Evaluation of the MTHFR C677T Polymorphism as a Risk Factor for Colorectal Cancer in Asian Populations

Background: Genetic and environmental factors play important roles in pathogenesis of digestive tract cancers like those in the esophagus, stomach and colorectum. Folate deficiency and methylenetetrahydrofolate reductase (MTHFR) as an important enzyme of folate and methionine metabolism are considered crucial for DNA synthesis and methylation. MTHFR variants may cause genomic hypomethylation, which may lead to the development of cancer, and MTHFR gene polymorphisms (especially C677T and A1298C) are known to influence predispositions for cancer development. Several case control association studies of MTHFR C677T polymorphisms and colorectal cancer (CRC) have been reported in different populations with contrasting results, possibly reflecting inadequate statistical power. Aim: The present meta-analysis was conducted to investigate the association between the C677T polymorphism and the risk of colorectal cancer. Materials and Methods: A literature search of the PubMed, Google Scholar, Springer link and Elsevier databases was carried out for potential relevant articles. Pooled odds ratio (OR) with corresponding 95 % confidence interval (95 % CI) was calculated to assess the association of MTHFR C677T with the susceptibility to CRC. Cochran’s Q statistic and the inconsistency index (I 2 ) were used to check study heterogeneity. Egger’s test and funnel plots were applied to assess publication bias. All statistical analyses were conducted by with MetaAnalyst and MIX version 1.7. Results: Thirty four case-control studies involving a total of 9,143 cases and 11,357 controls were retrieved according to the inclusion criteria. Overall, no significant association was found between the MTHFR C677T polymorphism and colorectal cancer in Asian populations (for T vs. C: OR=1.03; 95% CI= 0.92-1.5; p= 0.64; for TT vs CC: OR=0.88; 95%CI= 0.74-1.04; p= 0.04; for CT vs. CC: OR = 1.02; 95%CI= 0.93-1.12; p=0.59; for TT+ CT vs. CC: OR=1.07; 95%CI= 0.94-1.22; p=0.87). Conclusions: Evidence from the current meta-analysis indicated that the C677T polymorphism is not associated with CRC risk in Asian populations. Further investigations are needed to offer better insight into any role of this polymorphism in colorectal carcinogenesis.


Introduction
Colorectal cancer (CRC) is the second most common cancer worldwide and 60% cases occur in developed regions. The incidence varies considerably among different ethnicities (Parkin et al., 1999;Ferlay et al., 2008;Center et al., 2009;Jemal et al., 2009;Yousef et al., 2014). Although incidence rates are lower in Asian than Caucasian populations (Jang et al., 2013). Fifty percentage of the people diagnosed with colorectal cancer will die of the disease (Boyle and Ferlay, 2005;Li et al., 2011). CRC is a multifactorial disease with involvement of both genetic and environmental factors. Epidemiological studies have indicated that diets plays an important role in colorectal malignancy, high intake of alcohol and red meat and low intake of folate i.e green vegetables, fruits and dietary fibres are increased the risk of CRC (Russo et al., 1998;Bedine, 1999;Boutron- Ruault et al., 2001;Giovannucci, 2001;Giovannucci et al., 1995). High folate intake has protective effect against CRC (Giovannucci et al., 1995;Wang et al., 2006). Folate metabolism is regulated by several enzymes and out of which methylenetetrahydrofolate reductase (MTHFR) is the critical enzyme and genetic polymorphism of MTHFR enzyme is considered as a potential marker that may influence the risk of CRC.

