Methylenetetrahydrofolate Reductase C677T and A1298C Polymorphisms and Nonsyndromic Orofacial Clefts Susceptibility in a Southern Chinese Population

Introduction

O rofacial clefts are one of the most common congenital anomalies in humans. Nonsyndromic orofacial clefts (NSOC) are those occurring without any recognizable anomalies and almost 70% of orofacial clefts cases are this kind (Cobourne, 2004). Based on the surveillance data from the largest birth defects registry in China (CBDMN, Chinese Birth Defects Monitoring Network), Chinese newborns suffer these defects with a prevalence of 1.42/1000 (Dai et al., 2010).

NSOC are commonly categorized into cleft lip only (CLO), cleft palate only (CPO), and cleft lip with palate (CLP). Traditionally, CLO and CLP are collapsed to a single form, namely, cleft lip with or without cleft palate (CL/P); however, recent studies indicated that these might have distinct genetic origins and should be analyzed separately (Rahimov et al., 2008). In addition, CL/P and CPO are usually separated in studies due to differences in embryologic origin and recurrence risks.

Folic acid plays an important role in embryogenesis. Previous studies had strongly confirmed its preventive effect on both recurrence and occurrence of neural tube defects, which are related to orofacial clefts. Further, its role in orofacial clefts had been evaluated by many observational and nonrandomized interventional studies (Wehby and Murray, 2010), the majority of which suggested that maternal folic acid supplementation during early pregnancy might have a protective effect against orofacial clefts; however, the process underlying this was still unknown. One of the possible mechanisms by which low folate levels predispose some individuals to orofacial clefts could be the presence of polymorphisms in the genes encoding enzymes of the folate pathway, such as methylenetetrahydrofolate reductase (MTHFR). It catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and the carbon donor for the remethylation of homocysteine into methionine (Shotelersuk et al., 2003). MTHFR is mapped to 1p36.3 and two of its functional polymorphisms, C677T (rs1801133) and A1298C (rs1801131), have been extensively investigated. The C677T polymorphism, leading to the replacement of alanine by valine at codon 222 (A222V), is responsible for enzyme activity, thermolability, and mild to moderate hyperhomocysteinemia. Similarly, the A1298C variant, resulting in the substitution of glutamate for alanine at codon 429 (G429A), is also associated with lower enzymatic activity but does not seem to influence homocysteine plasma levels (Tong et al., 2010).

In light of the role these two polymorphisms seem to play in the pathogenesis of orofacial clefts, accumulated studies have explored their associations with risk of NSOC but provided conflicting results, which therefore impeled us to study further. In the present study, we carried out a case–control study consisting of 200 NSOC cases and 213 healthy controls from southern China to analyze whether the two polymorphisms might contribute to the risk of these anomalies.

Materials and Methods

Results

Discussion

MTHFR is an important enzyme involved in folate metabolism, making it particularly attractive as a candidate gene for NSOC. Since two of its functional polymorphisms, C677T and A1298C, had been identified, accumulated studies from different ethnic groups were subsequently employed to investigate their associations with susceptibility of NSOC and its subgroups.

The first study regarding MTHFR C677T and risk of NSOC was performed by Shaw et al. in 1998. With data from the California Birth Defects Registry, however, they failed to find any evidence that C677T contributed to elevated risk of NSOC in Caucasian, Hispanic, or black children (Shaw et al., 1998). Afterward, replication studies from different populations emerged, providing inconsistent results. Specifically, consistent with the results of Shaw et al., studies from Brazil (Gaspar et al., 1999; Brandalize et al., 2007), Italy (Martinelli et al., 2001), Netherlands (van Rooij et al., 2003), Central Europe (Reutter et al., 2008), United Kingdom (Little et al., 2008), Poland (Mostowska et al., 2010), and Venezuela (Sözen et al., 2009) did not support a role of the child's MTHFR C677T polymorphism with risk of NSOC. Nevertheless, some investigations reported a potential correlation between the proband's MTHFR C677T polymorphism and elevated risk of NSOC (Mills et al., 1999; Jugessur et al., 2003; Zhu et al., 2006, 2010; Ali et al., 2009). The inverse effect had also been identified by previous research (Chevrier et al., 2007; Boyles et al., 2008). Similarly, studies of associations between NSOC and MTHFR A1298C polymorphism also yielded inconsistent results. Shotelersuk et al. first conducted a case–control study but found no association between the patients' MTHFR A1298C genotypes and NSOC risk (Shotelersuk et al., 2003). Their observation was confirmed by other studies (Mills et al., 1999; van Rooij et al., 2003; Ali et al., 2009; Sözen et al., 2009). Interestingly, Pezzetti et al. (2004) and Jagomagi et al. (2010) found that the presence of MTHFR A1286C (known as A1298C) polymorphism seemed to provide some protection against NSOC.

