Methylenetetrahydrofolate Reductase Genetic Polymorphisms and Esophageal Squamous Cell Carcinoma Susceptibility : A Meta-analysis of Case-control Studies

Esophageal squamous cell and adenocarcinoma are common malignancies worldwide (Jemal et al., 2007), which is the sixth most commonly occurring cancer and sixth most common cause of cancer-realted death in the world (Jemal et al., 2007). The five-year survival rate for all stages combined was 15.6% from 1996 to 2003, which was much lower than most of other cancer types (ACS, 2008). Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancer in China, and it is estimated 250,000 cases were diagnosed yearly. Possible risk factors for ESCC include cigarette smoking, alcohol drinking, hot-temperature food, low intake of vegetable, salted food, pickled vegetables, chronic mucosal irritation and a family history of cancer (Wang et al., 2007; Falk, 2009; De et al., 2009; Morita et al., 2010; Yu et al., 2010). Deficiency of nutrients, such as vitamins and microelements, was suggested to be associated with an increased risk for ESCC (Yu et al., 2010). Folate is a water-soluble vitamin and naturally found in green leafy vegetables, cereals, legumes and fruits (Aune et al., 2011). Deficiency of folate could induce defective DNA repair and chromosomal fragile site expression, leading to chromosomal breaks and micronucleus


Introduction
Esophageal squamous cell and adenocarcinoma are common malignancies worldwide (Jemal et al., 2007), which is the sixth most commonly occurring cancer and sixth most common cause of cancer-realted death in the world (Jemal et al., 2007).The five-year survival rate for all stages combined was 15.6% from 1996 to 2003, which was much lower than most of other cancer types (ACS, 2008).Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancer in China, and it is estimated 250,000 cases were diagnosed yearly.Possible risk factors for ESCC include cigarette smoking, alcohol drinking, hot-temperature food, low intake of vegetable, salted food, pickled vegetables, chronic mucosal irritation and a family history of cancer (Wang et al., 2007;Falk, 2009;De et al., 2009;Morita et al., 2010;Yu et al., 2010).Deficiency of nutrients, such as vitamins and microelements, was suggested to be associated with an increased risk for ESCC (Yu et al., 2010).
Folate is a water-soluble vitamin and naturally found in green leafy vegetables, cereals, legumes and fruits (Aune et al., 2011).Deficiency of folate could induce defective DNA repair and chromosomal fragile site expression, leading to chromosomal breaks and micronucleus

Methylenetetrahydrofolate Reductase Genetic Polymorphisms and Esophageal Squamous Cell Carcinoma Susceptibility: A Meta-analysis of Case-control Studies
Yuan-Yuan Wen & , Shu-Juan Yang & *, Jian-Xing Zhang & , Xin-Yue Chen formation (Aune et al., 2011).Methylenetetrahydrofolate reductase (MTHFR) C677T in the gene encoding the MTHFR enzyme, which converts dietary folate to its active cofactor in Hcy catabolism, has been studies as candidate genetic risk factor for esophageal cancer (Song et al., 2001).As T allele dose increases, this functional polymorphism causes a graded elevation in individuals with low dietary folate consumption (Frosst et al., 1995).Therefore, several previous studies have investigated the association of MTHFR C677T and folate intake with esophageal cancer risk, but the results are conflicting (Song et al., 2001;Stolzenberg-Solomon et al., 2003;Yang et al., 2005).The variation of these results might be induced by difference in ethnicities, sample size, study design and background of patients as well as random error.Therefore, we conducted a systematic review to investigate the association of MTHFR C677T and folate intake with esophageal cancer risk by reducing random error and obtaining precise estimates for some potential genetic associations (Egger et al., 1997).

Searching strategy
We searched MEDLINE (from Jan. 1966

Data extraction
Two reviewers independently evaluated the retrieved articles, and the disagreements were resolved by discussion.Data retrieved from selected articles included In instances where the data were insufficient or missing, we attempted to contact the authors of the articles in order to request the relevant data.From those studies finally selected, we extracted the following data: first author's name, year of publication, country of origin, numbers of cases and controls, genotype frequencies of MTHFR C677T.

Quality score assessment
The quality of studies was evaluated by predefined scale in previous studies (Jiang et al., 2010) (Table 1).
The quality score assessment criteria were evaluated by traditional epidemiological considerations and cancer genetic issues.The quality scores ranged from 0 to 15. Score<10 was defined as low quality, and score≥10 was defined as high quality.

Statistical analysis
Statistical analysis was conducted by using STATA statistical package (version 9, STATA, College Station, TX).The distributions of genotypes in controls were tested by Hardy-Weinberg equilibrium (HWE) using the Chi-square test.The association of polymorphisms   Funnel plot with pseudo 95% confidence limits of MTHFR C677T and folate intake and ESCC risk was estimated by Odds ratio (ORs) with 95% confidence intervals (CIs).The heterogeneity was tested by the Q-statistics with p-values < 0.1, and its possible sources of heterogeneity were assessed by subgroup analysis.If there was heterogeneity, the random effect model would be used.Otherwise, a fixed-effect model was applied to obtain the summary OR and their 95% CI.One-way sensitivity analysis was performed to explore robustness of the results.All P values were two-sided and a P value of less than 0.05 was deemed statistically significant.

