American Journal of Epidemiology Copyright 2006 by the Johns Hopkins Bloomberg School of Public Health All rights reserved;

; printed in U.S.A.

Vol. 164, No. 9 DOI: 10.1093/aje/kwj285 Advance Access publication August 25, 2006

Original Contribution The C677T Polymorphism in the Methylenetetrahydrofolate Reductase Gene (MTHFR), Maternal Use of Folic Acid Supplements, and Risk of Isolated Clubfoot: A Case-Parent-Triad Analysis

Linda Sharp1, Zosia Miedzybrodzka2, Amanda H. Cardy3, Julie Inglis2, Londale Madrigal2, Simon Barker4, David Chesney5, Caroline Clark2, and Nicola Maffulli6
1 2

National Cancer Registry Ireland, Cork, Ireland. Medical Genetics Group, Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, Scotland. 3 Department of Public Health, University of Aberdeen, Aberdeen, Scotland. 4 Department of Orthopaedic Surgery, Aberdeen University School of Medicine, Aberdeen, Scotland. 5 Department of Orthopaedic Surgery, Freeman Hospital, Newcastle upon Tyne, England. 6 Department of Trauma and Orthopaedic Surgery, Keele University School of Medicine, Stoke on Trent, England. Received for publication July 15, 2005; accepted for publication April 7, 2006.

Worldwide, 14 per 1,000 births are affected by clubfoot. Clubfoot etiology is unclear, but both genetic and environmental factors are thought to be involved. Low folate status in pregnant women has been implicated in several congenital malformations, and folate metabolism may be affected by polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR). Using a case-parent-triad design, the authors investigated whether the MTHFR C677T polymorphism, and maternal periconceptional folic acid supplement use, inuenced risk of isolated clubfoot. Three hundred seventy-ve United Kingdom case-parent triads were recruited in 19981999. Among the children, there was a signicant trend of decreasing clubfoot risk with increasing number of T alleles: relative risk for CT vs. CC 0.75, 95% condence interval: 0.57, 0.97; relative risk for TT vs. CC 0.57, 95% condence interval: 0.35, 0.91; p trend 0.006. This association was not modied by maternal folic acid use. Maternal MTHFR genotype did not inuence clubfoot risk for the offspring overall, although a possible interaction with folic acid use was found. This is the rst known report of a specic genetic polymorphism associated with clubfoot. The direction of the association is intriguing and suggests that DNA synthesis may be relevant in clubfoot development. However, clubfoot mechanisms are poorly understood, and the folate metabolism pathway is complex. Further research is needed to elucidate these relations. clubfoot; dietary supplements; folic acid; genes; 5,10-methylenetetrahydrofolate reductase (FADH2); polymorphism, genetic; pregnancy

Abbreviations: CI, condence interval; MTHFR, 5,10-methylenetetrahydrofolate reductase; RR, relative risk.

Congenital talipes equinovarus (clubfoot) is a common developmental disorder of the lower limb, with a prevalence of 14 per 1,000 births worldwide (1). It is a three-dimensional deformity immediately recognizable at birth; the ankle is in the plantar exed (equinus) position, the heel is inverted (varus), and the midfoot and forefoot are inverted and ad-

ducted (varus) (2). Treatment can be protracted through childhood, and disability may persist into later life. Although some cases of clubfoot occur in association with other features or congenital anomalies as part of a genetic syndrome, the majority arise in isolation (3, 4). The etiology of isolated clubfoot has been relatively little studied and

The rst two authors contributed equally to this work, which was performed at the University of Aberdeen. Correspondence to Linda Sharp, National Cancer Registry Ireland, Elm Court, Boreenmanna Road, Cork, Ireland (e-mail: linda.sharp@ncri.ie).

