The correlation between methylenetetrahydrofolate reductase gene 677C > T polymorphism and fetal congenital defects: A meta-analysis

Abstract: Objective To investigate the correlation between the methylenetetrahydrofolate reductase (MTHFR) gene 677C> T polymorphism and fetal congenital defects. Method Original studies relevant to the MTHFR gene 677C>T single nucleotide polymorphism and fetal congenital defects were systematically searched in the electronic databases of Medline, EMBSE and China National Knowledge Infrastructure (CNKI). All relevant publications were screened for inclusion in the present work. The correlation between the MTHFR gene 677C > T single nucleotide polymorphism and the occurrence of fetal congenital defects was expressed as an odds ratio (OR) and its 95% confidence interval (95% CI). Publication bias was assessed by Begg’s funnel plot and Egger’s line regression test. Results Nineteen case-control studies were ultimately included in the present meta-analysis. The pooled results indicated that the general risk of fetal congenital defects was significantly elevated in subjects with the 677T allele of the MTHFR gene in dominant (OR=1.07,95%CI:1.03-1.12, P<0.05), homozygous (OR=1.17,95%CI:1.06-1.30, P<0.05) and recessive genetic models (OR=1.16,95%CI:1.03-1.31, P<0.05) through the random effect method. However, significant publication bias was identified upon pooling the individual data and evaluating the correlation. Conclusion According to the present evidence, the MTHFR gene 677C>T single nucleotide polymorphism is correlated with poor pregnancy outcomes, and subjects with the T allele have an increased risk of developing general fetal congenital defects.


Introduction
The incidence of birth defects in China is about 5.6%, making them the main cause of perinatal and infant deaths [1]. More and more studies have shown that genetic and environmental factors are involved in the occurrence of birth defects [2]. Maternal genetic susceptibility also increases the incidence of both spontaneous abortion and birth defects [3,4]. In recent years, attention has been paid to polymorphisms in genes closely related to folic acid metabolism, such as MTHFR and MTRR, and their relationship with fetal congenital malformations, spontaneous abortion and congenital cardiovascular diseases. This field has become a research hotspot in recent years.
The MTHFR 677C>T single nucleotide polymorphism is a common polymorphism. The 667 cytosine of MTHFR is replaced by thymine, which transforms the highly conserved alanine (Ala) into valine (Val). This change can affect the protein's activity, resulting in abnormal methylation metabolism of Hcy, hyperhomocysteinemia and other diseases. Previous studies on the MTHFR 677C>T single nucleotide polymorphism and birth defects have been reported [5], but due to the small sample size and low statistical efficiency of each report, there are some contradictions in the results. Therefore, in this study, we used the meta-analysis method to analyze the aforementioned studies and further clarify the correlation between the MTHFR 677C > T single nucleotide polymorphism and birth defects.

Study identification in the databases
Original studies relevant to the MTHFR gene 677C>T single nucleotide polymorphism and fetal congenital defects were systematically searched in the electronic databases of Medline, EMBSE and China National Knowledge Infrastructure (CNKI). Case-control or cohort studies relevant to the above topic and published in English or Chinese were systematically searched using the keywords "methylenetetrahydrofolate reductase," "MTHFR," "pregnancy," "fetal congenital defects" and "polymorphism." The references of the relevant publications were also reviewed in order to find additional potential studies related to this topic.

Study inclusion and quality assessment
Included studies should meet the following criteria: (1) The publication type is case-control or cohort study; (2) The study was published in Chinese or English; (3) The single nucleotide C and T distributions can be extracted from the original study; (4) Correct single nucleotide polymorphism detection methods were used. Study exclusion criteria were: (1) Other study design besides case-control or cohort; (2) Duplicated publication or data; (3) Single nucleotide C and T distributions were not provided or can't be calculated from the original studies.
The general methodological quality of the included studies was assessed using the Newcastle-Ottawa scale [6], which was used for quality assessment of observation studies by two reviewers (DX Li and EX Wang) independently.

Statistical analysis
Stata/SE 11.0 statistical software was applied for analysis of the data. The correlation between the MTHFR gene 677C> T polymorphism and fetal congenital defects was expressed as an odds ratio (OR) and its 95% confidence interval (95%CI). Statistical heterogeneity across the included nineteen publications was evaluated by the I 2 test. Publication bias was assessed through Begg's funnel plot and Egger's line regression test.

