Methylenetetrahydrofolate reductase C677T polymorphism and colorectal cancer susceptibility: a meta-analysis

The association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and colorectal cancer (CRC) susceptibility has been researched in numerous studies. However, the results of these studies were controversial. Therefore, the objective of this meta-analysis was to offer a more convincible conclusion about such association with more included studies. Eligible studies published till May 1, 2017 were searched from PubMed, Embase, Web of Science, and CNKI database about such association. Pooled odds ratios (ORs) together with 95% confidence intervals (CIs) were calculated to evaluate such association. And the Begg’s funnel plot and Egger’s test were applied to assess the publication bias. This meta-analysis contained 37049 cases and 52444 controls from 87 publications with 91 eligible case–control studies. Because of lack of data for a particular genotype in several studies, all the included studies were analysed barely in the dominant model. Originally, there was no association between MTHFR C677T polymorphism and CRC susceptibility (OR =0.99, 95% CI =0.94–1.05). After excluding 13 studies according to their heterogeneity and publication bias, rs1801133 polymorphism was found to reduce the risks of CRC significantly (OR =0.96, 95% CI =0.94–0.99). In the subgroup analysis of ethnicity, there was a significant association in Asians (OR =0.94, 95% CI =0.89–1.00). Furthermore, when stratified by the source of controls and genotyping methods, the positive results were observed in population-based control group (OR =0.97, 95% CI =0.93–1.00) and PCR-restriction fragment length polymorphism (PCR-RFLP) method (OR =0.95, 95% CI =0.91–0.99. The results of the meta-analysis suggested that MTHFR C677T polymorphism was associated with CRC susceptibility, especially in Asian population.


Introduction
Colorectal cancer (CRC) is a critical public health problem, which is the third most commonly diagnosed cancer and the third common cause of cancer deaths in both males and females. There were 134490 new CRC cases and 49190 mortalities by estimation in the United States in 2016 [1]. The colorectal carcinogenesis is a complex multistep progress (a benign adenomatous polyp -an advanced adenoma with high-grade dysplasia -an invasive cancer) with altered expression of oncogenes, tumor suppressor genes and DNA repair genes [2]. However, the etiology of CRC is still unclear. It is known to all that CRC is a multifactorial and multigenic disease, and is influenced by environment conditions, diet habits, genetic mutations, and Escherichia coli infection [3,4]. With increasing numbers of studies, more gene polymorphisms were found to contribute to CRC [5]. These single nucleotide polymorphisms (SNPs) can be used as makers for improving cancer diagnosis and determination of treatment plans [6].
As a key enzyme and an important regulator for the metabolism of folate/vitamin B 9 , methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate [7]. Simultaneously, the 5-methyltetrahydrofolate is the main circulatory form of folate in the body and provides a methyl group to convert the amino acid homocysteine into methionine, which is the precursor of S-adenosylmethionine (SAM). SAM is the major methyl donor in the cell and takes part in DNA methylation [8]. Therefore, MTHFR not only plays a role in making proteins and other important compounds, but also is an important factor in DNA methylation, synthesis, and repair [9]. The enzyme is encoded by the MTHFR gene located on the short arm of chromosome 1-1p36.3 [10]. Previously, several mutations of MTHFR gene have been found and MTHFR C677T (rs1801133) is the most common type amongst them. MTHFR C677T represents an alanine-to-valine substitution at nucleotide position 677 in exon 4 resulting in thermolability and concurrent decreased activity of the enzyme [11,12]. MTHFR gene mutations lead to MTHFR enzyme dificiency, low plasma folate levels, hyperhomocysteinemia [13,14] and certain diseases such as cardiovascular disease, pregnancy complications, neural defect, and several cancers including CRC [15][16][17][18][19][20][21]. With a growing number of studies conducted to explore such association, we hypothesized that rs1801133 was likely to relate to colorectal carcinogenesis.
Many researchers have carried out a large number of studies to examine the potential association between MTHFR C677T polymorphism and CRC susceptibility. But, the results are still inconclusive so far. Thus, the aim of this meta-analysis including all available case-control studies was to investigate a more reliable association.

Materials and methods
We searched several databases including PubMed, Embase, Web of Science, and CNKI database for published studies about exploring the association between MTHFR C677T polymorphism and CRC susceptibility till May 1, 2017. The search strategy included listed key words: 'methylenetetrahydrofolate reductase' , 'MTHFR polymorphism' , 'C677T' , 'rs1801133' , and 'risk or susceptibility' and 'colorectal or colon or rectal cancer' . Furthermore, we manually searched the reference lists of clinical trials and former meta-analyses for more relevant studies. When duplicate data appeared in different publications, this meta-analysis only adopted the most recent study or the study with the most complete information. The meta-analysis was on the basis of the preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) [22]. The eligible studies needed to accord with the following inclusion criteria: (i) case-control studies; (ii) the language was not restricted to English; (iii) investigating the association between MTHFR C677T polymorphism and CRC susceptibility; (iv) offering enough raw data to calculate odds ratio (OR) with 95% confidence interval (CI). Additionally, exclusion criteria were as follows: (i) non-case-control studies; (ii) lack of sufficient data for calculating genotype frequency; (iii) case-control studies about examining the relationship between MTHFR C677T polymorphism and colorectal adenoma; (iv) duplicated publications.

