]
  • Tolarova MM, van Rooij IALM, Pastor M, van der Put NMJ, Goldberg AC, Hol F, Capozzi A, Thomas CMG, Paster L, Mosby T, Ferrari C, Eskes TKAB, Steegers-Theunissen RPM. A common mutation in the MTHFR gene is a risk factor for nonsyndromic cleft lip and palate anomalies [Abstract] Am J Hum Genet. 1998;63:A27.
  • Ulvik A, Vollset SE, Hansen S, Gislefoss R, Jellum E, Ueland PM. Colorectal cancer and the methylenetetrahydrofolate reductase 677C -> T and methionine synthase 2756A -> G polymorphisms: A study of 2,168 case-control pairs from the JANUS cohort. Cancer Epidemiol Biomarkers Prev. 2004;13:2175–2180. [PubMed]
  • van der Linden IJ, Afman LA, Heil SG, Blom HJ. Genetic variation in genes of folate metabolism and neural-tube defect risk. Proc Nutr Soc. 2006;65:204–215. [PubMed]
  • van der Put NM, Steegers-Theunissen RP, Frosst P, Trijbels FJ, Eskes TK, van den Heuvel LP, Mariman EC, den Heyer M, Rozen R, Blom HJ. Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. Lancet. 1995;346:1070–1071. [PubMed]
  • van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocke MC, Zielhuis GA, Goorhuis-Brouwer SM, van der Biezen JJ, Kuijpers-Jagtman AM, Steegers-Theunissen RP. Does the interaction between maternal folate intake and the methylenetetrahydrofolate reductase polymorphisms affect the risk of cleft lip with or without cleft palate? Am J Epidemiol. 2003;157:583–591. [PubMed]
  • van Rooij IA, Ocke MC, Straatman H, Zielhuis GA, Merkus HM, Steegers-Theunissen RP. Periconceptional folate intake by supplement and food reduces the risk of non-syndromic cleft lip with or without cleft palate. Prev Med. 2004;39:689–694. [PubMed]
  • Verkleij-Hagoort A, Bliek J, Sayed-Tabatabaei F, Ursem N, Steegers E, Steegers-Theunissen R. Hyperhomocysteinemia and MTHFR polymorphisms in association with orofacial clefts and congenital heart defects: A meta-analysis. Am J Med Genet Part A. 2007;143A:952–960.
  • Vieira AR, Murray JC, Trembath D, Orioli IM, Castilla EE, Cooper ME, Marazita ML, Lennon-Graham F, Speer M. Studies of reduced folate carrier 1 (RFC1) A80G and 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms with neural tube and orofacial cleft defects. Am J Med Genet. 2005;135A(Part A):220–223.
  • Weinberg CR, Wilcox AJ, Lie RT. A log-linear approach to case-parent-triad data: Assessing effects of disease genes that act either directly or through maternal effects and that may be subject to parental imprinting. Am J Hum Genet. 1998;62:969–978. [PubMed]
  • Werler MM, Hayes C, Louik C, Shapiro S, Mitchell AA. Multivitamin supplementation and risk of birth defects. Am J Epidemiol. 1999;150:675–682. [PubMed]
  • Wilcox AJ, Lie RT, Solvoll K, Taylor J, McConnaughey DR, Abyholm F, Vindenes H, Vollset SE, Drevon CA. Folic acid supplements and risk of facial clefts: National population based case-control study. BMJ. 2007;334:464–470. [PMC free article] [PubMed]
  • Wong WY, Eskes TK, Kuijpers-Jagtman AM, Spauwen PH, Steegers EA, Thomas CM, Hamel BC, Blom HJ, Steegers-Theunissen RP. Nonsyndromic orofacial clefts: Association with maternal hyperhomocysteinemia. Teratology. 1999;60:253–257. [PubMed]
  • Zhu H, Curry S, Wen S, Wicker NJ, Shaw GM, Lammer EJ, Yang W, Jafarov T, Finnell RH. Are the betaine-homocysteine methyltransferase (BHMT and BHMT2) genes risk factors for spina bifida and orofacial clefts? Am J Med Genet Part A. 2005;135A:274–277.
  • Zhu J, Ren A, Hao L, Pei L, Liu J, Zhu H, Li S, Finnell RH, Li Z. Variable contribution of the MTHFR C677T polymorphism to non-syndromic cleft lip and palate risk in China. Am J Med Genet. 2006;140A(Part A):551–557.