Associations Between MTR A2756G,MTRR A66G,and TCN2 C776G Polymorphisms and Risk of Nonsyndromic Cleft Lip With or Without Cleft Palate: A Meta-Analysis

Abstract

Objective:

We conducted a meta-analysis to investigate the associations of methionine synthase (MTR) A2756G, methionine synthase reductase (MTRR) A66G, and transcobalamin 2 (TCN2) C776G gene polymorphisms with nonsyndromic cleft lip with or without cleft palate (NSCL/P).

Materials and Methods:

The PubMed, Web of Science, Embase, and Wiley Online Library databases and the China Biomedical Literature Service System (SinoMed) were searched for relevant articles to explore the associations between the MTR A2756G, MTRR A66G, and TCN2 C776G polymorphisms and the risk of NSCL/P. We performed overall comparisons and stratified analyses according to the ethnicity, type of NSCL/P, and Hardy-Weinberg equilibrium (HWE) of the control group. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were applied to estimate the associations of these gene polymorphisms with NSCL/P risk using fixed-effects or random-effects models incorporating five genetic models.

Results:

Ultimately, 12 articles were included in this study. The pooled results did not reveal a significant association of the MTR A2756G polymorphism with NSCL/P risk (G vs. A: OR = 0.95, 95% CI = 0.82-1.11, p = 0.55). Similar results were observed for the MTRR A66G polymorphism (G vs. A: OR = 0.99, 95% CI = 0.82-1.18, p = 0.72) and the TCN2 C776G polymorphism (G vs. C: OR = 0.95, 95% CI = 0.86-1.06, p = 0.37).

Conclusion:

In summary, the MTR A2756G, MTRR A66G, and TCN2 C776G polymorphisms might not be associated with NSCL/P risk.

Introduction

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is among the most prevalent congenital birth defects in humans, including cleft lip only (CLO), cleft lip and palate (CLP), and cleft palate only (CPO) (Cobourne, 2004). These defects occur in ∼1 in 700-1000 neonates, with ethnic and geographic variations (Yuan et al., 2011). Available data indicate higher incidences in Asia, including China and Japan, and in Latin America, whereas southern Europe, South Africa, and Israel have the lowest incidences (Mossey et al., 2012). NSCL/P not only has a serious impact on the development of a patient's maxillofacial region, pronunciation, and swallowing function but also imposes a heavy psychological and economic burden on the family and society (Mossey et al., 2009). NSCL/P is a disease with multiple aetiologies that are influenced by both genetic and environmental factors. Various environmental risk factors, including smoking, drinking, and vitamin deficiency, are closely related to oral clefts (Jia et al., 2011; Wu et al., 2012; Gunnerbeck et al., 2014).

Folic acid plays an important role in one-carbon metabolism in DNA methylation and in the synthesis of nucleotides and amino acids, which is necessary for chromatin dynamics and subsequent gene expression (de Arruda et al., 2013). Previous studies have reported that folic acid supplementation decreased the risk of NSCL/P during early pregnancy (Rouget et al., 2005; Wehby et al., 2010, 2012). It is widely believed that the occurrence of NSCL/P is related not only to folic acid deficiency (Prescott and Malcolm, 2002) but also to hyperhomocysteinemia (Hcy) caused by a genetic defect in the folate metabolism pathway (Bhaskar et al., 2011). Evidence shows that Hcy is associated with a high risk of NSCL/P incidence (Wong et al., 1999). Folic acid metabolism is a complex process that requires certain key enzymes, such as methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and transcobalamin 2 (TCN2) (Martinelli et al., 2006; Pan et al., 2012; Murthy et al., 2015; Wang et al., 2016).

MTR, which is encoded by the MTR gene on chromosome 1q43 (Leclerc et al., 1996), is also called methionine synthase and catalyses the remethylation of homocysteine to form methionine. A common polymorphism, the A2756G transition of the MTR gene, results in the conversion of an aspartic acid residue to a glycine residue in the MTR enzyme (Leclerc et al., 1996; Chen et al., 1997). Li et al. (1996) reported that the A2756G polymorphism in the MTR gene led to a reduction in enzyme activity and caused increased concentration of plasma homocysteine. The MTRR gene is located on chromosome 5p15.2-15.3 and has a length of 32021 kb (Olteanu et al., 2001). This gene encodes MTRR, which promotes methylcobalamin regeneration and maintains MTR activation. MTRR A66G is a common polymorphism in the MTRR gene that converts isoleucine to methionine (66 A→G) at codon 22 (Bhaskar et al., 2011). The G allele produces an enzyme with less affinity for methionine synthase (Han et al., 2012). The TCN2 gene is located on chromosome 22q12.2, and its translated product is a plasma protein that transfers vitamin B12 into cells (Carmel, 2002; Herrmann et al., 2003). The C776G genetic variant in TCN2 results in the substitution of proline for arginine at codon 259 (Namour et al., 2001). A result of the C776G polymorphism in the TCN2 gene is a decrease in the transcription and cellular and plasma concentrations of transcobalamin (Gueant et al., 2007).