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remains unclear. Several strands of evidence suggest environmental triggers acting in a genetically predisposed individual (47). Neither the environmental nor the genetic factors have been elucidated. The B vitamin folate is crucially involved in several important metabolic processes, including DNA synthesis and repair and DNA methylation (8). Low folate status in pregnant women has been observed to affect risk of several congenital malformations (9, 10). Regarding clubfoot, a small reduction in the birth prevalence of isolated clubfoot has been observed in the United States after fortication of grains with folic acid was introduced (11). In addition, in an intervention study in Denmark, the rate of clubfoot-affected births was lower among women taking folic acid supplements than those not, although the number of clubfoot births was very small (12). The mechanisms by which maternal folate status affects pregnancy outcome are not established. Supplementation with folic acid might overcome a genetically inherited block in folate metabolism (13). The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5-10 methylenetetrahydrofolate into 5-methyltetrahydrofolate, thereby directing the folate pool toward either DNA synthesis and repair or DNA methylation. The C677T polymorphism in the MTHFR gene causes an alanine-to-valine change in the protein (14). Enzyme activity is reduced in a dose-response fashion in heterozygotes and homozygotes for the variant T allele (15). MTHFR has been implicated in the etiology of several congenital malformationsincluding neural tube defects (10) and orofacial clefts (16)but, to our knowledge, has not previously been investigated in clubfoot. We used a case-parent-triad design to investigate, rstly, whether the MTHFR C677T polymorphism affects risk of isolated clubfoot and, secondly, whether associations between genotype and clubfoot risk are modied by maternal periconceptional use of folic acid supplements.
MATERIALS AND METHODS Recruitment of case-parent triads

consumption of alcohol during the index pregnancy. Fiftytwo percent of eligible families took part. The other series was identied via registers maintained by orthopedic surgeons treating clubfoot in Scotland and northeast England. Families of all children recorded on the register were approached and were invited to take part in the study. Recruitment took place during 19981999 (7, 17). Families were asked to attend a hospital clinic appointment; if they agreed, mouthwash samples were collected from the child and parents, and a questionnaire similar to the one described above was completed during the visit. If the family declined to attend the clinic, they were sent the mouthwash kits and questionnaire by mail and were asked to complete and return them. Forty-two percent of eligible families took part. Only biologically related triads were included. Checks were made to ensure that no family was included in both series. Recruited were 289 triads from the rst series and 98 from the second. None of the rst series were excluded on the basis of having foot deformity that was not talipes equinovarus. Of the 387, 12 cases were assessed by a clinical geneticist (Z. M.) to be syndromic and were excluded, leaving 375 triads in the analysis. The study was approved by the Multi-Centre Research Ethics Committee for Scotland and the Grampian Research Ethics Committee.
Genotyping methods

We recruited two series of children affected by clubfoot and their parents. The source of one series was the United Kingdom support group for children with lower limb deformities, STEPS. All families registered with STEPS as having a child affected by clubfoot were invited to take part in the study. Parents were approached by mail and were asked whether they, and their affected child, would participate. Recruitment took place during 20012002. Participants provided a buccal DNA sample, collected by mouthwash or a cytocell cheek smear. Parents, and children who were old enough, provided their own sample. Samples from young children were collected by their parents by using a cytocell brush. Mothers completed a questionnaire on socioeconomic factors, ethnicity, the pregnancy and birth of the index child, the nature of the childs clubfoot (laterality, etc.), the childs other medical conditions (to enable assessment of syndromic status and exclusion of children with other foot deformities), family history of clubfoot, maternal reproductive history, and maternal use of supplements and
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DNA was extracted from the cheek smears and mouthwashes by using Instagene matrix (Bio-Rad, Hercules, California) and sodium hydroxide, respectively. The C677T polymorphism was detected by using restriction fragment length polymorphism methods devised by Frosst et al. (18) for mothers, fathers, and children from all 375 triads (i.e., 1,125 genotype determinations were undertaken). DNA was amplied by polymerase chain reaction using anking primers. Polymerase chain reaction products were digested with HinfI, separated by 3 percent agarose gel electrophoresis, and visualized by ethidium bromide staining and ultraviolet transillumination. Genotyping was undertaken without knowledge of exposure status (e.g., maternal folic acid use) of the triad. Gels were double-read (i.e., two people read each gel and assigned a genotype; discrepancies were discussed and resolved). Genotyping was repeated in a random sample of 130 samples (11.6 percent of 1,125), blind to the original genotyping result. No differences were found between the original and repeated genotypes.
Classication of folic acid, alcohol consumption, and family history