Vandana Rai
MTHFR enzyme is involved in DNA synthesis, repair and methylation (Sameer et al., 2011). It is responsible for determining whether reduced folates are directed towards DNA methylation pathways or DNA synthesis (Ryan and Weir, 2001). The MTHFR locus has been mapped to chromosome 1p36.3 in humans (Goyette et al., 1994). The most well-studied polymorphism in MTHFR gene is the replacement of the nucleotide thymine by cytosine at position 677 (Ala222Val) (Frosst et al., 1995). This variant, which is relatively common in many populations worldwide, produces a form of methylenetetrahydrofolate reductase that reduces activity at higher temperatures, leading to lower levels of circulating folate (5-methyl-THF), an accumulation of 5, 10-methylene-THF, and increased plasma homocystein levels (Ma et al., 1997;Yan et al., 2012). The frequency of T allele varies in different ethnic population. According to HapMap database, the highest frequency i.e 0.48 is reported from Chinese Han population and T allele frequency in Europeans, Africans, Japanese and Indians is reported as 0.31, 0.08 to 0.12, 0.38 and 0.16 respectively (http:// hapmap.ncbi.nlm.nih.gov). Previous studies have suggested that T allele frequency can be higher in regions where food and vitamins are sufficient, as higher homocysteine and low-folate levels are compensated (Kennedy et al., 2012). MTHFR C677T had been suggested as a possible risk factor for congenital anomalies, heart disease, stroke, diabetes, psychiatric disorders and certain types of cancer (Fu et al., 2013;Rai et al., 2014;Rai 2014a, b).
DNA strand breaks, impaired DNA methylation and repair have been associated with folate deficiency and CRC (Duthie, 1999;Ames, 2001;Fenech, 2001;Kim, 2003). Several studies from around the globe have indicated that the common polymorphisms in the MTHFR gene might play a critical role in increasing susceptibility to CRC (Park et al., 1999;Yin et al., 2004). However, some other studies suggested that the MTHFR C677T polymorphisms were not associated with susceptibility to CRC (Chen et al., 1996;Ma et al., 1997;Slattery et al., 1999;Sachse et al., 2002;Shannon et al., 2002). In view of the conflicting results from previous studies, present metaanalysis of all available data was performed to evaluate the association between MTHFR C677T polymorphisms and susceptibility to CRC.

Literature search strategy and inclusion criteria:
Meta-analysis was performed according to MOOSE guidelines (Stroup et al., 2000). A literature search of the PubMed, Google Scholar, Elsevier and Springer Link databases was conducted to identify articles published up to July, 2014, with following keywords: using the following terms: (''genetic polymorphism'' or ''polymorphism'' or ''SNP'' or ''single nucleotide polymorphism'' or ''gene mutation'' or ''genetic variants'') and (''Colorectal cancer'' or ''colon cancer'' or ''rectal cancer'') and (''MTHFR'' or ''methylenetetrahydrofolate reductase'' or ''C677T''). Bibliographies of review articles were manually searched to find additional eligible studies. If more than one study by the same author using the same case series was published, either the studies with the largest sample size or the most recently published study was included. Included studies had to fit the following criteria:(1) sufficient data regarding allele frequency; (2) an association analysis between the MTHFR C677T polymorphism and CRC risk; and (3) independent casecontrol studies.

Data extraction
The following information was extracted from the each identified studies: the first author family name, year of publication, sample size, country name, genotyping method, the numbers of patients and controls, MTHFR C677T genotypes information and frequencies of alleles in all study. For data not provided in the main text, the relevant information was obtained by contacting corresponding authors.

Statistical analysis
The strength of the association between MTHFR C677T polymorphisms and colorectal risk was measured by using crude odds ratio (OR) with 95% confidence interval (CI). The pooled ORs were estimated in following genetic models: allele contrast (T vs. C), codominant model (CT vs. CC), homozygote model (TT vs. CC), dominant model (TT+CT vs. CC), and recessive model (TT vs. CT+CC). Heterogeneity among studies was examined with the χ 2 test-based Q statistics and P < 0.05 was considered significant. Heterogeneity was quantified with the I 2 metric, which is independent of the number of studies in the meta-analysis (Zintzaras and Lau, 2008). I 2 takes values between 0% and 100%, with higher values indicating a greater degree of heterogeneity. Fixedeffects summary ORs were calculated using the Mantel-Haenszel method (Mantel and Haenszel, 1959), and the DerSimonian and Laird method was used to calculate random-effects summary ORs (DerSimonian and Laird, 1986).
Egger's test and an inverted funnel plot was used to assess publication bias (Begg and Mazumdar, 1994;Egger et al., 1997). HWE was checked in the control group of the eligible studies by the chi-square test (p ≤ 0.05). Sensitivity analysis was performed including studies that deviated from HWE and with small sample size. Methodological quality was assessed according to the Newcastle-Ottawa Scale (NOS) criteria (Stang, 2010). The NOS criteria includes three aspects: (1) subject selection: 0, 4 points; (2) comparability of subject: 0, 2 points; (3) clinical outcome: 0, 3 points. NOS scores range from 0 to 9 with a score >7 indicating good quality (Yan et al., 2013). All statistical analysis was undertaken using the program MetaAnalyst and MIX version 1.7 (Bax et al., 2006). All P values were two-sided.

