Meta-analysis of methylenetetrahydrofolate reductase ( MTHFR ) A 1298 C polymorphism and risk of orofacial cleft

Polymorphisms in key genes involving the folate pathway have been reported to be associated with the risk of orofacial cleft (OFC) and several studies were published with conflicting results. A meta-analysis of the previous studies of allelic association between OFC with A1298C polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene was carried out. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the association between MTHFR A1298C polymorphism and OFC risk. A total of 11 studies including 1628 cases and 2676 controls were involved in this meta-analysis. No statistical relationship was found with any genetic model (C vs. A (Additive): OR = 1.14, 95%CI = 0.76-1.65, P = 0.47; CC vs. AA (homozygote): OR = 0.90, 95%CI = 0.72-1.15, P = 0.0.41; AC vs. AA (co-dominant): OR = 0.97, 95%CI = 0.85-1.11, P = 0.0.63; CC+AC vs. AA (Dominant): OR = 0.96, 95%CI = 0.84-1.1 , P = 0.51; CC vs. AC+AA (Recessive): OR = 0.93, 95%CI = 0.74-1.16, P = 0.52). The present meta-analysis supports that the common A1298C polymorphism of MTHFR gene is not risk factor for OFC.


Approximately
90% of craniofacial congenital abnormalities comprised orofacial cleft (OFC) or cleft lip and/or palate (CL/P).According to the world health organization (WHO) data, the frequency of this pathology in the world is 0.6 to 1.6 cases per 1000 newborns (Shaw et al., 2001;Chorna et al., 2011).Prevalence rate varies according to geographical origin, sex, racial background, ethnicity, and socio-economic status (Vanderas et al., 1987;Croen et al., 1998;Clark et al., 2003;Brito et al., 2011;Aslar et al., 2013).Prenatal folic acid supplementation to pregnant women has been shown to reduce the incidence of CL in many (van Rooij et al., 2004;Badovinac et al., 2007;Rouget et al., 2005;Yazdy et al., 2007;Wilcox et al., 2007), but not all (Ray et al., 2003) populations studied (Sozen et al., 2009).Several studies established that polymorphisms in genes implicated in folate metabolism may play a significant role in the OFC etiology.Among several genes that take part in folate metabolism, the methylenetetrahydrofolate reductase gene (MTHFR) has been the most frequently reported to be associated with OFC.MTHFR (EC.1.5.1.20)is a key enzyme in folate and homocysteine metabolism and catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5methyltetrahydrofolate, which provides the methyl group for the remethylation of homocysteine to methionine.Methionine is in turn converted to S-adenosylmethionine (SAM), the common methyl donor for the methylation processes of DNA, proteins, phospholipids and neurotransmitters (Finkelstein, 1990;Bailey and Gregory, 1999;Ozarda et al., 2009).The MTHFR gene is localized on chromosome 1p36.3and two common and clinically important polymorphisms (C677T and A1298C) identified in the MTHFR gene (Frosst et al., 1995;Weisberg et al., 1998) are implicated in the development of OFC.MTHFR C677T polymorphism is very well studied but A1298C polymorphism is less explored.A1298C influences specific activity of the enzyme, homocysteine levels, and plasma folate concentration, but to a lesser extent than the C677T polymorphism does (Blount et al., 1997;Shen et al., 2005).
Substitution at nucleotide 1298 (exon 7) results in an amino acid substitution of glutamate for alanine at codon 429.A1298C (glutamate to alanine) polymorphism, has been associated with decreased enzyme activity (40%), although to a lesser extent than C677T (Weisberg et al., 1998).Those who have the AC or CC genotype present with a decreased ability to produce the methyl form of folate, which together with cobalamin, is essential for the remethylation of homocysteine to methionine.The resulting abnormality in folate metabolism and the resultant increase in homocysteine levels may be a direct cause of the observed teratogenicity, homocysteine itself may be toxic to the embryo or it may be an indicator of reduced availability of SAM for the methylation of DNA.Animal studies suggest that a decreased conversion of homocysteine to methionine could be a crucial step in causing neural tube defects.It has been shown that rat embryos in culture require methionine for neural tube closure (Mills et al., 1999).
A1298C allele frequency differs greatly in various ethnic groups of the world.The prevalence of the A1298C homozygote variant genotype ranges from 7 to 12% in White populations from North America and Europe.Lower frequencies have been reported in Hispanics (4 to 5%), Chinese (1 to 4%) and Asian populations (1 to 4%) (Botto and Yang, 2000;Rabein and Ulrich, 2003).A number of molecular epidemiological studies have been conducted to investigate the associations of the MTHFR A1298C polymorphism with OFC.However, the results remain conflicting rather than conclusive.Hence, a meta-analysis to derive a more precise estimation of this association is needed.In light of the above facts, a meta-analysis of all available studies relating the A1298C polymorphism of MTHFR gene to the risk of having cleft lip was conducted.

Selection of studies
All studies that investigate the association of the A1298C polymorphism in the MTHFR gene with CLP published before October 2013 were considered in the meta-analysis.The studies were identified by extended computer-based searches of the PubMed, Google Scholar, Elsevier and Springer Link databases.As a search criterion, the following terms were used: 'MTHFR', 'orofacial cleft', 'OFC', 'cleft lip', 'cleft lip and palate', 'A1298C'.
The following inclusion criteria were used: (i) studies must have a case-control study, (ii) study must be published as full papers, (iii) authors must investigate patients with cleft lip and palate cases and healthy control subjects, (iv) authors must provide information on genotype frequencies of the MTHFR A1298C polymorphism or sufficient data for the calculation, (iv) studies with overlapping cases and/or controls, the largest study with extractable data was included.The major reasons for exclusion of studies were (1) only case studied, (2) review papers, editorial, letter to editor and (3) containing overlapping data.