Given that the MTR, MTRR, and TCN2 genes play a significant role in folate/homocysteine metabolism and methionine synthesis, these three common polymorphisms (MTR A2756G, MTRR A66G, and TCN2 C776G) cause increased levels of plasma homocysteine. To date, a considerable number of studies have assessed the associations of the MTR A2756G, MTRR A66G, and TCN2 C776G polymorphisms with NSCL/P risk, but their conclusions have been contradictory. Therefore, we conducted the present meta-analysis to perform a more accurate assessment of the associations between these three single nucleotide polymorphisms (SNPs) and NSCL/P risk.

Materials and Methods

Search strategy

We performed a systemic literature search in PubMed, Embase, Wiley Online Library, Web of Science, and the China Biomedical Literature Service System (SinoMed), updated on August 31, 2017, to screen for relevant articles. The following search terms were used: “methionine synthase,” “MTR,” “MTR A2756G,” “rs1805087,” “MTRR,” “methionine synthase reductase,” “MTRR A66G,” “rs1801394,” “TCN2,” “transcobalamin II,” “TCN2 C776G,” “rs1801198,” “gene polymorphism,” “cleft lip and palate,” “oral clefts,” “nonsyndromic cleft lip with or without palate,” and “NSCL/P.” Additionally, the reference lists of relevant articles were checked to identify additional potentially relevant publications. We limited the publication language to English and Chinese. Any differences of opinion between the two investigators were resolved through discussions with two other reviewers (Y.W. and G.F.).

Inclusion and exclusion criteria

The inclusion criteria for the eligible studies were as follows: (1) studies focused on the association between the MTR A2756G, MTRR A66G, or TCN2 C776G polymorphism and the risk of NSCL/P; (2) case-control studies; (3) the availability of adequate data to calculate odds ratios (ORs), 95% confidence intervals (CIs), and p-values; and (4) a publication language of English or Chinese. We eliminated studies that met the following exclusion criteria: (1) non-case-control studies; (2) duplicate studies; and (3) reviews, editorials, and other non-original studies.

Data extraction

The available data were independently extracted from all eligible publications by two reviewers (W.L. and Y.X.) using a predetermined strategy. The following items were collected: publication year, the surname of the first author, ethnicity, country, NSCL/P type, source of controls, numbers of cases and controls, genotyping method, and p-value for Hardy-Weinberg equilibrium (HWE) of the control group.

Methodological quality evaluation

Two investigators (W.L. and Y.X.) independently estimated the quality of the eligible studies using the Newcastle-Ottawa Scale (NOS) criteria, which are recommended for studies estimating the quality of case-control studies. Differences were resolved by consultation with the other two reviewers (Y.W. and G.F.). The NOS criteria include the following three aspects: (1) population selection, (2) comparability of subjects, and (3) measurement of exposure factors. The NOS scores range from 0 to 9. A study was considered high quality if the score was ≥5, whereas a study with a score of <5 was defined as having inferior quality.

Statistical analysis

The p-values for HWE of the control groups in the eligible articles were calculated using the chi-squared test to estimate the quality of the case-control studies. Genotype distributions were considered to deviate from HWE when the p-value was <0.05. The associations of the MTR A2756G, MTRR A66G, and TCN2 C776G polymorphisms with the risk of NSCL/P were assessed using ORs and 95% CIs. The significance of the pooled ORs was detected using the Z test, and a p-value of <0.05 was considered statistically significant. We assessed the association of these three SNPs with NSCL/P risk using the following five genetic models: the allele contrast model (A₁ vs. A₂), recessive genetic model (A₁A₁ vs. A₂A₂/A₁A₂), dominant genetic model (A₁A₂/A₁A₁ vs. A₂A₂), homozygous genetic model (A₁A₁ vs. A₂A₂), and heterozygous genetic model (A₁A₂ vs. A₂A₂). In addition, we conducted stratified analyses based on ethnicity, NSCL/P type, and HWE of the control group. Cochran's Q-statistic and the I² test were used to detect heterogeneity among the included publications. If the Q-test showed a p > 0.05 or I² < 50%, indicating low heterogeneity, a fixed-effects model was used. Otherwise, a random-effects model was applied. Furthermore, Begg's funnel plot and Egger's linear regression test were used to assess publication bias. A sensitivity analysis was conducted to detect the effect of each separate study on the pooled OR by successively removing each eligible study. All statistical analyses were conducted using RevMan 5.3 software (Cochrane Collaboration) and STATA software 12.0 (STATA Corp., College Station, TX).