Dominant genetic model (TT+CT vs CC)
Significant statistical heterogeneity was found to exist across the 19 publications for the dominant genetic model (I 2 =60.5%, P<0.05). The individual correlations between the MTHFR 677C>T gene polymorphism and general fetal congenital defects were pooled through the random effect model. The pooled results indicated that subjects with a TT or CT genotype had an increased risk of developing fetal congenital defects (OR=1.07,95%CI:1.03-1.12, P<0.05),

Homozygous genetic model (TT vs CC)
In the homozygous genetic model, the data was pooled by the random effect model for statistical heterogeneity (I 2 =40.3%, P<0.05). The pooled data indicated that the general risk of fetal congenital defects was significantly elevated in subjects with a TT genotype in a homozygous genetic model (OR=1.17, 95%CI:1.06-1.30, P<0.05),

Recessive genetic model (TT vs CT+CC)
For a recessive genetic model (TT vs CT+CC), the pooled results also showed subjects with the TT genotype had

Subgroup analysis
To investigate different fetal congenital defects and subject ethnicities, we performed subgroup analysis. The correlations between the methylenetetrahydrofolate reductase 677C>T gene polymorphism and different fetal congenital defects or different ethnicities are shown in Table 2. In different genetic models, the correlations between the methylenetetrahydrofolate reductase 677C>T gene polymorphism and fetal congenital defects were quite different. However, the MTHFR 677C>T gene polymorphism was significantly correlated with anencephaly in various genetic models.

Publication bias evaluation
Publication bias was evaluated by Begg's funnel plot and Egger's line regression test. The funnel plot was asymmetric at its bottom, indicating potential publication bias in all genetic models ( Figure 5). The Egger's line regression test indicated significant publication bias in the homologous and recessive genetic models, but not in the dominant genetic model, Table 3.

Discussion
The methylenetetrahydrofolate reductase protein primarily reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate in the folic acid metabolic pathway, thus participating in purine synthesis, pyrimidine synthesis and DNA synthesis in vivo as an indirect donor of methyl groups [26]. The normal function of the MTHFR protein is to maintain the efficacy of the folate methionine cycle and ensure the normal progress of DNA synthesis and methylation. If the MTHFR gene is mutated, this can lead to the disruption of many basic biochemical processes in the body, as well as hyperhomocysteinemia [27,28]. The 677C > T polymorphism in the MTHFR gene causes alanine to be replaced by valine, leading to a decrease in the heat resistance and activity of the enzyme. It has been reported that MTHFR gene mutations can induce elevated blood Hcy levels, leading to many adverse pregnancy outcomes, such as neural tube defects, congenital heart disease, premature delivery, pregnancy complications, recurrent abortion and Parkinson's disease [29]. To this end, some studies have shown that the MTHFR single nucleotide polymorphism can affect serum Hcy levels and is associated with adverse pregnancy outcomes [30]. However, other studies have shown that subjects with the T genotype have an increased risk of developing fetal congenital defects, such as neural tube deformity and congenital heart disease.     polymorphism was correlated with Down's syndrome and anencephaly. Finally, for the homozygous genetic model, the MTHFR 677C>T gene polymorphism was correlated with neural tube defects, anencephaly and premature birth. Therefore, under different genetic models, the MTHFR 677C>T polymorphism may affect different fetal congenital defects, which had not been evaluated in previous publications. However, the MTHFR 677C>T gene polymorphism was significantly correlated with anencephaly in all genetic models. This indicates that the MTHFR 677C>T gene polymorphism may be an important factor in the development of anencephaly. Fetal congenital defects or poor pregnancy outcomes represent a group of complex diseases, which are affected by multiple susceptibility genes and many environmental factors. Because of this, only assessing the effect of one single nucleotide polymorphism in one gene may be not enough to completely explain the disease etiology. Therefore, high-throughput analysis of folic acid metabolism-related genes and their correlations with fetal congenital defects or poor pregnancy outcomes is necessary.
The present work also had limitations: (1) Heterogeneity across the included studies was significant, which may decrease statistical power; (2) Only open studies published in English or Chinese were included in the work. Therefore, other suitable studies may have been omitted; (3) The general methodological quality of the included studies was moderate. Therefore, more highquality relevant studies will be needed to further validate our findings.