The folate metabolism pathway plays an important role in DNA methylation, DNA synthesis, cell division, and tissue growth, especially in the rapidly developing cells 
. Thus, a defective folate metabolism could result in an impaired DNA synthesis or DNA methylation involved in the neurulation process. MTHFR
is a key enzyme in the folate metabolism pathway. Although several single nucleotide polymorphisms (SNPs) in the MTHFR
gene have been characterised, the C677T
polymorphism is a widely described mutation. Heterozygotes (CT) for the polymorphism have 65% of the normal enzyme activity and 10% lower red blood cell folate level; patients with the homozygous variant (TT) have only 30% of normal enzyme activity and 18% lower red blood cell folate levels 
. Additionally, individuals with the TT variant also have lowered plasma folate and vitamin B12
levels and increased homocysteine levels 
. Overall, due to its potential role in decreasing MTHFR
activity, causing high plasma homocysteine and low plasma folate levels, it contributes to NTDs 
. Despite the potential implication of MTHFR C677T
in the pathogenesis of NTDs 
, the association between the MTHFR C677T
polymorphism and NTDs remains unclear.
Our meta-analysis, which included 2429 cases and 3570 controls, explored the associations between the maternal MTHFR
C667T polymorphism and susceptibility to NTDs. Overall, we found that mothers with the homozygous TT genotype showed a significantly increased NTD risk compared with homozygous CC genotype carriers (with pooled odds ratio 2.022; 95% CI: 1.508, 2.712; P
<0.001). Our results were consistent with a previous report 
that showed an overall odds ratio of 2.04 (TT versus CC: 95% CI: 1.49, 2.81).
In subgroup analysis stratified by ethnicity, we found that the variant genotypes were associated with a significantly increased NTD risk in Asian, Caucasian and mixed populations. However, we did not found this association in African groups in any genetic model, possibly due to the limited studies and a small sample size. Moreover, the pooled odds ratios of mixed populations were higher than those of Caucasian populations and the overall populations in mothers (TT versus CC: OR
3.595, 1.596, and 2.022, respectively). Many factors may contribute to the finding that the same polymorphism affects different ethnic populations to a different extent. First, the frequency of the T-allele varies in different ethnicities with different genetic backgrounds 
. Second, different populations may have different dietary patterns, such as intake of folic acid, vitamin B12
, and vitamin B6
, some of which may affect NTD development. Finally, analysis of the data from the various ethnic groups might eliminate some bias caused by language because only papers written in English or Chinese were included. Thus, large-scale studies should be performed to validate ethnic differences in the effect of this functional polymorphism on NTD risk.
When stratified by study design, significantly increased risks were also found in both population-based and hospital-based studies. Nevertheless, population-based studies have a higher risk than hospital-based studies. Hospital-based studies usually have a high risk of producing unreliable results because hospital-based controls may not always accurately represent the general population, especially when the genotypes under investigation are expected to affect disease conditions that might be observed in the hospital-based controls 
. Thus, in genetic association studies, the selection of controls and matching status should be carefully considered. To reduce the bias, well-designed, population-based studies should be performed to explore the association.
Because heterogeneity is a potential problem when interpreting the results of all meta-analyses, we detected the source of heterogeneity by ethnicity, publication year, control source, and sample size and found that none substantially contributed to the heterogeneity. One possible reason might be the matching status.
It is assumed that MTHFR
genetic polymorphisms play an important role in the development of NTDs; however, only 13% of NTDs were attributed to the MTHFR C677T
, suggesting that the MTHFR C677T
polymorphism alone cannot be responsible for NTDs. Thus, potential gene-gene, maternal-foetal, genetic-nutritional interactions 
, and other SNPs in the MTHFR
gene may have an association with NTD risk.
1) Gene-gene interactions
Folate metabolism is complex and involves several regulatory mechanisms. Genetic variations affecting protein function at any step may alter the balance of metabolites, and gene-gene interactions 
. The combination of MTHFR
and cystathione-β-synthase (CBS) mutations was reported to have a fivefold increase in the risk for spina bifida compared with each variant alone 
, indicating the presence of gene-gene interactions.
2) Maternal-foetal interactions
Maternal variant genotypes were associated with NTD risk, indicating possible maternal-foetal interactions. Using family-based approaches, researchers have found that the OR increased to 4.1 (95% CI: 1.5, 11.1) if the mother had a TT genotype and her child a CT genotype and to 6.1 (95% CI: 1.0, 35.5) if both the mother and her child had TT genotypes 
3) Genetic-nutritional interactions
The combination of MTHFR
mutations and low folate concentrations could lead to a hypomethylation of homocysteine to methionine, enhancing the impairment of folate metabolism and increasing the risk for NTDs 
, suggesting a strong genetic-nutritional interaction. This interaction was found in a previous study that showed the combination of MTHFR
TT genotype and RBC folate level in the lowest quartile conferred an odds ratio of 13.43 (95% CI: 2.49, 72.33) for an NTD case and an odds ratio of 3.28 (95% CI: 0.84, 12.85) for having offspring with NTDs 
Although these studies were hampered by small sample sizes, they illustrate the existence of potential interactions. Thus, further large-scale studies focusing on these complex interactions with NTD risk should be performed.
4) Other SNPs in the MTHFR gene
Some researchers have demonstrated that other SNPs in MTHFR
gene showed increased NTD risks, such as A1298C
, and were linkage disequilibrium with C677T
polymorphism. All these suggest these SNPs can be additional genetic factors for NTDs.
Several potential limitations of this meta-analysis should be discussed. 1) Although the funnel plot and Egger's test showed no publication bias, selection bias may have occurred because only studies in English or Chinese were selected. 2) Our results were based on unadjusted estimates due to the absence of available information, such as supplementation of folic acid, maternal use of drugs, and other factors that are associated with NTD risk. 3) We did not consider the foetal MTHFR C677T polymorphisms due to the limited data. Despite the limitations listed above, our meta-analysis has some clear advantages. 1) The well-designed search and selection method significantly increased the statistical power of this meta-analysis. 2) No publication bias was detected, indicating that our pooled results are likely to be reliable.
In conclusion, our meta-analysis provided evidence that the MTHFR C677T polymorphism is a genetic risk factor for NTDs. Because NTDs are the most common congenital malformations worldwide and have a multifactorial aetiology, various genetic and environmental factors as well as their complex interactions implicated in the pathogenesis should be taken into account. Further functional studies to investigate folate-related gene polymorphisms, periconceptional multivitamin supplements, complex interactions and their role in development of NTDs are warranted.