NSCL/P is a multifactorial disease caused by genetic and environmental factors. In previous years, various genomic susceptibility regions have been identified in association studies, linkage studies, family sequencing studies, and animal experiments suggesting that gene mutations influence the development of maxillofacial area. However, the underlying biological mechanisms remain unclear.
Folic acid is an important factor that influences the metabolism and the synthesis of nucleotides and amino acids. Previous studies have suggested that folic acid plays an important role in decreasing the risk of NSCL/P [2, 3]. Folic acid metabolism is a complex process and many genes are involved in the pathway, such as MTHFR, MTRR, TCN2, and BHMT. However, there are no consistent results regarding the association between the genetic variations of these genes and NSCL/P in different populations. To clarify these inconsistent results, we carried out the meta-analysis in this study.
The present meta-analysis results demonstrated a significant association between rs1801133 and NSCL/P risk in two genetic models: TT allele vs CC allele (OR=1.333 95%CI=1.062-1.674, P=0.013) and recessive model (OR=1.325 95%CI=1.075-1.634, P=0.008). There were a significant association found between rs1801394 and NSCL/P risk in Asian (GG allele vs AA allele, OR=0.520 95%CI=0.321-0.841, P=0.008).
TCN2, encode transcobalamin2, transports vitamin B12 to cells, have been reported to be associated with multiple diseases, such as cancer, Alzheimer and other congenital abnormalities [39-41]. In 2006, Martinelli et al. found that the C776G in TCN2 was associated with risk of cleft lip, but subsequent studies didn’t get the significant results [10]. Similarly, the present study didn’t find the significant association between the C776G and NSCL/P.
BHMT, a zinc dependent cytosolic enzyme, is important for homocysteine metabolism and methionine synthesis. In 2010, Mostowska et al. first found that rs3733890 of the BHMT was associated with NSCL/P, and other studies also indicated its association with coronary artery disease and neural tube defects [23]. In the present study, we found no evidence showing rs3733890 playing any significant role [23]. We inferred several factors may contribute to the result. First, we found a relative high value of heterogeneity among studies, which cause a different distribution of genotype. Second, the number of included studies and sample size are relatively small. So the subgroup analysis was not conducted based on ethnicity.
MTRR plays a vital role in functional regeneration of methionine synthase, and it may be associated with increasing the congenital heart disease risk [18]. But the meta-analysis conducted by Zhang et al. in 2013 and Lei et al. in 2018 showed no association between rs1801394 and the risk of NSCL/P [42, 43]. In the present study, we found a significant association between rs1801394 and the NSCL/P risk in Asian, but no association in Caucasian. The data from 1000 genomes and ExAC database was shown in Supplementary table 1.
MTHFR is an important enzyme in homocysteine metabolism and C677T rs1801133 is one of the most important functional polymorphisms. Prediction by bioinformatics tools showed that the change of genetic variant will influence the protein function and predispose to cause the disease (Table 1). However, the association between rs1801133 and the risk of NSCL/P is inconsistent. The allelic frequencies vary in different ethnic groups and the minor allele frequency (MAF) of MTHFR rs1801133 in Asian are lower than that in European and American, so it is very valuable to summarize and analyze by systematic statistical methods. In the present study, we included 30 studies including 5517 cases and 7770 controls and found T allele can increase the risk of NSCL/P.
The strength of this meta-analysis is that it expands to a large number of related studies, and the most updated publications were included. To guarantee the quality of the included studies, a strict procedure for search strategy, literature inclusion, data extraction, and quality assessment was performed, which could avoid potential influences and increase the strength of the results. Meta-regression and sensitivity analysis were also performed to strengthen the conclusions. We confirmed the previous investigation by summarizing a larger number of closely related studies.
There are some limitations in the present meta-analysis. Firstly, studies published only in English were included in the meta-analysis, and studies published in other languages were excluded. Secondly, environmental factors also contribute to NSCL/P, and in the present study, non-genetic factors and other potential interactions, including environment-gene interactions, were limited. Additionally, some potential covariates (e.g., age, sex, folate etc) were not included in the analysis due to insufficient information.