Data extraction
Following information was extracted from each study: first author, journal, year of publication, racial descent of study population, demographics, matching, validity of the genotyping method, and the number of cases and controls for MTHFR A1298C.The frequencies of A and C alleles were calculated for cases and controls from the corresponding genotype distributions.

Meta-analysis
The meta-analysis examined the overall association of the C allele with the risk of OFC relative to the A allele, the additive model for C allele (C vs. A), the co-dominance model (AC vs. AA), the homozygote model for allele C (CC vs. AA), the dominant model for C allele (CC + AC vs. AA), and the recessive model for C allele (CC vs. AC + AA).All associations were indicated as odds ratios (OR) with the corresponding 95% confidence interval (CI).A pooled OR was estimated based on the individual ORs.Heterogeneity between studies was tested using the Q statistic (Cochran, 1954).Heterogeneity was considered statistically significant if P<0.05.Heterogeneity was quantified with the I 2 metric, which is independent of the number of studies in the meta-analysis (I 2 <25%: no heterogeneity; I 2 =25-50%: moderate heterogeneity; I 2 =50-75%: large heterogeneity; I 2 >75%: extreme heterogeneity (Higgins and Thompson, 2002).The pooled OR was estimated using fixed effects (Mantel and Haenszel, 1959) and random effects (Dersimonian and Laird, 1986) models (Whitehead, 2002).Random effects modeling assume a genuine diversity in the results of the studies, and it incorporates the calculations of between-study variability; it therefore tends to provide wider CIs (Zintzaras and Hadjigeorgiou, 2005).

Publication bias
An estimate of the potential publication bias was carried out by funnel plot, in which the standard error (SE) of log (OR) of each study was plotted against its log (OR).An asymmetric plot suggested a possible publication bias.The funnel plot asymmetry was assessed by Egger's test, and P=0.05 was considered representative of statistically significant publication bias (Egger et al., 1997).All analyses were performed using the computer program MIX version 1.7 (Bax et al., 2006).A p value less than 0.05 was considered statistically significant, and all the p values were two sided.

Statistical analysis
Overall, eleven studies provided 1628/2676 cases/controls for MTHFR A1298C.The frequencies of the genotypes MTHFR 1298AA and AC were the highest in both OFC cases and controls, and allele A was the most common.In all eleven studies, total cases were 1628 with AA (825), AC (668) and CC (135), and controls were 2676 with AA (1285), AC (1140), and CC (251) genotypes.In controls genotypes, percentage of AA, AC and CC were 48, 42.6, and 9.38%, respectively.In total cases, genotype percentage of AA, AC, and CC was 50.6, 41 and 8.3%, respectively.The genotype and allele distributions are as shown in Table 2.Only in four studies (Shoteresuk et al., 2003;van Roij et al., 2003;Semic-Jusufagic et al., 2012;Kumari et al., 2013), OR was above one and in other seven studies did not show any association between MTHFR A1298C polymorphism and OFC.The distribution of genotypes in the control groups were in Hardy-Weinberg equilibrium (HWE) in all studies.Lack of HWE indicates possible genotyping errors and/or population stratification (Zintzaras, 2007).

Sensitivity analysis
Sensitivity analysis was performed by sequential omission of individual studies from various contrasts.
The associations of the A1298C polymorphism with cleft lip did not change during the sensitivity analysis.

Publication bias
Funnel plots using standard error and precision values for allele and genotypes using fixed effect model were generated (Figure 3).Symmetrical distribution of studies in the funnel plots suggests absence of publication bias.This is also supported by Beggs and Eggers test (Begg's  3).

DISCUSSION
Normal MTHFR activity is crucial to maintain the pool of circulating folate and methionine and to prevent the accumulation of homocysteine (Frosst et al., 1995).Homocysteine considered as a useful and important metabolic marker of the overall folate status.Folic acid derivatives provide essential single carbon units fro nucleic acid synthesis and methylation reactions both of which are essential for cell division, gene expression and maintenance of chromosome structure during fetal development.It is interesting to note that the case control studies have indicated an effect of the maternal MTHFR genotype rather than that of the affected child (Martinelli et al., 2001;Prescott et al., 2002;Pezzetti et al., 2004).
This study has some limitations and strength also.The main strength was the absence of publication bias and except additive model, low heterogeneity was observed.The insignificant and inconclusive result of the present meta-analysis may be due to (i) small number of studies (only eleven studies), (ii) small sample size, (iii) different ethnic backgrounds of the individuals included in the study, (iv) widely spread exclusion and inclusion criteria which might complicate the comparison between the studies.
In conclusion, result of the present meta-analysis demonstrated that MTHFR A1298C polymorphism did not show any association with CLP and is not a risk factor for oral facial cleft.Oral facial cleft has not been studied as extensively.Further research on facial cleft associations with this MTHFR polymorphism is needed.

Figure 1 .Figure 2 .
Figure 1.Forest plots for the association between MTHFR C677T polymorphism and orofacial cleft for additive model (C vs. A) with random effect model

Table 1 .
Characteristics of eleven studies included in the present meta-analysis.

Table 2 .
The distributions of MTHFR A1298C genotypes and allele frequencies for CLP cases and controls.

Table 3 .
Summary estimates for the odds ratio (OR) of MTHFR A1298C in various allele/genotype contrasts, the significance level (p value) of heterogeneity test (Q test), and the I 2 metric: overall analysis, subgroup analyses, and publication bias p-value (Egger test).
Table 3 lists the main results of the meta-analysis.