Results

Study characteristics

Initially, 523 publications were identified in the electronic database search. Based on the inclusion and exclusion criteria, 19 articles were identified after the titles and abstracts were scanned. However, four articles were discarded because of unavailable data, and three studies were excluded as non-case-control studies after the full text was read. Thus, 12 articles were ultimately included in this meta-analysis (Martinelli et al., 2006; Brandalize et al., 2007; Mostowska et al., 2010; Hu, 2011; Wang and Nan, 2011; Yuan and Nan, 2013; Aşlar and Hakkı, 2014; Bezerra et al., 2015; Jin et al., 2015; Murthy et al., 2015; Waltrick-Zambuzzi et al., 2015; Wang et al., 2016). Among these 12 studies, 8 articles focused on the MTR A2756G polymorphism, 7 articles on the MTRR A66G polymorphism, and 5 articles on the TCN2 C776G polymorphism. The detailed process used to retrieve the relevant publications is shown in Figure 1. The main characteristics, including the surname of the first author, publication year, country, ethnicity, NSCL/P type, source of controls, genotyping method, numbers of cases and controls, and p-value for HWE of the control group, are presented in Table 1. The estimation of the quality of the included articles is shown in Table 2.

FIG. 1.

Flow diagram for literature selection.

Table 1.

Characteristics of Studies Included in the Meta-Analysis

						TCN2 C776G		MTR A2756G		MTRR A66G
Study	Country	Ethnicity	Types of disease	Source of controls	Genotyping method	Case	Control	Case	Control	Case	Control	p of HWE
Wang et al. (2016)	China	Asian	CL/P	NA	PCR-RFLP	—	—	147	129	147	129	0.01/0.68
Waltrick-Zambuzzi et al. (2015)	Brazil	Mixed	CLP, CLO, CPO	HB	PCR	359	440	—	—	342	401	0.86/0.97
Murthy et al. (2015)	India	Asian	CLP, CPO	NA	PCR-RFLP	—	—	142	141	142	141	0.25/—
Jin et al. (2015)	China	Asian	CL/P	HB	M-TMS	429	461	—	—	—	—	0.77
Bezerra et al. (2015)	Brazil	Mixed	CL/P	PB	PCR-RFLP	—	—	140	175	140	175	0.61/0.28
Aşlar and Hakkı (2014)	Turkey	Caucasian	CL/P	NA	PCR-RFLP	—	—	100	125	100	125	0.98/—
Yuan and Nan (2013)	China	Asian	CL/P, CLO	HB	PCR-RFLP	—	—	150	150	—	—	0.9
Hu et al. (2011)	China	Asian	CL/P	HB	PCR-RFLP	105	184	—	—	—	—	0.85
Wang and Nan (2011)	China	Asian	CLP, CLO, CPO	HB	PCR-RFLP	—	—	174	169	—	—	0.56
Mostowska et al. (2010)	Poland	Caucasian	CL/P	NA	PCR-RFLP	163	181	160	169	164	166	0.13/0.88/0.24
Brandalize et al. (2007)	Brazil	Mixed	CL/P, CPO	NA	PCR-RFLP	—	—	114	110	114	110	0.92/0.01
Martinelli et al. (2006)	Italy	Caucasian	CL/P	NA	PCR	218	289	—	—	—	—	0.62

CLO, cleft lip only; CL/P, cleft lip with or without palate; CLP, cleft lip and palate; CPO, cleft palate only; HB, hospital based; HWE, Hardy-Weinberg equilibrium; M-TMS, MALDI-TOF mass spectrometry; NA, not available; PB, population based; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

Table 2.

Newcastle-Ottawa Quality Assessment Scores for the Studies Included in the Meta-Analysis

Study	Selection	Comparability	Exposure	Score
Wang et al. (2016)	⋆⋆	⋆	⋆⋆⋆	6
Waltrick-Zambuzzi et al. (2015)	⋆⋆⋆	⋆⋆	⋆⋆	7
Murthy et al. (2015)	⋆⋆⋆	⋆	⋆⋆	6
Jin et al. (2015)	⋆	⋆	⋆⋆⋆	5
Bezerra et al. (2015)	⋆⋆⋆⋆	⋆⋆	⋆⋆	8
Aşlar and Hakkı (2014)	⋆⋆⋆	⋆	⋆⋆⋆	7
Mostowska et al. (2010)	⋆⋆⋆	⋆	⋆⋆⋆	7
Brandalize et al. (2007)	⋆⋆⋆	⋆⋆	⋆⋆⋆	8
Martinelli et al. (2006)	⋆⋆⋆	⋆	⋆⋆	6
Hu et al. (2011)	⋆⋆	⋆	⋆⋆	5
Yuan and Nan (2013)	⋆⋆⋆	⋆	⋆⋆⋆	7
Wang and Nan (2011)	⋆⋆⋆	⋆	⋆⋆⋆	7

Quantitative data synthesis

Eight studies including 1127 patients and 1168 controls were identified to evaluate the association between the MTR A2756G polymorphism and NSCL/P risk. In the overall comparison, there was no statistically significant evidence of an association of this SNP with NSCL/P risk in any of the genetic models (G vs. A: OR = 0.95, 95% CI = 0.82-1.11, I² = 47%, p = 0.55; Fig. 2); the other pooled results are shown in Table 3. Similarly, in subgroup analyses based on ethnicity, type of NSCL/P, and HWE of the control group, the MTR A2756G polymorphism was not significantly associated with the risk of NSCL/P (Table 3).

