Genetic Polymorphism of MTHFR A 1298 C and Esophageal Cancer Susceptibility : A Meta-analysis

In all human cancers, esophageal cancer (EC), with the fiveyear survival rate less than 20%, is regarded as one of the most common lethal malignancies worldwide. Because of highly incidence and mortality, even in Southern and Eastern Africa and Eastern Asia, EC has been listed as eighth and sixth in all cancers respectively in the world (Jemal et al., 2011). Squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) are the main histological types, In the “esophageal cancer belt” of the ESCC even has reached 90% (Wheeler et al., 2012). It is a multi-factor and multistep process to develop EC, such as poor nutritional status, drinking of alcohol, smoking, ethnic group, hot beverage and high-temperature cooking methods, serious shortage of vegetable and fruit intake (Yu et al., 1988; Farrow et al., 2000; Lin et al., 2011). However, not all exposed factors develop EC, it may be shown that genetic factors play an important role for developing EC. Methylenetetrahydrofolate reductase (MTHFR) located in 1p36.3, acting as a critical enzyme, which catalyzed reduction of 5, 10-methylene-tetrahydrofolate to 5-methyltetra hydrofolate, as a methyl donor for the remethylation of homocysteine to methionine. MTHFR


Introduction
In all human cancers, esophageal cancer (EC), with the five-year survival rate less than 20%, is regarded as one of the most common lethal malignancies worldwide.Because of highly incidence and mortality, even in Southern and Eastern Africa and Eastern Asia, EC has been listed as eighth and sixth in all cancers respectively in the world (Jemal et al., 2011).Squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) are the main histological types, In the "esophageal cancer belt" of the ESCC even has reached 90% (Wheeler et al., 2012).It is a multi-factor and multistep process to develop EC, such as poor nutritional status, drinking of alcohol, smoking, ethnic group, hot beverage and high-temperature cooking methods, serious shortage of vegetable and fruit intake (Yu et al., 1988;Farrow et al., 2000;Lin et al., 2011).However, not all exposed factors develop EC, it may be shown that genetic factors play an important role for developing EC.

Genetic Polymorphism of MTHFR A1298C and Esophageal Cancer Susceptibility: A Meta-analysis
Xiang Tan, Yong-Yong Wang, Lei Dai, Xu-Qiang Liao, Ming-Wu Chen* Glu 429 Ala (AgC)variants have been identified in protein coding sequences.The MTHFR mutation led to 5 -methyltetrahydrofolate reduction and homocysteine accumulation in the blood, Which made the methyl donor of the methionine dyssynthesis, eventually caused hypomethylation of DNA and decreased the activity of the enzyme, leading to increase the cancer susceptibility (Chen et al., 1996;Ekiz et al., 2012).Besides, it has been suggested that a significant function of folate is to provide the required methyl groups for intracellular methylation reaction and denovo deoxynucleotide triphosphate synthesis, folate deficienc might cause carcinogenic by disruption of DNA methylation (Umar et al., 2010).
Given know the Enzyme activity of the MTHFR A1298C, individuals mutational homozygous compared for the common, had been reduced approximately60% of enzyme activity (Ekiz et al., 2012).And the variations of MTHFR A1298C, it has been shown that these polymorphisms may strongly affect cancer risk, such as lung cancer, colorectal cancer, gastric cancer, breast cancer (Dong et al., 2008;Li et al., 2011;de et al., 2012;Zhang et al., 2012).However, recently a meta-analysis has also suggested that MTHFR A1298C polymorphism might correlate with ESCC by alcohol status (Fang et al., 2011), but the results were conflicting and inconclusive.Therefore, a meta-analysis was conducted to investigate the relationship between both A1298C polymorphism and susceptibility to EC.

Data extraction
Two investigators (X.Tan and Y.Y.Wang) were carefully to extract data independently from the above selection criteria.Disagreement must be discussed and decided by third investigators (M.W.Chen).The data items included: First author, publication date, country,race, source of controls, genotyping method, and different genotype numbers in all studies.

Statistical analysis
The association between MTHFR A1298C polymorphism and EC risk was calculated by ORs with 95% CIs.In order to avoid the use of a specific genetic model and lead to the outcome bias, at least three possible genotypes was performed comparing in the meta-analysis.We estimated the OR of a cancer associated with Codominant model (CCversusAA, CA versusAA), Dominant model (CC+CAversus AA)and Recessive model (CC versus CA+AA), respectively.
Chi-square based Q statistic test and p value was used to assess the between-study heterogeneity (Higgins et al., 2003).The studies heterogeneity did not exist when P -value > 0.1and I 2 < 25%.If there was no heterogeneity, The fixed-effects model was performed using the Mantel-Haenszel's method (Mantel et al., 1959).The heterogeneity was considered to be of statistical significance when I 2 > 50% or P -value <0.1, sensitivity analysis and subgroup analysis were used to explore the source of the heterogeneity , so the random effects model was performed using DerSimonian and Laird method when the heterogeneity still existed (Lau et al., 1997).
Begg's funnel plot was test for Potential publication bias,and funnel plot unweighted was by applied regression test (Peters et al., 2006).Due to different geographic, ethnic, and pathological type of esophageal cancer, in order to assess the effects of covariance, we performed subgroup analyses by ethnic subgroup (Caucasian and Asian) and pathological type subgroup (ESCC and EAC).all analyses were carried out by the Stata software version 11.1 (Stata Corporation.USA).All of P values were two-sided, less than 0.05 was considered statistically significant.