The questionnaires sought information on whether the mother had taken supplements containing folic acid in the 3 months prior to and/or in the rst trimester of the index pregnancy. Folic acid containing supplements were dened as specic folic acid preparations intended for the periconceptional period, vitamin B-complex, or multivitamins. Period of use was classied as use during the 1) 3 months

854 Sharp et al.

preconception and/or the rst trimester, 2) 3 months preconception, or 3) rst trimester. Data were missing on preconception use for seven triads (1.9 percent) and on rst-trimester use for six (1.6 percent). Because alcohol has an adverse effect on folate metabolism (19), mothers were classied dichotomously according to whether they had consumed alcohol at any time during the index pregnancy. This information was missing for two triads (0.5 percent). A positive family history of clubfoot was dened as a report of clubfoot in any rst-, second-, or third-degree relative of the index child. These data were missing for two triads (0.5 percent).
Statistical analysis

TABLE 1. Characteristics of 375 case-parent triads including a child with isolated clubfoot, United Kingdom
No. %*,y

Sex of the index child Male Female Affected foot Left only Right only Both Data missing Period of birth of the index child 19631979 19801989 19901994 19951999 20002002 Data missing Age of the mother (years) at birth of the index child 19 2024 2529 3034 3539 40 Data missing Age of the father (years) at birth of the index child 19 2024 2529 3034 3539 40 Data missing Family history of clubfootz Yes No Data missing Maternal alcohol consumption during the index pregnancy Yes No Data missing Maternal folic acid use in the 3 months prior to, or during the rst trimester of, the index pregnancy Yes No Data missing 188 182 5 51 49 174 199 2 46 54 64 309 2 17 83 3 16 81 138 75 58 4 1 4 22 37 20 16 3 36 124 140 55 14 3 1 10 34 37 15 4 15 52 131 152 24 1 4 14 35 40 7 62 80 211 22 18 23 60 261 114 70 30

The analysis was conducted by using Stata 8 software (20). The Pearson v2 test was used to determine whether genotype frequencies in mothers, fathers, and children were in Hardy-Weinberg equilibrium. Log-linear methods were used to calculate relative risks associated with maternal and child alleles (21, 22). These methods test for asymmetric distribution of the variant allele among affected offspring and their parents. The genotypes of cases and parents were stratied into 15 possible mating types, from which the relative risks for the childs and the mothers genotypes were computed by tting a Poisson model. The initial analysis estimated separate relative risks for heterozygous (CT) and homozygous (TT) variant genotypes in both children and mothers, using homozygous wild-type (CC) as the reference category. Trend tests were applied to assess whether there was a dose-response pattern in risk with increasing number of variant alleles (23). Because it is not clear what model of inheritance might be postulated a priori, this analysis was repeated by assuming both dominant (i.e., CT/TT vs. CC) and recessive (i.e., TT vs. CC/CT) transmission. Interactions between genotype and folic acid use were determined by stratifying on whether the mother used supplements during the index pregnancy and comparing the strength of the associations with genotype between the strata by using a v2 -based test of heterogeneity (24). This assessment was done for the three categorizations of folic acid use. To increase statistical power in the interaction analysis, a dominant effect of the variant allele was assumed. Interactions between genotype and maternal alcohol consumption during the index pregnancy were investigated in the same way. To consider the possibility of etiologically distinct subgroups of clubfoot, the effects of maternal and child genotypes were compared in families with and without a family history of clubfoot. The statistical models can be tted with or without an assumption of Hardy-Weinberg equilibrium. The goodness of t of both sets of models was compared; those without the assumption of Hardy-Weinberg equilibrium provided the better t and were used in all analyses.

RESULTS

The characteristics of the 375 case-parent triads are summarized in table 1. The series included more male than

* Excluding triads for which the relevant data were missing. y Percentages may not total 100 because of rounding. z Includes up to third-degree relatives of the index child.