FIG. 2.

Forest plot of association between the MTR A2756G polymorphism and NSCL/P risk in allelic model (G vs. A). NSCL/P, nonsyndromic cleft lip with or without cleft palate.

Table 3.

Overall and Subgroup Results of the Association Between the MTR A2756G Polymorphism and NSCL/P Risk

		G vs. A			AG/GG vs. AA			GG vs. AA/AG			GG vs. AA			AG vs. AA
	Studies (n)	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	p	I²
Total	8	0.95 (0.82-1.11)	0.55	47%	0.92 (0.68-1.25)	0.6	64%	1.04 (0.63-1.72)	0.87	0%	1.01 (0.61-1.68)	0.96	0%	0.91 (0.65 − 1.27)	0.57	69%
Ethnicity
Asian	4	0.96 (0.77-1.21)	0.76	49%	0.98 (0.65-1.49)	0.93	60%	0.66 (0.24-1.82)	0.42	0%	0.72 (0.26-1.98)	0.52	0%	1.00 (0.66-1.51)	1	59%
Caucasian	3	0.93 (0.73-1.19)	0.55	73%	0.84 (0.40-1.75)	0.64	83%	1.09 (0.58-2.06)	0.79	0%	1.01 (0.53-1.93)	0.97	0%	0.79 (0.33-1.90)	0.6	86%
Mixed	1	0.99 (0.65-1.51)	—	—	0.90 (0.56-1.47)	—	—	2.12 (0.50-9.04)	—	—	2.02 (0.47-8.66)	—	—	0.84 (0.51-1.39)	0.53	0%
Type of disease
CLO	2	0.82 (0.47-1.44)	0.49	0%	0.85 (0.47-1.52)	0.58	0%	0.43 (0.02-9.15)	0.59	—	0.43 (0.02-9-18)	0.59	—	0.89 (0.49-1.59)	0.68	0%
CPO	2	1.10 (0.63-1.91)	0.74	0%	1.11 (0.59-2.12)	0.74	0%	1.99 (0.36-11.05)	0.43	33%	2.41 (0.40-14.42)	0.33	13%	1.08 (0.56-2.07)	0.83	42%
CLP	2	1.28 (0.65-2.51)	0.47	54%	1.37 (0.84-2.22)	0.20	48%	0.36 (0.02-7.59)	0.51	—	0.36 (0.02-7.67)	0.51	—	1.43 (0.88-2.32)	0.15	34%
HWE
HWE (yes)	7	1.01 (0.86-1.20)	0.88	36%	0.99 (0.72-1.35)	0.95	60%	1.09 (0.65-1.80)	0.75	0%	1.06 (0.63-1.77)	0.83	0%	0.97 (0.68-1.39)	0.87	67%

OR, odds ratio; 95% CI, 95% confidence interval; p, p-value for test of association; I², a measure of heterogeneity expressed in %; NSCL/P, nonsyndromic cleft lip with or without cleft palate.

Seven studies of 1149 patients with NSCL/P and 1247 controls were included to assess the association between the MTRR A66G polymorphism and NSCL/P risk. Overall, we failed to observe any significant association of this SNP with NSCL/P risk using five genetic models (G vs. A: OR = 0.99, 95% CI = 0.82-1.18, I² = 52%, p = 0.72; Fig. 3); the pooled results from the remaining four genetic models are shown in Table 4. Additionally, the results of the stratified analysis based on ethnicity, type of NSCL/P, and HWE of the control group did not reveal any association of this SNP with NSCL/P risk (Table 4). Statistical heterogeneity was detected in the overall comparison using three genetic models (allele contrast model: I² = 52%; dominant model: I² = 80%; heterozygous genetic model: I² = 86%; Table 4); however, this heterogeneity was eliminated after excluding the study by Wang et al. (2016), which indicates that the heterogeneity originated from this study.

FIG. 3.

Forest plot of association between the MTRR A66G polymorphism and NSCL/P risk in allelic model (G vs. A).

Table 4.

