We examined four SNPs from genes related to folate directly or indirectly via vitamin B12. MTHFR
677 C→T and 1298 A→C have been studied extensively, but with inconsistent results. MTHFD1
1958 G→A and TC II
776C→G have only been examined in a few studies (Boyles et al., 2008
), also with conflicting results.
The most interesting results appeared in our investigation of MTHFD1
1958 G→A. As has been reported by us previously (Brody et al., 2002
), the control population was not in HWE. This finding complicates the interpretation of the case-control and mother-control analyses. We found that mothers of isolated (p
= .02) and multiple defect CPO cases were significantly (p
= .007) more likely to be homozygous for the A (Q) variant than controls, although A was not a significant risk factor in the log-linear analysis. In the case-control analysis, adding multiple cases increased the case control OR from 1.31 (p
= .13) to 1.50 and produced a statistically significant result (p
= .02). As in the mothers, the log-linear analysis was not significant. In the CLP group, both cases and mothers showed a significant association with the variant allele (both p
= .03). The log-linear analysis showed at most a borderline association (p
= .09) in the mothers and no significance in the cases. In contrast to our findings, the other studies that looked at cases (Boyles et al., 2008
; Palmieri et al., 2008
) found no effect, as did the two studies that looked for an effect in mothers (Boyles et al., 2008
; Mostowska et al., 2006
has been shown by our group to be a maternal, but not an embryonic, risk factor for NTDs in two studies of Irish subjects (Brody et al., 2002
; Parle-McDermott et al., 2006
is a logical candidate gene to explore for possible associations with birth defects. It catalyzes the conversion of tetrahydrofolate to the corresponding 10 formyl, 5,10 methenyl, and 5,10 methylene derivatives. 10-Formyltetrahydrofolate and 5,10 methenyltetrahydrofolate act as carbon donors for the de novo synthesis of purines and pyrimidines and hence are required for DNA synthesis. Insufficient DNA synthesis by the mother could damage the developing embryo.
In contrast to our smaller, earlier study, we found that the MTHFR 677C→T variant was not a risk factor in CPO or CLP cases in any of our analyses. The same was true in mothers of CLP cases. In mothers of isolated CPO cases, however, the MTHFR 677 C→T variant was significantly more common in the mother-control analysis and a borderline risk factor (p = .07) in the log-linear analysis. Thus, the MTFHR 677 C→T variant in mothers may play a role in CPO in the Irish population, although these findings could be due to chance.
Results of previous investigations have been mixed. Some studies of MTHFR
677 C→T have shown no association with CLP in either cases or mothers while others have reported associations in mothers, or in mothers who did not use folic acid periconceptionally (Jugessur et al., 2003
; Gaspar et al., 1999
; Blanton et al., 2002
; Martinelli et al., 2001
; Prescott et al., 2002
; Shotelersuk et al., 2003
; Shaw et al., 1998
; van Rooij et al., 2003
; Mills et al., 1999
). One recent study (Chevrier et al., 2007
) found that the TT genotype was significantly protective in their case control analysis, although the authors noted that the sample size was small and that the TT rate in controls was higher than expected. CPO has not been studied as extensively. Shaw et al. (1999)
found no association between MTHFR
677 C→T and CPO. Jugessur et al. (2003)
found no association in mothers but an increased risk when the mother was heterozygous CT.
1298 A→C variant was not found to be a risk factor for CPO or CLP in any of our analyses. Neither mothers nor cases showed any associations in the case-control, mother-control, log-linear, or TDT analyses. These results are consistent with the findings of a recent meta-analysis (Verkleij-Hagoort et al., 2007
) that showed ORs close to 1 for CLP in both mothers and cases. CPO was not included in the meta-analysis. The only previous study (Jugessur et al., 2003
) of 1298 A→C in CPO found, as we did, no association in either cases or mothers.
Our investigation of the role of the B12 transporter gene variant 776 C→G showed no association with either CPO or CLP. Only two other studies have examined this variant. Boyles et al. (2008)
found no association except in the parent of origin test, and this was based on only 10 cases in which the allele was inherited from the father and six in which it was inherited from the mother. Martinelli et al. (2006)
found a positive association with CLP. There are several possible reasons for these discordant results. Their subjects came from genetically different populations. Martinelli et al. (2006)
had a higher proportion of familial cases (87/218) and chance may have played a role; the authors noted that their findings required confirmation. Thus, our data and data from the other large study, Boyles et al. (2008)
, do not suggest that TC II
776C→G is an important risk factor in CLP, but additional investigation would help to clarify the situation.
Our study has numerous strengths. It included a large number of triads and controls. The Irish population is genetically homogeneous, which reduces concerns that stratification could have occurred and increases our power. We were able to document other potential effect modifiers and confounders including smoking, alcohol, and folic acid use. The study has some limitations as well. Folate levels during the pregnancy of interest could not be measured. Our study included many tests of genotype-phenotype association so that our statistically significant findings could have occurred by chance. Syndromic cases were identified by the attending surgeons and excluded, but clinical geneticists did not examine the cases. We did, however, record the other defects seen in cases with multiple defects and there were no patterns of defects suggestive of syndromes present.
In summary, ours is the first study to find that the MTHFD1 1958G→A variant is a maternal risk factor for CLP. MTHFR 677 C→T was not a risk factor in cases; however, there was modest evidence that it was a maternal risk factor in CPO by both mother-control and log-linear analyses. In light of the fact that multiple comparisons were made, the positive findings require additional investigation. Nonetheless, our findings suggest that the MTHFD1 1958 G→A variant may be important in the etiology of CPO and CLP and this variant merits additional attention.