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TABLE 2. Results of log-linear analysis of maternal and child alleles and risk of clubfoot under different models of inheritance, United Kingdom, children born in 19632002
Model and genotype No. % RR* 95% CI* LRT*,y

Primary analysis Mother CC CT TT Child CC CT TT Assumed recessive model Mother CC/CT TT Child CC/CT TT Assumed dominant model Mother CC CT/TT Child CC CT/TT 182 193 48.5 51.5 1.0 0.72 0.56, 0.94 v2 5.82, p 0.016 1 160 215 42.7 57.3 1.0 0.89 0.67, 1.20 v2 0.56, p 0.453 1 336 39 89.6 10.4 1.0 0.70 0.46, 1.08 v2 2.75, p 0.097 1 337 38 89.9 10.1 1.0 1.0 0.59, 1.69 v2 0.00, p 1.00 1 182 154 39 48.5 41.1 10.4 1.0 0.75 0.57z 0.57, 0.97 0.35, 0.91 v2 7.56, p 0.024 2 160 177 38 42.7 47.2 10.1 1.0 0.88 0.94 0.65, 1.20 0.55, 1.62 v2 0.61, p 0.736 2

* RR, relative risk; CI, condence interval; LRT, likelihood ratio test. y Chi-squared statistic and p value from likelihood ratio test for the overall contribution of the mother or child to the statistical model. z Likelihood ratio test for linear trend in risk with number of variant alleles, v2 7.45, 1 p 0.006.

female cases (male:female ratio 2.3:1). Sixty percent of the children had bilateral clubfoot, and, of those affected unilaterally, the right foot was involved slightly more often than the left. The median maternal and paternal ages at the birth of the affected child were 30 (range, 1845) and 32 (range, 1758) years, respectively. Seventeen percent of children had a positive family history of clubfoot. The homozygous variant genotype (TT) was present in 10 percent of children and in the same proportions of mothers and of fathers. Forty-one percent of children were heterozygous compared with 47 percent of mothers and 50 percent of fathers. Genotype frequencies in fathers, mothers, and children did not depart from Hardy-Weinberg equilibrium (p > 0.05). Table 2 shows the results of the log-linear analysis of maternal and child genotypes. For children, compared with CC individuals, heterozygotes (CT) had a modest, statistically signicant, reduced risk of clubfoot (relative risk (RR) for CT vs. CC 0.75, 95 percent condence interval (CI): 0.57, 0.97). Risk was substantially, and signicantly, decreased in homozygous variant children (RR for TT vs. CC 0.57, 95 percent CI: 0.35,0.91), and there was a strong
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linear trend in risk with number of variant T alleles (p 0.006). When the analysis was repeated by assuming either dominant or recessive models, the association was still evident, but the relative risks were somewhat ameliorated (as would be expected based on the observed trend with number of variant alleles). There was no strong association between maternal genotype and risk of clubfoot in the offspring. Compared with offspring of CC mothers, offspring of CT or TT mothers had a slight, nonsignicant, decreased risk of clubfoot. Folic acidcontaining supplements were taken by 24 percent of mothers in the 3 months prior to the index pregnancy, by 49 percent in the rst trimester, and by 51 percent in either time period. The effects of genotype stratied by maternal folic acid use in either time period are shown in gure 1. For mothers, carrying the T allele was associated with a modest reduced risk of clubfoot in the offspring among those who had used folic acid (RR for CT/TT vs. CC 0.72, 95 percent CI: 0.47, 1.09) but not among those who had not used folic acid (RR 1.01, 95 percent CI: 0.68, 1.51). However, the test for interaction

856 Sharp et al.

10

A
RR of clubfoot with one or two copies of T allele

1.01 0.72 0.69 0.74

0.1

No

Yes

No

Yes

Folic acid use 3 months prior to pregnancy or during first trimester

FIGURE 1. Relative risk (RR) of clubfoot associated with having one or two copies versus no copies of the methylenetetrahydrofolate reductase gene (MTHFR) variant T allele, within strata of maternal folic acid use during the periconceptional period of the index pregnancy, United Kingdom, children born in 19632002. A, mothers alleles; B, childs alleles; vertical lines, 95% condence intervals for the risk estimates.