Overall and Subgroup Results of the Association Between the MTRR A66G Polymorphism and NSCL/P Risk

		G vs. A			AG/GG vs. AA			GG vs. AA/AG			GG vs. AA			AG vs. AA
	Studies (n)	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	P	I²
Total	7	0.99 (0.82-1.18)	0.72	52%	0.92 (0.60-1.40)	0.68	80%	1.01 (0.79-1.27)	0.97	49%	0.94 (0.71-1.25)	0.68	40%	0.85 (0.50-1.44)	0.55	86%
Ethnicity
Asian	2	0.93 (0.42-2.03)	0.85	90%	0.75 (0.13-4.15)	0.74	96%	1.11 (0.67-1.83)	0.69	—	0.50 (0.27-0.91)	0.02	—	0.57 (0.06-5.42)	0.63	97%
Caucasian	3	1.02 (0.83-1.25)	0.84	0%	1.00 (0.70-1.43)	0.99	0%	1.07 (0.74-1.57)	0.71	74%	1.17 (0.72-1.91)	0.53	9%	1.01 (0.70-1.46)	0.95	0%
Mixed	2	1.03 (0.86-1.24)	0.55	83%	1.04 (0.60-1.79)	0.89	73%	0.89 (0.62-1.30)	0.56	32%	1.11 (0.73-1.69)	0.63	0%	1.03 (0.51-2.07)	0.93	82%
Type of disease
CPO	2	1.19 (0.83-1.71)	0.34	37%	1.75 (0.71-4.35)	0.22	51%	0.85 (0.38-1.89)	0.69	—	1.01 (0.41-2.49)	0.99	—	1.69 (0.96-2.98)	0.07	42%
HWE
HWE (yes)	4	0.87 (0.69-1.11)	0.28	60%	0.78 (0.42-1.46)	0.44	87%	0.90 (0.70-1.16)	0.43	0%	0.87 (0.64-1.17)	0.34	42%	0.71 (0.31-1.64)	0.42	91%

p, p-value for test of association; I², a measure of heterogeneity expressed in %.

We identified five studies including 1274 patients with NSCL/P and 1555 controls to investigate the association between the TCN2 C776G polymorphism and NSCL/P risk. We did not observe an obvious association of this SNP with NSCL/P risk using the five genetic models (G vs. C: OR = 0.95, 95% CI = 0.86-1.06, I² = 36%, p = 0.37; Fig. 4); the pooled results from the remaining four genetic models are shown in Table 5. In addition, the outcome of the stratified analysis based on ethnicity clearly showed that this SNP was not significantly associated with NSCL/P risk (Table 5).

FIG. 4.

Forest plot of association between the TCN2 C776G polymorphism and NSCL/P risk in allelic model (G vs. C).

Table 5.

Overall and Subgroup Results of the Association Between the TCN2 C776G Polymorphism and NSCL/P Risk

		G vs. C			CG/GG vs. CC			GG vs. CC/CG			GG vs. CC			CG vs. CC
	Studies (n)	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	p	I²	OR (95% CI)	p	I²
Total	5	0.95 (0.86-1.06)	0.37	36%	0.93 (0.78-1.10)	0.39	0%	0.95 (0.79-1.13)	0.55	29%	0.91 (0.73-1.14)	0.42	44%	0.93 (0.78-1.12)	0.46	0%
Ethnicity
Asian	2	0.96 (0.81-1.13)	0.59	0%	0.95 (0.70-1.29)	0.74	0%	0.93 (0.73-1.20)	0.59	0%	0.91 (0.65-1.29)	0.6	0%	0.97 (0.71-1.34)	0.87	0%
Caucasian	2	0.83 (0.68-1.01)	0.06	56%	0.77 (0.58-1.03)	0.08	0%	0.76 (0.52-1.11)	0.16	64%	0.66 (0.43-1.02)	0.06	64%	0.81 (0.60-1.10)	0.18	0%
Mixed	1	1.10 (0.90-1.35)	0.35	—	1.09 (0.82-1.44)	0.57	—	1.22 (0.83-1.80)	—	—	1.25 (0.82-1.89)	—	—	1.04 (0.76-1.40)	—	—

p, p-value for test of association; I², a measure of heterogeneity expressed in %.

Sensitivity analysis

We performed sensitivity analyses to detect the impact of each study on the pooled estimates by sequentially omitting the eligible studies. The significance of the pooled ORs was not affected by excluding any eligible article. Therefore, we obtained stable and credible results from the present meta-analysis (Supplementary Figs. S1-S3; Supplementary Data are available online at www-liebertpub-com.web.bisu.edu.cn/gtmb).

Publication bias

In our study, Begg's funnel plots suggested symmetric distributions (Supplementary Figs. S4-S6), and we did not detect notable publication bias for these three SNPs in any genetic model (Table 6), with one exception: slight publication bias was observed for MTRR A66G in the recessive effect model (Egger's test, p = 0.045). All p-values from Begg's test and Egger's test are shown in detail in Table 6.

Table 6.

