Methylenetetrahydrofolate reductase C677T polymorphism is not associated with the risk of nonsyndromic cleft lip/palate: An updated meta-analysis

Both genetic and environmental factors affect the risk of orofacial clefts. The present meta-analysis aimed to evaluate the association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and risk of nonsyndromic cleft lip/palate (NSCL/P) in cases-control studies. The PubMed/Medline, Scopus, Web of Science, and Cochrane Library databases were searched up to April 2019 with no restrictions. The odds ratios (ORs) and 95% confidence intervals (CIs) in all analyses were calculated by Review Manager 5.3 software. The funnel plot analysis was carried out by the Comprehensive Meta-Analysis version 2.0 software. Subgroup analysis, meta-regression, and sensitivity analysis were performed for the pooled analyses. Thirty-one studies reviewed in this meta-analysis included 4710 NSCL/P patients and 7271 controls. There was no significant association between MTHFR C677T polymorphism and NSCL/P susceptibility related to allelic model (OR = 1.04; P = 0.49), homozygote model (OR = 1.11; P = 0.35), heterozygote model (OR = 0.99; P = 0.91), dominant model (OR = 1.00; P = 0.96), or recessive model (OR = 1.08; P = 0.23). There was no significant association between MTHFR C677T polymorphism and NSCL/P susceptibility based on the ethnicity or the source of cases. There was a significant linear relationship between the year of publication and log ORs for the allele model. The results of the present meta-analysis failed to show an association between MTHFR C677T polymorphism and NSCL/P susceptibility. The subgroup analyses based on the ethnicity and the source of cases further confirmed this result.

. Distribution of MTHFR C677T polymorphism genotype and allele in NSCL/P patients and controls. Abbreviation: HWE, Hardy-Weinberg equilibrium.
Subgroup analysis was performed according to the ethnicity and the source of cases to explore potential heterogeneity. The meta-regression analysis is a technique used to assess heterogeneity between the studies. This statistical approach determines whether there is a significant association between the study period and number of individuals with the pooled OR. The Begg's funnel plot was carried out by the Comprehensive Meta-Analysis version 2.0 software identifying the standard error of log (OR), and the precision of each study was plotted against its log (OR) 25 . In addition, the results of Egger's linear regression were retrieved using this software 26 . To estimate the consistency or stability of the results, we used sensitivity analysis namely cumulative analysis and one study was removed. P-value (2-tailed) <0.05 was statistically significant.

Results
A total of 353 records were retrieved from the databases and after removing the duplicates, 187 records were screened (Fig. 1). Next, 117 records were excluded considering the eligibility criteria. Then, the full-texts of 70 articles were evaluated and 36 articles were excluded with reasons (five studies were systematic reviews/ meta-analyses; one study was erratum; two studies were letter to editors/research letters; one study was editorial comment; three studies were family-based studies; five studies had irrelevant data; ten studies were case-control parents; three studies had no control group; five studies were case-parent triads; one study reported complex birth defects). On the other hand, by searching the references of meta-analyses, two other articles 27,28 were found. Totally, 36 articles were included in this systematic review 6,27-61 out of which, 5 studies 6,58-61 had a deviation from the HWE for the control group and were excluded from the meta-analysis. Therefore, 31 articles were included and analyzed in this meta-analysis. In addition, one study 62 was excluded for reducing bias compared with other previous meta-analyses, because it was a conference paper and therefore didn't involve the eligibility criteria. Table 1 shows the characteristics of each study included in this meta-analysis. The articles had been published from 1998 to 2019. Overall, the studies included 4,710 NSCL/P patients and 7,271 controls. Out of 31 studies, 10 studies were reported in mixed ethnicities, 10 studies had been conducted on Asians, and 11 studies had been    www.nature.com/scientificreports www.nature.com/scientificreports/ conducted on Caucasians. In addition, the source of cases (patients) was population-based in 13 studies and hospital-based in 18 studies. Table 2 shows the distribution of MTHFR C677T polymorphism genotype and allele in NSCL/P patients and controls. All studies followed the HWE for the control group.    Subgroup analysis. The subgroup analysis was performed based on the ethnicity and the source of cases for the association between MTHFR C677T polymorphism and NSCL/P risk (Table 3). There was no significant association between MTHFR C677T polymorphism and NSCL/P susceptibility with regard to the ethnicity (Asian, Caucasian, and mixed ethnicities) or the source of cases (population-based and hospital-based).