was not statistically signicant (p interaction 0.218). Among children, the relative risk of clubfoot for CT/TT versus CC genotypes was reduced irrespective of maternal folic acid use (folic acid not used: RR 0.69, 95 percent CI: 0.48, 0.99; folic acid used: RR 0.74, 95 percent CI: 0.52, 1.05; p interaction 0.778). When the analysis was repeated for preconception and rst-trimester use of folic acid separately, the results were very similar (data not shown). Forty-six percent of mothers reported consuming alcoholic drinks at some point during the index pregnancy. We found no interaction between maternal alcohol consumption and childs genotype (p 0.883; gure 2). Regarding maternal genotype, the risk of clubfoot in the offspring was reduced for only those women with the T allele who consumed alcohol (RR for CT/TT vs. CC 0.65, 95 percent CI: 0.42, 1.00); the test for interaction was borderline signicant (p 0.060). There was no evidence of an interaction between family history of clubfoot and either maternal (p interaction 0.522) or child (p 0.479) genotype (gure 3).

associated with a signicantly reduced risk of isolated clubfoot, and risk decreased with increasing number of variant alleles. This association was not affected by whether the mother took supplemental folic acid in the periconceptional period. There was a suggestion of an interaction between maternal genotype and folic acid use and risk of clubfoot in the offspring, but the tests of interaction were not statistically signicant.
Strengths and limitations of the study

DISCUSSION

Although MTHFR has been investigated in several congenital malformations (10, 16, 25), to our knowledge this is the rst study of genetic variation in folate metabolism in clubfoot. For children, carrying the variant 677T allele was

The mains strengths of our study are its size and ability to investigate combinations of maternal and child genotype and maternal folic acid use. We included 375 complete triads, which provided 80 percent power to detect a relative risk of 0.67 associated with a genotype occurring in 10 percent, assuming dominant transmission and a type I error of 0.05. A further strength was the case-parental-control design, which is efcient and has greater power than the caseunrelated-control approach for investigating associations between polymorphic genes and disease (26, 27). Moreover, it avoids the possibility of population stratication (i.e., that case-control differences are due to selection of controls whose genetic background differs systematically from that of cases (28)). A priori, it was not clear whether the childs or the mothers genotype, or both, were etiologically relevant. Confounding occurs if the childs genotype is measured when the mothers genotype was actually the relevant one (29). To
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857

A
RR of clubfoot with one or two copies of T allele

1.10 0.65 0.71 0.74

0.1

No

Yes

No

Yes

Maternal alcohol consumption during pregnancy

FIGURE 2. Relative risk (RR) of clubfoot associated with having one or two copies versus no copies of the methylenetetrahydrofolate reductase gene (MTHFR) variant T allele, within strata of maternal alcohol consumption during the index pregnancy, United Kingdom, children born in 1963 2002. A, mothers alleles; B, childs alleles; vertical lines, 95% condence intervals for the risk estimates.

10

A
RR of clubfoot with one or two copies of T allele

1.08 0.85 0.75 0.60

0.1

No

Yes

No

Yes

Family history of clubfoot

FIGURE 3. Relative risk (RR) of clubfoot associated with having one or two copies versus no copies of the methylenetetrahydrofolate reductase gene (MTHFR) variant T allele, within strata of family history of clubfoot, United Kingdom, children born in 19632002. A, mothers alleles; B, childs alleles; vertical lines, 95% condence intervals for the risk estimates.

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858 Sharp et al.