Data extraction
In order to guarantee the accuracy of extracted information, two authors individually reviewed each publication and extracted useful data on the basis of the inclusion criteria listed above. When disagreements arose in the course of data extraction, discussion was carried out with other authors until the agreements were reached. The following information were extracted from each study to accomplish a standardized sheet: first author's name, year of publication, ethnicity of population, source of controls (hospital based or population based), genotyping method, sample size of cases and controls, genotype frequency of rs1801133 in cases and controls, and the results of the Hardy-Weinberg equilibrium (HWE) test.

Statistical analysis
The relationship between MTHFR C677T polymorphism and CRC susceptibility was analyzed by using five models including the dominant model (CT + TT compared with CC), the recessive model (TT compared with CT + CC), the homozygous model (TT compared with CC), the heterozygous model (CT compared with CC), and the allele model (T compared with C). The goodness-of-fit χ 2 test was conducted to evaluate the HWE in control groups and P<0.05 was regarded as significant disequilibrium [23]. Stratified analysis were performed by ethnicity, source of controls, and genotyping method. Besides, the pooled OR together with 95% CI were measured to bring out the strength of such association. The fixed effects model (Mantel-Haenszel method) and the random effects model (Dersimonian-Laird method) were selected to use based on heterogeneity in the meta-analysis. If there was no or little heterogeneity, the fixed effects model was used; otherwise, the random effects model was used. Due to only particular genotypes extracted in several studies, the dominant model analysis were carried out for all the included studies [84]. Galbraith graph was performed to explore the impossible cause of heterogeneity [24]. A sensitivity analysis was conducted to assess the stability of the results. Begg's funnel plot was performed for potential publication bias and Egger's linear regression test was executed to assess funnel plot asymmetry statistically. If P<0.05, publication bias existed [25]. All statistical data analyses were carried out by using Stata software (version 12.0, StataCorp LP, College Station, TX, U.S.A.).

Characteristics of the studies
According to PRISMA-P, this meta-analysis contained 37049 cases and 52444 controls that were combined from 87 publications with 91 eligible case-control studies to examine the relationship between rs1801133 polymorphism and CRC risks . The literature retrieval and selection process are shown in the flowchart in Figure  1. Detailed information of each study were listed in Table 1. The distribution of genotypes in controls was consistent with HWE except 15 studies [33][34][35]37,39,47,63,71,76,80,87,88,106,110,111]. In these studies, four ethnicities of population were included: Asian, Caucasian, African, and mixed ethnic group. Nine genotyping methods were applied: PCR-restriction fragment length polymorphism (PCR-RFLP), real-time PCR (RT-PCR), PCR-single     89-0.99) were significantly correlated with risks of decreased CRC. However, RT-PCR method was not relevant to significant associations for all genetic models. In conclusion, the present meta-analysis suggested that MTHFR C677T polymorphism was connected with CRC susceptibility.

Publication bias
The Begg's funnel plot and Egger's test were performed to assess the publication bias. Initially, the Begg's funnel plot was asymmetrical obviously with all the included studies and it suggested a potential publication bias (Begg's test: P=0.103; Egger's test: P=0.058; Figure 5A). After the removal of 13 studies mentioned above [27][28][29]38,41,45,46,50,53,59,61,75,105], the plots seemed to have a symmetrical distribution in the funnel plot and then Egger's test was used to provide statistical evidence (Begg's test: P=0.369; Egger's test: P=0.136; Figure 5B). No significant publication bias was observed in the present studies.

Sensitivity analysis
In order to distinguish the impact of each study on the pooled ORs, we conducted one-way sensitivity analysis. Each time one study was omitted, meta-analysis was repeated and the statistical significance of the results was not changed. Therefore, the results confirmed that the present meta-analysis was relatively stable and reliable.