The Results of Begg Test and Egger Test for Publication Bias

	Comparisons	Begg test p-value	Egger test p-value
TCN2 C776G	G vs. C	0.806	0.777
	CG/GG vs. CC	1.000	0.881
	GG vs. CC/CG	1.000	0.692
	GG vs. CC	0.806	0.672
	CG vs. CC	0.806	0.921
MTR A2756G	G vs. A	0.386	0.503
	AG/GG vs. AA	0174	0.446
	GG vs. AA/AG	0.386	0.319
	GG vs. AA	0.386	0.331
	AG vs. AA	0.386	0.313
MTRR A66G	G vs. A	0.548	0.769
	AG/GG vs. AA	0.548	0.362
	GG vs. AA/AG	0.133	0.045
	GG vs. AA	0.133	0.274
	AG vs. AA	0.072	0.187

Discussion

To the best of our knowledge, this meta-analysis represents the first comprehensive investigation of possible associations between the MTR/MTRR/TCN2 variants and NSCL/P risk. However, our results did not reveal significant associations of these three SNPs with NSCL/P risk. In addition, similar results were observed in stratified analyses based on the type of NSCL/P (CLO, CPO, or CLP), ethnicity, and the HWE of the control group. These results still should be interpreted with caution.

In the past few decades, the roles in NSCL/P epidemiology played by folate and genetic polymorphisms in genes encoding enzymes related to folate metabolism have attracted considerable attention (Badovinac et al., 2007; Zhao et al., 2014). As one of the most important enzymes in folate metabolism, MTR, encoded by the MTR gene, catalyses the methylation of homocysteine to methionine with the simultaneous conversion of 5-methylenetetrahydrofolate to 5, 10-methylenetetrahydrofolate (THF). The synthesis of S-adenosylmethionine requires THF and is necessary for nucleotide synthesis and methylation reactions (Platek et al., 2009). Functional regeneration of MTR requires the participation of another enzyme, MTRR, which is encoded by the MTRR gene (Leclerc et al., 1998; Wilson et al., 1999). In a previous meta-analysis, Cai et al. (2014) suggested that the MTRR A66G polymorphism, but not MTR A2756G, may be associated with an increase in congenital heart disease risk. However, a meta-analysis conducted by Zhang et al. (2013) showed no significant effect of the MTR A2756G or MTRR A66G polymorphism on the risk of neural tube defects. In the present meta-analysis, our results showed that the MTR A2756G and MTRR A66G polymorphisms are not associated with NSCL/P risk. Interestingly, in a study by Mostowska et al. (2010), where the MTR A2756G polymorphism was considered separately, no significant association with NSCL/P risk was detected. However, a gene-gene interaction analysis showed a significant epistatic interaction leading to NSCL/P susceptibility among the MTHFR (rs1801133), PEMT (rs4646406), and MTR (rs1805087) genotypes (Mostowska et al., 2006). In addition, a recent study reported that the interaction of the MTR and BHMT genes plays a vital role in the pathogenesis of NSCL/P in the Chinese population (Jin et al., 2015). Waltrick-Zambuzzi et al. (2015) found only a borderline association between the MTRR A66G gene polymorphism and the risk of CLP. Therefore, they hypothesized that the MTRR A66G polymorphism specifically plays a role in the CLP type of NSCL/P, in which it plays a minor role in the establishment of clefts. In addition, they found that smoking during pregnancy may be associated with MTRR A66G (Waltrick-Zambuzzi et al., 2015). Therefore, in future research, quantifying the effects of gene-gene and gene-environment interactions will facilitate a more accurate understanding of the roles of these genetic polymorphisms in the pathogenesis of NSCL/P.

Transcobalamin 2 is encoded by the TCN2 gene, and it transports vitamin B12 into cells. Specific membrane receptors recognize the protein portion of the transcobalamin 2-vitamin B12 complex, whereas other complexes or free vitamin B12 are not taken up by cells. Within cells, vitamin B12 is necessary for normal carbon metabolism (Martinelli et al., 2006). The C776G variant (rs1801198) in the transcobalamin gene (TCN2) decreases the cellular and plasma concentrations of transcobalamin and thereby influences the cellular availability of vitamin B12 (Gueant et al., 2007). Martinelli et al. (2006) provided the first evidence supporting the involvement of the TCN2 C776G polymorphism in NSCL/P, but subsequent studies concluded that the TCN2 C776G polymorphism was not associated with NSCL/P susceptibility (Mostowska et al., 2010; Jin et al., 2015; Waltrick-Zambuzzi et al., 2015). Similarly, our meta-analysis did not find a significant association between TCN2 and the risk of NSCL/P. Several factors may have contributed to this finding. First, the associations between gene mutations and NSCL/P susceptibility differ among populations (Mangold et al., 2011). Second, only five available articles on TCN2 were included, and the relatively small sample size may have limited the statistical power. Third, NSCL/P is a disease with multiple aetiologies, and the possible impact of gene-gene or gene-environment interactions should be considered.

Several limitations of our study should be noted when interpreting these findings. First, because of a lack of original information, we could not assess the impacts of gene-gene and gene-environment interactions, which may affect the risk of NSCL/P. Thus, the potential role of these genetic polymorphisms may be masked by other gene-gene/gene-environment interactions. Second, the total sample size was relatively small, although we performed a systematic search to screen for all related studies. Finally, despite the lack of obvious publication bias, as determined through Begg's funnel plots or Egger's test, we could not completely eliminate potential sources of bias because only published studies were examined.