avoid this problem, we used the log-linear approach developed by Wilcox et al. (21) and Weinberg et al. (22), which permits investigation of the independent effects of both maternal and child genotypes. Several assumptions underpin the case-parent design. Firstly, there must be mendelian transmission of alleles in the population. It seems unlikely that this assumption was violated. Secondly, survival of the index child from fertilization must not be associated with genotype. Studies of MTHFR and fetal loss are inconsistent (3033), and there is no evidence on MTHFR and survival after birth. Finally, in respect to interactions, it is essential that the genetic and environmental factors are independent. There is no evidence that periconceptional folic acid use and MTHFR genotype are related. The main limitation of the study is that the cases were not drawn from a population-based sampling frame. However, for the results to be seriously biased, the probability of participation would need to have been associated with maternal or child genotype. This seems unlikely, particularly because there is no known MTHFR-clubfoot association, and participants were not aware of the study hypotheses when they agreed to take part. Ten percent of mothers and fathers in the study had the TT genotype, which is consistent with other United Kingdom series (10). The male excess and dominance of bilateral disease in our study population mirrors patterns in other European and US studies of children with clubfoot (3441). The frequency of children with a family history of clubfoot (17 percent) falls between estimates from two US investigators (14 percent and 24 percent) (41, 42). Altogether, this nding suggests that our series is unlikely to be seriously biased. A further limitation of the study is that we did not have detailed information on the dose, frequency, and duration of maternal use of either folic acid supplements or alcohol during the index pregnancy. In the United Kingdom, the proportions of pregnant women taking folic acid supplements varies by time period, maternal age, and social class (43), and it is probable that exposure levels among those who used supplements vary considerably. This variation is likely to be reected in our study population, making it possible that our results are biased, most likely toward the null.
Interpretation of interaction analyses

stratum-specic relative risks was slightly more pronounced (nonusers: RR for CT/TT vs. CC 1.06, 95 percent CI: 0.72,1.57; users: RR for CT/TT vs. CC 0.67, 95 percent CI: 0.44, 1.03). Although not statistically signicant, these results suggest an interaction between maternal folic acid supplement use and maternal genotype in relation to clubfoot in the offspring. Caution is needed in interpretation, however, because the case-parent design does not enable comparison of absolute levels of risk, only comparison of relative effects of the genetic variant in each exposure stratum (45). Hence, we cannot discriminate between the situation in which clubfoot risk is reduced by folic acid use among mothers with the T allele and that in which clubfoot risk is increased by folic acid use among mothers without the T allele. It is well established that alcohol interferes with folate absorption and utilization (19, 46), and this knowledge provided the rationale for investigating interactions between maternal alcohol consumption and maternal and child genotype. Although we observed a borderline signicant interaction between maternal alcohol consumption and maternal genotype, interestingly, this interaction was in the same direction as the maternal genotypefolic acid association (i.e., the risk reduction associated with the T allele was observed among only those mothers who reported drinking alcohol). This nding may reect a positive association between alcohol consumption and folic acid use in our study population; 44 percent of mothers who did not have any alcoholic drinks reported taking folic acid supplements compared with 58 percent who did drink alcohol (v2 7.10, p 0.008). 1 Alternatively, the result may reect an interrelation between alcohol, folate, and folate-metabolizing genes. When the maternal genotypefolic acid analysis was repeated by stratifying on maternal alcohol consumption, the genotypefolic acid association was observed for only that group who reported drinking alcohol. Honein et al. (47) reported an interaction between family history and maternal smoking during pregnancy, suggesting that the etiology of clubfoot in cases with and without a family history of the disease might differ. We found no notable difference in the effects of genotype according to whether the child had a positive or negative family history. However, only 64 triads had a positive family history, so the possibility of an MTHFRfamily history interaction cannot be excluded.
Possible mechanisms

When all triads were analyzed together, we found no association between maternal genotype and the risk of having a clubfoot-affected child. However, when mothers were stratied by folic acid use in the periconceptional period, the T allele was associated with reduced risk of clubfoot in the offspring for mothers who had used folic acid (RR 0.72) but not for mothers who had not used folic acid (RR 1.01). In the normal embryo, the foot is formed in the vertical plane, and rotation into the plantar (sole on the ground) position commences between 9 and 13 weeks and continues throughout gestation (44), suggesting that rst-trimester exposuressuch as folic acid supplementationmay inuence the process of foot development. When the maternal genotypefolic acid use analysis was repeated by limiting folic acid use to the rst trimester, the difference in the