In sum, whether these two polymorphisms play important roles in NSOC remained controversial. In the present study, we found that MTHFR C677T might be a risk factor of NOSC, whereas MTHFR A1298C might be protective. Their distinctive roles in cleft pathogenesis may be due to the following: MTHFR C677T mutants were associated with reduced MTHFR activity, elevated plasma homocysteine levels, and lowered plasma folate, which consequently contributed to orofacial clefts (Frosst et al., 1995). Nevertheless, the MTHFR A1298C polymorphism could alter one-carbon metabolism within cells to prevent aberrant methylation patterns that would lead to chromosome instability, thus protecting against NSOC (Wiemels et al., 2001).We subsequently catagorized all cleft cases into four subgroups (CL/P, CLO, CLP, and CPO) and found that MTHFR C677T was associated with increased risk of CL/P and CLO, whereas MTHFR A1298C was associated with decreased risk of CL/P and CLP.

For MTHFR C677T, it is worth noting that Zhu et al. found that this polymorphism was associated with increased NSOC susceptibility in northern Chinese rather than southern Chinese (Zhu et al., 2006, 2010), which seemed to be contrary with our observation. However, given the complicated, heterogeneous nature of NSOC and the number of confounding factors, this was not unexpected. The MTHFR C677T genotype and allele distributions in the present study were similar to those in southern Chinese from other studies. The frequencies of CC, CT, and TT were 34.7%, 51.7%, and 13.6%, respectively, in our controls, which were comparable to those in 508 southern Chinese (34.1%, 50.6%, and 15.3%, respectively) by Tong et al. (2010) and 430 southern Chinese (39.0%, 53.0%, and 8.1%, respectively) by Wilcken et al. (2003). The frequency of the 677 T allele was also highly similar among them (0.39, 0.406, and 0.347, respectively).

To our knowledge, no confirmative studies had been conducted to explore the association between MTHFR A1298C and NSOC in the Chinese population, and we found that MTHFR A1298C was protective against NOSC, consistent with two previous studies (Pezzetti et al., 2004; Jagomägi et al., 2010). Further, its protection was also apparent on another type of congenital disease, congenital heart diseases, which share similar underlying pathogenesis with NSOC (Verkleij-Hagoort et al., 2007). There are at least four possible interpretations of our findings. First of all, it is likely that this polymorphism, by itself, was not associated with NSOC, but rather in LD with other causative variants responsible for these defects. Second, genetic selection might lead to a higher frequency of mutated individuals in the population. Particularly of interest was a study performed by Reyes-Engel et al. (2002). They found that increased frequency of the 1298 C allele in children coincided with a generally increased folate intake by pregnant women, which has also been occurring among pregnant Chinese women in recent years (Zhu and Ling, 2008). A third possible explanation was the selective survival of those with the 1298 A allele. Decreased viability of fetuses had been found in association with 1298 C allele (Zetterberg et al., 2002). Thus, selective survival could have influenced our findings. Finally, as is the case for some cancers, the 1298 C allele could be protective (Lightfoot et al., 2008). It was proposed that the slightly lower activity associated with the C allele provided a better balance between methylation and DNA synthesis, which was important during embryo development.

Taken together, our results provide further evidence that these two polymorphisms are involved in the development of NSOC in a southern Han Chinese population; however, two major limitations should be addressed. First of all, borderline significance of the p-values for association tests was obtained, which indicated that these two MTHFR polymorphisms could modify an individual's risk of NSOC. However, given our limited sample size, our findings should be carefully interpreted. With the current sample size in our study, we only had 70% power at 0.05 or lower to detect an OR of 1.70 or greater and 0.55 or lower with an exposure frequency of 30%. Second, some important information, such as in utero environmental factors of the developing fetus, maternal periconceptional use of folic acid as well as information on the genotypes of their other folic acid metabolism genes was not available in our study. Therefore, other well-designed studies with different ethnic populations are warranted to verify our findings.