Meta-analysis results
The main results of the meta-analysis on the association between MTHFR A1298C polymorphism and EC risk was shown in Table 2.When all studies were pooled, we performed analysis using fixed-effects models if the Q -test of heterogeneity was considered no significant, otherwise, using random-effects models.In the A1298C polymorphism, we found that a stronger risk for EC (CCversus AA, OR=1.951, 95%CI=1.475-2.581,P=0.000; Dominant model CC+CA versus AA, OR=1.790, 95%CI=1,312-2.444,P=0.000; Recessive model CCversusCA+AA, OR=1.843, 95%CI=1.414-2.402,P=0.000) (Figure 1).

Test for heterogeneity, sensitive analysis and test for publication bias
When we overall pooled analysis the heterogeneity was existence.To explore the sources of heterogeneity, ethnicity and histological type were performed by subgroup analyses.No heterogeneity was reduced.Sensitivity analysis was performed in our meta-analysis.When we omitted every study at each time, the results of reanalyse for A1298C polymorphism was stabily, which suggested that our meta-analysis results was reliable (data not show).
Begg's funnel plot and the Egger's test were conducted to estimate the publication bias of articles.Both the results of Begg's and Egger's test did not show any evidence of publication bias (CC vs et al., 2010;Ekiz et al., 2012.Our meta-analysis included six studies with a total of 1302 cases and 2391controls have investigated the A1298C polymorphism with EC.Our meta-analysis provided evidence that the A1298C polymorphism might play an important role in EC.No publication bias was found by the funnel plots which proved our meta-analysis conclusion more credible. When we performed the subgroup analyses by ethnicity, significant with susceptibility for the development of EC among Asian and Caucasian population.There might be some reasons could be explained that.First, the relationship between genes and genes might be susceptible in different ethnicity.In addition, gene-environmental interaction might play an important role in susceptibility to EC.Each people stays with different lifestyle and environment.Inadequate folate intake might be also susceptible to EC (Zhao et al., 2011).Besides, it also seems to show association of polymorphism A1298C with increased risk of ESCC and EAC.And the exact mechanism for the incidence of ESCC and EAC is unclear.At last, it might be low sample size or some other potentially suspected factors such as smoking status, alcohol consumption, occupational and lifestyle to influence our research.There were only two studies for Caucasian population and two studies for EAC, so further study with large samples must to be performed.
Despite we have made great efforts to collect all possible data for investigating between MTHFR A1298C polymorphism and EC, but several potential limitations inherent in our meta-analysis should be known.First, present research associations between MTHFR A1298C polymorphism and EC risk was still fewer, there were only six studies with full-text articles relation of MTHFR A1298C polymorphism with EC, so the sample of participants included in our meta-analysis was comparatively small.results should be interpreted with caution.Second, it is well-known that publication bias is a problem for interpretating the results in metaanalysis.Although Begg's and Egger's test did not show any evidence of publication bias in our research, we only collected full-text articles in our research, excluded abstract and unpublished articles, and positive results may be easily accepted by journals, potential publication bias may occur.Besides, all of the studied objects may be not matched for the age, race and smoking status, living habits and environment was also different.As stated above, it might be sources of heterogeneity.At last, the gene-gene and gene-environmental interactions we did not make further research, there may be some predisposing factors effect on esophageal cancer.
Although these limitations, our meta-analysisof the association of MTHFR A1298C polymorphism with Esophageal Cancer risk is significantly more powerful than any single study.In all, our research suggeste that MTHFR A1298C polymorphism may increase risk for Esophageal Cancer in Asians and Caucasians.In addition, we also found that MTHFR A1298C polymorphism might be risk for ESCC and EAC.Folate intake, gene-gene and gene-environmental interactions on Esophageal Cancer risk, owing most data were insufficient, therefore, further studies with more comprehensive, larger sample, well-designed and high quality case-control studies are required to investigate the associated with MTHFR and Esophageal Cancer.

Figure 1 .
Figure 1.The Forest Plot Describing the Meta-analysis with a Fix-effect Model (CCvs.AA) for the Association of MTHFR A1298C Polymorphism and Esophageal Cancer Risk in Overall Studies.Each study is depicted with size inversely proportional to its variance, accompanied by the respective 95% confidence intervals

Figure 2 .
Figure 2. The Forest Plot Describing the Meta-analysis with a Fix-effect Recessive Model (CCvs.CA+AA) for the Association of MTHFR A1298C Polymorphism and Esophageal Cancer in Ethnicity.Each Study is Depicted with size inversely proportional to its variance, accompanied by the respective 95% confidence intervals

Table 2 . Main Results of the Meta-analysis MTHFRA1298Cpolymorphism Relation with Esophageal Cancer
n, number of studies in each analysis; Dominant model, CC+CA vs. AA; Recessive model, CC vs. CA+AA; OR, odds ratio; CI, confidence interval; a the pool p value; b p value for heterogeneity test; c esophageal squamous cell carcinoma; d esophageal adenocarcinoma