Our observation of a reduced risk of clubfoot in children with the variant T allele is intriguing, particularly in view of the fact that the T allele is associated with reduced enzyme activity (15). The product of the MTHFR reaction, 5-methyltetrahydrofolate, is a major methyl donor in the remethylation of homocysteine to methionine. Homocysteine, or its derivatives, in high doses may have toxic effects on developing tissues (48), and two studies have reported an association between increased maternal homocysteine levels and clubfoot (49, 50). In several (although not all) studies (5153), carriers of the T allele have been found to have raised homocysteine levels. At rst glance,
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these observations would appear to be inconsistent with the results of the current study. However, the nature of the homocysteine-MTHFR relation has not yet been fully claried. Several investigators have found that the relation holds only when folate status is low (5459). Others have suggested that the relation might be present in some age groups but not others (60, 61) and/or might vary by gender (57, 59), smoking status (57), weight (62), alcohol intake (63), or riboavin status (64). In addition, the two studies of homocysteine and clubfoot were awed; both included relatively small numbers of clubfoot-affected births (10 in one study and 62 in the other) and measured maternal homocysteine levels sometime after the relevant pregnancies (49, 50). Thus, it is possible that the effects of MTHFR in clubfoot may not operate through the homocysteine pathway (although further evidence is needed before this pathway can be denitely excluded). The substrate for the MTHFR enzyme, 5,10-methylenetetrahydrofolate, is required as a methyl donor for the conversion of deoxyuridine monophosphate to thymidine monophosphate, which is then used for DNA synthesis and repair. When folate is depleted, the conversion is blocked, deoxyuridine monophosphate accumulates, and uracil may be misincorporated into DNA in place of thymine, leading to catastrophic DNA repair, DNA double strand breakage, and chromosome damage (65, 66). Decreased MTHFR activity provides increased methylenetetrahydrofolate for DNA synthesis, reducing uracil misincorporation, DNA instability, and chromosome breaks. The decreased risk of clubfoot with the low-activity T allele suggests that this pathway could be relevant in clubfoot development, such that the presence of the MTHFR T allele in the fetus provides increased levels of circulating methylenetetrahydrofolate, thus maintaining (or raising) DNA repair capacity and protecting against clubfoot. We acknowledge, however, that this potential mechanism is speculative, and further investigation is needed to conrm (or refute) these speculations. Many of the genes involved in repair of strand breaks are essential for normal embryo development, and DNA repair capacity is thought to be involved in the response of the conceptus to genotoxic agents that could induce malformations (67). However, current knowledge regarding DNA repair systems in organogenesis is incomplete (67), and human evidence on the roles of MTHFR and folate in maintaining DNA integrity is limited and inconclusive (6873). Interestingly, our results are broadly compatible with what has been reported for colorectal cancer, with regard to both the main effect of C677T and interactions with folate (reviewed by Sharp and Little (74)). In most of the available studies, colorectal cancer risk is reduced in those with the TT genotype, and the group at lowest disease risk are T allele carriers with high folate levels (refer, for example, to Ma et al. (51), Chen et al. (75), and Le Marchand et al. (76)). These relations were not what might have been expected a priori (on the basis of the reported inverse relation between folate and colorectal cancer) and suggest that the roles of folate and folate metabolism genes in the disease etiology are complex (74). By analogy, this conclusion is also likely to apply to clubfoot.
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Conclusions

We found that children who carry the 677T variant of the MTHFR gene are at decreased risk of clubfoot. Moreover, risk may be affected by an interaction between maternal genotype and supplement use during the index pregnancy. These ndings suggest that folate status could be relevant in clubfoot etiology. The importance of replication of ndings of genetic association studies has been emphasized in recent years (75, 77), and we recognize that our novel results require conrmation. Folate metabolism is complex, and several other nutrients and polymorphic genes are involved (74, 78). In addition, despite numerous hypotheses, the pathogenesis of clubfoot is very poorly understood (4). Further etiologic and mechanistic research is warranted to elucidate the role of the folate pathway in this common developmental disorder.

ACKNOWLEDGMENTS

The research was funded by Sports Action Research for Kids (SPARKs). L. M. was funded by BBSRC. The authors are grateful to STEPS (National Association for Children with Lower Limb Abnormalities) for facilitating the recruitment of families affected by clubfoot, to Martine Barnes and Hazel Hailey for administrative support, and to Ewan Stronach for assistance with DNA extraction. They also thank the orthopedic surgeons who agreed to let the authors approach their patients. Conict of interest: none declared.

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