In summary, the MTR A2756G, MTRR A66G, and TCN2 C776G polymorphisms might not be associated with NSCL/P risk. Considering the above-mentioned limitations, larger well-designed studies of multiple populations should be conducted to confirm our findings. In addition, in-depth research investigating the effects of gene-gene and gene-environment interactions might provide a more precise view of the associations between these three SNPs and NSCL/P risk.

Footnotes

Acknowledgments

We sincerely thank Professor Qin Liu from the Chongqing Medical University for providing assistance with the statistical analysis. This study was supported by grants from the Program for Innovation Team Building at Institutions of Higher Education in Chongqing in 2016 (no. CXTDG201602006) and the Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education.

Authors' Contributions

W.L. and Y.X. made equal contributions to the literature search, study selection, data collection, statistical analysis, and drafting of the manuscript. Y.W. and G.F. were involved in the study selection and data collection. A.R. conceived and designed the study and revised the manuscript.

Author Disclosure Statement

No competing financial interests exist.

References

Aşlar

, Hakkı

(2014) Prevalence of MTHFR, MTR and MTRR gene polymorphisms in Turkish patients with nonsyndromic cleft lip and palate. Gene Ther Mol Biol, 16:115-129.

Badovinac

, Werler

, Williams

, et al. (2007) Folic acid-containing supplement consumption during pregnancy and risk for oral clefts: a meta-analysis. Birth Defects Res A Clin Mol Teratol, 79:8-15.

Bezerra

, Oliveira

, Soares

, et al. (2015) Genetic and non-genetic factors that increase the risk of non-syndromic cleft lip and/or palate development. Oral Dis, 21:393-399.

Bhaskar

, Murthy

, Venkatesh Babu

(2011) Polymorphisms in genes involved in folate metabolism and orofacial clefts. Arch Oral Biol, 56:723-737.

Brandalize

, Bandinelli

, Borba

, et al. (2007) Polymorphisms in genes MTHFR, MTR and MTRR are not risk factors for cleft lip/palate in South Brazil. Braz J Med Biol Res, 40:787-791.

Cai

, Zhang

, Zhong

, et al. (2014) Genetic variant in MTRR, but not MTR, is associated with risk of congenital heart disease: an integrated meta-analysis. PLoS One, 9:e89609.

Carmel

(2002) Measuring and interpreting holo-transcobalamin (holo-transcobalamin II). Clin Chem, 48:407-409.

Chen

, Liu

, Hwang HY et al. (1997) Human methionine synthase. cDNA cloning, gene localization, and expression. J Biol Chem, 272:3628-3634.

Cobourne

(2004) The complex genetics of cleft lip and palate. Eur J Orthod, 26:7-16.

10.

de Arruda

, Persuhn

, de Oliveira

(2013) The MTHFR C677T polymorphism and global DNA methylation in oral epithelial cells. Genet Mol Biol, 36:490-493.

11.

Gueant

, Chabi

, Gueant-Rodriguez

, et al. (2007) Environmental influence on the worldwide prevalence of a 776C->G variant in the transcobalamin gene (TCN2). J Med Genet, 44:363-367.

12.

Gunnerbeck

, Edstedt Bonamy

, Wikstrom

, et al. (2014) Maternal snuff use and smoking and the risk of oral cleft malformations—a population-based cohort study. PLoS One, 9:e84715.

13.

Han

, Shen

, Meng

, et al. (2012) Methionine synthase reductase A66G polymorphism contributes to tumor susceptibility: evidence from 35 case-control studies. Mol Biol Rep, 39:805-816.

14.

Herrmann

, Schorr

, Obeid

, et al. (2003) Vitamin B-12 status, particularly holotranscobalamin II and methylmalonic acid concentrations, and hyperhomocysteinemia in vegetarians. Am J Clin Nutr, 78:131-136.

15.

, Chen

, et al. (2011) Association of TCN2 gene with non-syndromic cleft lip with or without cleft palate. Beijing J Stomatol, 19:4-7.

16.

Jia

, Shi

, Chen

, et al. (2011) Maternal malnutrition, environmental exposure during pregnancy and the risk of non-syndromic orofacial clefts. Oral Dis, 17:584-589.

17.

Jin

, Chen

, Hou

, et al. (2015) The Association between folate pathway genes and cleft lip with or without cleft palate in a Chinese population. Biomed Environ Sci, 28:136-139.

18.

Leclerc

, Campeau

, Goyette

, et al. (1996) Human methionine synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum Mol Genet, 5:1867-1874.

19.

Leclerc

, Wilson

, Dumas

, et al. (1998) Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc Natl Acad Sci U S A, 95:3059-3064.

20.

, Gulati

, Baker

, et al. (1996) Cloning, mapping and RNA analysis of the human methionine synthase gene. Hum Mol Genet, 5:1851-1858.

21.

Mangold

, Ludwig

, Nothen

(2011) Breakthroughs in the genetics of orofacial clefting. Trends Mol Med, 17:725-733.