Sensitivity analysis
Sensitivity analyses by sequential omission of individual studies or studies with low quality (studies departed from HWE or with small sample size) did not alter the overall pooled ORs. The genotype distribution of the control population in four studies (Haghighi et al., 2008;Kang et al., 2011;Sameer et al., 2011;Kim et al., 2012) was not in HWE. Overall, the results from sensitivity analysis after exclusion of these four studies were similar to those from nonsensitivity analysis, and MTHFR C677T allele or TT genotype was not associated with CRC susceptibility(OR TvsC =0.96; 95%CI=0.91-1.00; p=0.008; I 2 =83.6%; P heterogeneity <0.0001).

Bias diagnosis
Begg funnel plot and Egger test were performed to assess publication bias. Egger test was used to provide statistical evidence for funnel plot symmetry. The Funnel plots' shape of all contrasts did not reveal obvious evidence of asymmetry ( Figure 4A-D), and all the P values of Egger's test were more than 0.05, providing statistical evidence for the funnel plots' symmetry (Table 2).

Discussion
In present study, the possible association between the C677T polymorphisms of MTHFR gene and CRC was investigated with a meta-analysis. The results showed that MTHFR C677T polymorphism is not associated with CRC. Colorectal cancer is an important clinical problem which has been well-studied but its mechanism is still relatively unclear. DNA methylation is an important epigenetic process for transcriptional control of human genome including those genes involved in cancer initiation and progression. Clinical studies have suggested that biological explanation to the protective effect of some nutrients could be linked with the DNA methylation (Galvan-Portillo et al., 2009). The metabolism of folate is significant for the maintenance of genome integrity due to its role in DNA synthesis, repair, and methylation (Fowler, 2005). Defects in folate metabolism can arise from a poor dietary intake of folates and other nutrients or may be determined genetically as a result of the combined influence of many low-penetration mutations or single nucleotide polymorphisms (SNPs). These genetic polymorphisms could modify the activity, stability, or level of the corresponding enzymes and thereby impair folate absorption or disturb the balance between folate derivatives (Weiwei et al., 2014).
Several large-scale meta-analyses combining data from multiple studies have been published investigating the association between MTHFR C677T polymorphism and various cancers such as gastric, lung cancer, breast cancer, cervical cancer, and liver cancer (Zintzaras, 2006;Bai et al., 2009;Jin et al., 2009;Ding et al., 2012;Mei et al., 2012;Niu et al., 2012;Tu et al., 2012;Zhuo et al., 2012;Wen et al., 2013;You et al., 2013;Rai, 2014, a, b). Some reports demonstrated association between MTHFR C677T polymorphism and cancer (You et al., 2013;Rai, 2014a), whereas others reported contradictory results i.e no association (Ding et al., 2012;Niu et al., 2012). These inconsistent and confusing conclusions can be attributed to several factors like (i) different selection criteria and selection bias might account for the diversity of the results, (ii) folate metabolic pathway is complex pathway and MTHFR is only one of many enzymes involved in the pathway, (iii) the studies with small sample size were included in the meta-analyses and these studies have lower statistical power than those with large sample size and (iv) finally different mechanisms of carcinogenesis of different cancers might due to gene-variant associations vary in different kinds of diseases (Qin et al., 2014). This contradiction suggests that the effect of the C677T polymorphism of MTHFR on the susceptibility to cancer may vary in different cancer type in different populations. Heterogeneity is a critical issue in any meta-analysis, and an important aim of a meta-analysis is to determine the source and causes of heterogeneity. Evidence of betweenstudy heterogeneities was observed in allelic contrast, co-dominant, homozygote, dominant and recessive models, therefore the random effect models were adopted and sensitivity analyses were done . However, author failed to find the source of heterogeneity by conducting sensitivity analyses.
The present meta-analysis on CRC and MTHFR C677T association is more reliable than previous metaanalysis studies because it included the latest published articles and a larger sample size. However it has some limitations also. First, the results were based on unadjusted ORs, while a more precise evaluation should be adjusted by potentially confounding factors, including age, gender, body mass index, smoking status, drink abuse, and environmental factors. Second, the controls were not uniformly defined. Some studies used population-based controls, while others used hospital based controls. Third, single gene of folate pathway was considered in present meta-analysis. Finally, the effect of gene-gene and geneenvironment interactions was not considered.
In summary, present meta-analysis indicated that C677T polymorphism in the MTHFR gene may not be associated with CRC susceptibility in Asians. Welldesigned prospective studies with large sample size should be conducted to validate findings of the present meta-analysis..