Discussion
MTHFR is a key enzyme in the folate metabolism and may play a role in the CRC carcinogenesis. It is an essential enzyme in the catalytic reaction that converts 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate. On one hand, 5,10-methylenetetrahydrofolate takes part in the thymidylate synthesis. On the other hand, 5-methyltetrahydrofolate promotes methionine synthesis and SAM-mediated methylations. In brief, MTHFR has an influence on DNA synthesis, methylation, and repair [113]. The MTHFR polymorphisms result in the decreased enzyme activity and then low levels of plasma folate and high homocysteine come to light. Folate is one of water-soluble B vitamins that takes part in various biochemical reactions with its activity to provide or accept one-carbon units [13]. Folate deficiency is likely to contribute to the development of CRC, and several mechanisms may explain how it leads to CRC, including DNA strand breaks, abnormal DNA methylation, and impaired DNA repair [114].   [29,30,31,40,43,47,48,52,55,61,63,77,107].  Several polymorphisms have been reported about the MTHFR gene coding relevant enzyme, and MTHFR C677T polymorphism is the most common one. Heretofore, various studies conducted to detect such association and obtained inconsistent results. Chen et al. [112], first reported that MTHFR variant homozygous (TT) genotype was closely linked to reduced incidence of CRC with low consumption of alcohol. In the next few years, similar results were replicated by several other studies [109][110][111]. However, another study of a homogeneous northern European population obtained different conclusions that MTHFR CT heterozygote had a significantly increased risk of developing CRC and no increased cancer risk was observed in TT homozygotes [107]. In addition, a hospital-based case-control study conducted by Matsuo et al. [104] found no significant relativity between MTHFR C677T and the risks of CRC. Owing to the difference in study design and the sample size, the different ethnicity, and the diverse stratification, these controversial results were found in published studies. Hence, meta-analysis is essential to be carried out by combining all studies that meet the requirements to get more precise conclusions.
In recent years, there were several meta-analyses performed to elucidate the association of MTHFR C677T polymorphism and the susceptibility to CRC before [26,[115][116][117][118]. Compared with them, this meta-analysis included the most eligible reported studies with the largest sample size and had no restrictions in ethnicity. Since the quality of included documents were disequilibrium, our initial analysis achieved no significant results with all eligible studies. In order to obtain more reliable results, the final conclusion were obtained excluding 13 studies in accordance with the analysis of heterogeneity and publication bias. In this meta-analysis, the pooled conclusions revealed that rs1801133 polymorphism significantly reduced the risk of CRC in the dominant model. The findings agreed with the overwhelming majority results reported by the published studies.
When stratified by ethnicity, there was a significant association with reduced risks of CRC in Asians. The result was consistent with the two previous meta-analysis based on the Asians [116,117]. Zhong et al. [118], carried out a meta-analysis obtaining similar results in East Asians and further subgroup analyses by country identified such association in Korea and Japan. Nevertheless, the recent meta-analysis failed to identify that rs1801133 polymorphism was connected with CRC susceptibility in Iranian population [26]. By means of stratified analysis based on the source of controls and genotyping methods, the positive results were observed in population-based control group and PCR-RFLP method. In general, the source of controls included healthy individuals and patients without CRC. Since the risks of CRC varies amongst individuals over a few years, it might have an impact on the results of relevant studies and make them unreliable. Therefore, inclusion criteria should be improved and studies with large sample sizes should be accepted. In the subgroup of genotyping method, there were nine methods applied for genotyping such as PCR-RFLP, RT-PCR, PCR-SSCP, MS-PCR, MSP, MALDI-TOF-MS, Taqman, MassARRAY, and Sequenom in the including studies. Specific methods and steps were described in each article. Amongst these 87 studies, the majority method was PCR-RFLP. Different methods have their own merits, and when all included studies used the same method, the final results would be more reliable.
In the present meta-analysis, we had obtained weak associations significantly with a large sample size. However, the potential limitations of the meta-analysis should be acknowledged. First, this meta-analysis was based on unadjusted effect estimates and 95% CI, and the influence of multiple cofactors such as age, gender, diet habits including intake of alcohol and consumption of cigarette, the level of folate, and the other environmental factors should be taken into consideration. Second, because of incomplete data of some genotypes, only the dominant model was analyzed in all the included studies. Third, we did not perform stratification analysis by serum folate levels, locations of the tumor and so on, which might result in confounding bias. In addition, after excluding 13 studies according to the analysis of heterogeneity and publication bias, the heterogeneity decreased significantly and the publication bias seemed to disappear. However, the selection bias existed because all the studies were published. Furthermore, the gene-gene and gene-environment interactions were not mentioned in this meta-analysis. In addition, the potential roles of the gene polymorphism which were hidden or magnified by other interactions were omitted.

Conclusion
In summary, the present meta-analysis revealed that there was a significant association between MTHFR C677T polymorphism and susceptibility to CRC. Simultaneously, the TT genotype of MTHFR C677T polymorphism could reduce the risk of CRC. In addition, the associated risk of CRC was also reduced in Asians and those studies with population-based controls and used the PCR-RFLP method. Therefore, detection of the MTHFR C677T polymorphism might be used as markers for CRC prediction and treatment selection.