22.

Martinelli

, Scapoli

, Palmieri

, et al. (2006) Study of four genes belonging to the folate pathway: transcobalamin 2 is involved in the onset of non-syndromic cleft lip with or without cleft palate. Hum Mutat, 27:294.

23.

Mossey

, Little

, Munger

, et al. (2009) Cleft lip and palate. Lancet, 374:1773-1785.

24.

Mossey

, Modell

(2012) Epidemiology of oral clefts 2012: an international perspective. Front Oral Biol, 16:1-18.

25.

Mostowska

, Hozyasz

, Jagodzinski

(2006) Maternal MTR genotype contributes to the risk of non-syndromic cleft lip and palate in the Polish population. Clin Genet, 69:512-517.

26.

Mostowska

, Hozyasz

, Wojcicki

, et al. (2010) Associations of folate and choline metabolism gene polymorphisms with orofacial clefts. J Med Genet, 47:809-815.

27.

Murthy

, Gurramkonda

, Lakkakula

BVKS

(2015) Genetic variant in MTRR A66G, but not MTR A2756G, is associated with risk of non-syndromic cleft lip and palate in Indian population. J Oral Maxillofac Surg Med Pathol, 27:782-785.

28.

Namour

, Olivier

, Abdelmouttaleb

, et al. (2001) Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood. Blood, 97:1092-1098.

29.

Olteanu

, Banerjee

(2001) Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. J Biol Chem, 276:35558-35563.

30.

Pan

, Zhang

, Ma

, et al. (2012) Infants' MTHFR polymorphisms and nonsyndromic orofacial clefts susceptibility: a meta-analysis based on 17 case-control studies. Am J Med Genet A, 158a:2162-2169.

31.

Platek

, Shields

, Marian

, et al. (2009) Alcohol consumption and genetic variation in methylenetetrahydrofolate reductase and 5-methyltetrahydrofolate-homocysteine methyltransferase in relation to breast cancer risk. Cancer Epidemiol Biomarkers Prev, 18:2453-2459.

32.

Prescott

, Malcolm

(2002) Folate and the face: evaluating the evidence for the influence of folate genes on craniofacial development. Cleft Palate Craniofac J, 39:327-331.

33.

Rouget

, Monfort

, Bahuau

, et al. (2005) Periconceptional folates and the prevention of orofacial clefts: role of dietary intakes in France. Revue d'epidemiologie et de sante publique, 53:351-360.

34.

Waltrick-Zambuzzi

, Tannure

, Vieira

, et al. (2015) Genetic variants in folate and cobalamin metabolism-related genes in nonsyndromic cleft lip and/or palate. Braz Dent J, 26:561-565.

35.

Wang

, Nan

(2011) Association between Rs1805087 polymorphisms of MTR gene and non-syndromic cleft lip with or without cleft palate. Beijing J Stomatol, 19:88-91.

36.

Wang

, Jiao

, Wang

, et al. (2016) MTR, MTRR, and MTHFR gene polymorphisms and susceptibility to nonsyndromic cleft lip with or without cleft palate. Genet Test Mol Biomarkers, 20:297-303.

37.

Wehby

, Goco

, Moretti-Ferreira

, et al. (2012) Oral cleft prevention program (OCPP). BMC Pediatr, 12:184.

38.

Wehby

, Murray

. (2010) Folic acid and orofacial clefts: a review of the evidence. Oral Dis, 16:11-19.

39.

Wilson

, Platt

, Wu

, et al. (1999) A common variant in methionine synthase reductase combined with low cobalamin (vitamin B12) increases risk for spina bifida. Mol Genet Metabol, 67:317-323.

40.

Wong

, Eskes

, Kuijpers-Jagtman

, et al. (1999) Nonsyndromic orofacial clefts: association with maternal hyperhomocysteinemia. Teratology, 60:253-257.

41.

, Fallin

, Shi

, et al. (2012) Evidence of gene-environment interaction for the RUNX2 gene and environmental tobacco smoke in controlling the risk of cleft lip with/without cleft palate. Birth Defects Res A Clin Mol Teratol, 94:76-83.

42.

Yuan

, Nan

(2013) Association between polymorphism of MTR rs1805087 locus and nonsyndromic cleft lip with or without cleft palate. J Oral Maxillofac Surg, 23:248-252.

43.

Yuan

, Blanton

, Hecht

(2011) Genetic causes of nonsyndromic cleft lip with or without cleft palate. Adv Otorhinolaryngol, 70:107-113.

44.

Zhang

, Lou

, Zhong

, et al. (2013) Genetic variants in the folate pathway and the risk of neural tube defects: a meta-analysis of the published literature. PLoS One, 8:e59570.

45.

Zhao

, Ren

, Shen

, et al. (2014) Association between MTHFR C677T and A1298C polymorphisms and NSCL/P risk in Asians: a meta-analysis. PLoS One, 9:e88242.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.75 MB