Characteristics of studies
47 studies were initially identified after search, and 28 studies were excluded due to overlapping data and being without meeting the criteria.Finally, 19 studies (4239 cases and 5575 controls) were included for metaanalysis.The detaied characteristics of these studies are summarized in Table 2.Only two studies had high quality score, and the scores of other studies ranged from 7 to 10.Of the 19 case-control studies, 14 studies were conducted in China.
Subgroup analysis was taken according to folate intake, which indicated low intake of folate had significantly higher risk of esophageal cancer among individuals with CT/TT genotype [OR(95%)=1.65(1.1-2.49)](Table 3).However, high intake of folate did not find significant high risk of esophageal cancer among individuals with CT/TT genotype [OR(95%)=1.64 (0.82-3.26)].No significant heterogeneity was found between studies (P>0.05).These results indicated folate had a significant interaction with MTHFR C677T.
A single study in this meta-analysis was deleted each time to reflect the impact of the individual data on the pooled ORs, and most of the results did not altered (Data not shown).Funnel plot an Egger's test were used to assess the publication bias, and it provided evidence that there was no publication bias among studies regarding MTHFR 677 CT, but a significant publication bias was found in studies regarding MTHFR 677 TT genotype (P<0.05).The shape A of funnel plots was asymmetrical (Figure 1 and Figure 2).

Discussion
Although many epidemiologic studies investigated the role of folate intake and MTHFR C677T for EC risk provided inconsistent results.Most of those studies involved few cases, and these few sample size limited the genetic effect reliably.Our meta-analysis recognized as an important tool to more precisely define the effect  of selected genetic polymorphisms on risk of disease and to identify the potentially important sources of betweenstudy heterogeneity.A previous meta-analysis in Asian population included 13 case-control studies indicated MTHFR 677 CT and TT genotypes were significantly association with increased risk of esophageal cancer, especially in drinkers and smokers (Fang et al., 2011).However, this study did not explore the interaction between folate intake and MTHFR genotype.Therefore, we conducted an updated meta-analysis by critically reviewing 19 individual case-control studies on MTHFR C677T and folate intake with esophageal cancer risk.Compared with the last meta-analysis conducted in China by Fang et al, this updated meta-analysis included another 6 new case-control studies, and we explore the interaction between folate intake and MTHFR C677T.Our study indicated high intake of folate was a protective factor for esophageal cancer, and folate showed a significant interaction with polymorphism of MTHFR C677T.
Heterogeneity is a potential problems in the metaanalysis, and eliminating heterogeneity is an important role during meta-analysis (Higgins et al., 2003).In our study, we found there was significant heterogeneity between studies by using Q-statistics.However, after stratifying by the quantity of folate intake suggested folate was an important source of heterogeneity.
Previous studies indicated folate mediates the transfer of one-carbon moieties both in the synthesis of nucleotides necessary for DNA synthesis, replication, and repair and in DNA methylation reactions (Wang et al., 2008).These functions may play a critical role in carcinogenesis, and previous epidemiological studies indicated an abundant intake of food stuffs full of folate could protect the development of various cancers (Mason et al., 2009).Ours study indicated the folate intake was associated with a decreased risk of esophageal cancer, which proved previous hypothesis.Moreover, the activity of folate metabolic enzyme, such as MTHFR, are involved in the folate metabolic and DNA methylation process.As a key enzyme in folate metabolism, the product of MTHFR serves as the carbon donor for the methylation of homocysteine tomethionine, which is catalyzed by the enzyme MTR (Sabia et al., 2006).The MTHFR gene is high polymorphic in the general population, the mutation of most common functional variant of 677C to T. This polymorphism results in an alanine to valine substitution, leading to a reduction in enzyme activity (Langevin et al., 2009).The role of MTHFR in the folate metabolism decided the interaction between folate and polymorphisms of MTHFR, which was proved by our meta-analysis.Our study showed the MTHFR had strong risk of esophageal cancer in individuals with low intake of folate intake.
Possible limitations of this meta-analysis have to be considered in explaining our results.Firstly, most of the studies are conducted in China, and this could limit the power to find the difference in genotypes by different ethnicities.Secondly, publication bias may have occurred due to only published papers which included in the metaanalysis.Thirdly, there might be misclassification during our study.Some controls in our study were selected from non-cancer inpatients, and some were selected from residents.Finally, there might be gene-environment interaction for esophageal cancer, however, we did not perform subgroup analysis due to lack of data on environmental factors.Further studies are warranted to interpreted this interaction.
In conclusion, our meta-analysis indicated the folate intake and MTHFR 677CT/TT are associated with the risk of ESCC, and folate showed a significant interaction with polymorphism of MTHFR C677T.

Figure
Figure 1.Publication Bias on Studies of MTHFR 677CT vs CC