Meta-regression.
Considering the year of publication and the number of individuals as independent variables and the log (OR) as the dependent variable, the fixed-effect meta-regression results are presented in Table 4, Figs. 7 and 8. To estimate the functional relationship of the log OR with the year of publication and the number of individuals, the analysis showed only a significant relationship for the allele model (T vs. C) for the year of publication with a regression coefficient of −0.01346. Therefore, there was a significant linear relationship between the year of publication and log ORs for the allele model (T vs. C), but not for the genetic models.
Publication bias. Figure 9 shows the funnel plots of all genetic models to evaluate the association between the NSCL/P risk and MTHFR C677T polymorphism in a fixed-effect model. There was no publication bias between the NSCL/P risk and MTHFR C677T polymorphism in the genetic models. Sensitivity analysis. Two analyses (one study excluded and cumulative analysis) were performed and the pooled ORs did not change qualitatively. Therefore, the analyses showed that the pooled ORs under all genetic models were stable and trustworthy.

Discussion
NSCL/P is one of the most common congenital anomalies with high rate of mortality. Its pathogenesis is difficult to be attributed to either environmental or genetic factors. The pathway of folate metabolism plays a significant role in the synthesis, repair, and methylation of DNA involved in NSCL/P pathogenesis 63 . MTHFR enzyme plays an important role in folate intake, and mutations of MTHFR gene significantly impact the stability and thus the function of the enzyme; MTHFR C677T is the most common mutation of this gene 64 . MTHFR C677T polymorphism is related to a reduction in MTHFR activity, raised plasma homocysteine concentration, and lower plasma level of folic acid, which consequently contribute to NSCL/P 65 . The present meta-analysis was performed  www.nature.com/scientificreports www.nature.com/scientificreports/ to more precisely assess the relationship between MTHFR C677T polymorphism and NSCL/P susceptibility. In pooled analysis, the meta-analysis showed no significant association between MTHFR C677T polymorphism and NSCL/P risk.
Out of 31 studies included in the present meta-analysis, six studies 27,34,39,44,45,52 , five studies 34,39,44,46,52 , and four studies 34,39,46,52 reported significantly increased risk of T allele, TT genotype, and CT genotype in NSCL/P patients compared with controls, respectively. Also, five studies 36,47,49,55,57 , two studies 47,57 , and four studies 28,36,47,57 reported a significantly decreased risk of T allele, TT genotype and CT genotype in NSCL/P patients compared with controls, respectively. In addition, TT + CT genotype was reported to have a significantly increased risk in five studies 34,39,44,46,52 and significantly decreased risk in four studies 36,47,55,57 in NSCL/P patients compared with controls. Based on the recessive model, three studies 39,46,54 reported significantly increased risk of TT genotype and one study 57 reported its significantly decreased risk in NSCL/P patients compared with controls.
A recent meta-analysis with 24 case-control studies 14 investigating the relationship between NSCL/P and MTHFR C677T polymorphism showed that the TT genotype was a risk factor for NSCL/P in Asians in homozygote (OR = 1.96, P < 0.001) and recessive (OR = 1.45, P = 0.028) models. Also, based on mothers with NSCL/P progeny versus control mothers with healthy progeny in 10 studies, the TT genotype of Caucasian mothers may increase progeny NSCL/P morbidity. Another recent meta-analysis of 22 case-control studies 15 showed that MTHFR C677T polymorphism was associated with a higher risk of NSCL/P. Both meta-analyses also included studies with a deviation of HWE. However, in the present meta-analysis, we excluded such studies from the pooled analysis and therefore, reviewed 31 case-control studies and had lower heterogeneity compared with the meta-analysis with 22 studies 15 . Almost similar to the findings of a meta-analysis with 24 studies 14 , our results www.nature.com/scientificreports www.nature.com/scientificreports/ showed no association between MTHFR C677T and susceptibility to NSCL/P. In one meta-analysis 15 , definition of ethnicity was different from that in another study 14 and our meta-analysis. The meta-regression showed a linear relationship with a negative slope between the year of publication and log ORs for the allele model and therefore by increasing years of publication, the risk of T allele decreased in NSCL/P patients compared with controls. There were two other meta-analyses with eight 16 and nine 7 case-control studies related to our topic. One of them 16 reported no association and another one on Asian ethnicity showed a significant association between MTHFR C677T and susceptibility to NSCL/P. Luo et al. 17 on nine studies in a meta-analysis didn't show any evidence for significant association between infant or maternal MTHFR C677T polymorphism and NSCL/P risk, but suggested that maternal MTHFR 677TT polymorphism could increase the risk of having a NSCL/P offspring in the white population. Pan et al. 8 on seventeen studies showed that this polymorphism was a risk factor involved in the development of NSCL/P in Asians that definition of ethnicities in this meta-analysis was different from our meta-analysis. The results showed that the effect of each factor alone on the association was low, but such a high heterogeneity among the studies could be due to simultaneous effect of several factors such as differences in the ethnicity of the study populations, source of cases, and number of individuals.
This study had several important limitations including high heterogeneity across studies, unadjusted ORs used in the studies, and intake of folic acid and other supplements that were not considered. Nevertheless, the present study included more studies with meta-regression without any deviation of HWE for controls in all studies compared with other meta-analyses. It did not have publication bias, and the results were stable.