Quantitative assessment of the association between MHTFR C677T (rs1801133, Ala222Val) polymorphism and susceptibility to bladder cancer

Background The association between Methylenetetrahydrofolate reductase (MTHFR) Ala222Val (rs1801133) has been implicated to alter the risk of bladder cancer, but the results are controversial. Methods A comprehensive databases of Pubmed, Embase, Web of Science, and the Chinese Biomedical Database (CBM) were searched for case–control studies investigating the association between MTHFR Ala222Val polymorphism and bladder cancer susceptibility. Odds ratios (OR) and 95% confidence intervals (95%CI) were used to assess this possible association. A χ2-based Q-test was used to examine the heterogeneity assumption. Begg’s and Egger’s test were used to examine the potential publication bias. The leave-one-out sensitivity analysis was conducted to determine whether our assumptions or decisions have a major effect on the results of the review. Statistical analysis was performed with the software program Stata 12.0. Results A total of 15 independent studies were identified, including 3,570 cases and 3,926 controls. Our analysis suggested that Ala222Val was not associated with bladder cancer risk in overall population under additive model (OR=0.96, 95%CI=0.76-1.21, P=0.731), dominant model (OR=1.00, 95%CI=0.87-1.15, P=0.975), recessive model (OR=0.92, 95%CI=0.79-1.07, P=0.279), and Ala allele versus Val allele (OR=0.96, 95%CI=0.86-1.07, P=0.427). In the subgroup analysis stratified by ethnicity and sources of controls, there were also no significant associations detected among different descent populations, population-based studies and hospital-based studies. Conclusion This meta-analysis showed the evidence that MTHFR Ala222Val polymorphism was not contributed to the development of bladder cancer. Virtual slide The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2117182849994994.


Introduction
Bladder cancer is the second most common genitourinary malignant disease, with an expected 73,510 newly diagnosed cases and 14,880 deaths in 2012 in America [1]. Many studies have suggested that several environmental factors such as cigarette smoking, aromatic amines contained in dyes, chronic inflammation due to infection, anticancer drugs and radiation are thought to be risk factors for bladder cancer [2]. However, most individuals with these known smoking habits, chemical or environmental exposures never develop bladder cancer while many bladder cancer cases develop among individuals without those known risk factors, suggesting that genetic factors also play an important role in bladder carcinogenesis [3]. Host factors including genetic polymorphisms, have been suggested as risk factors for the development of a variety of cancers, including bladder cancer.
Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are two enzymes essential for maintaining folate homeostasis. MTHFR catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which serves as a substrate for the remethylation of homocysteine to methionine with subse-quent synthesis of S-adenosylmethionine (SAM). Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene are receiving increasing attention as variants that may potentially influence methyl group metabolism and, thereby, alter chromosome integrity. A common Ala222Val variant (an alanine to valine change) within the N-terminal catalytic domain results in a thermolabile enzyme with with 35-50% reduced activity [4]. Folate deficiency is associated with DNA strand breakage and uracil misincorporation into DNA [5]. Thus, if MTHFR polymorphic variants reduce folate levels by diminishing enzymatic activity, they could enhance the propensity for DNA strand breakage and cancer occurrence [6]. Alternatively, variant MTHFR activity could influence the availability of methyl donors by altering S-adeno-sylmethionine levels, and potentially, the methylation status of key tumor suppressor or promoter genes involved in bladder carcinogenesis [7]. Furthermore, functional polymorphisms in MTHFR could affect the metabolism of other carcinogenic substances that undergo one carbon metabolism such as arsenic [8,9].
Recently, many studies have investigated the role of the MTHFR Ala222Val polymorphism in the etiology of bladder cancer susceptibility. However, the results of these studies remain inconsistent. Therefore, we conducted a meta-analysis of all available case-control studies that have been published to assess the effect of the MTHFR Ala222Val polymorphism on the risk of bladder cancer.

Search strategy
We conducted a comprehensive search in the Pubmed, Embase, Web of Science, and Chinese Biomedical Database (CBM) databases updated on March 1, 2013. We combined search terms for MTHFR Ala222Val polymorphism and bladder cancer risk. Search terms included: (Methylene-tetrahydrofolate reductase, MTHFR, Ala222Val, or rs1801133) and bladder cancer. There was no sample size and language limitation. We evaluated all associated publications to retrieve the most eligible literatures. All references cited in the included studies were also hand-searched and reviewed to identify additional published articles not indexed in common databases. Of the studies with overlapping data published by the same authors, only the most recent or complete study was included in this meta-analysis.

Inclusion and exclusion criteria
Studies included in this meta-analysis had to meet the following criteria: (1) evaluate the MTHFR Ala222Val polymorphism and bladder cancer risk, (2) Only the case-control studies were considered; (3) The paper should clearly describe the diagnoses of bladder cancer and the sources of cases and controls; (4) sufficient reported genotypic frequencies in both cases and controls for estimating an odds ratio (OR) with a 95% confidence interval (95%CI). The exclusion criteria were: (1) none case-control studies; (2) control population including malignant tumor patients; and (3) duplicated publications.

Data extraction
Information was carefully extracted from all eligible publications independently by two investigators according to the selection criteria. In case of disagreement, a third reviewer assessed the articles. For each of the eligible case-control studies, the following data were collected: the first author's last name, year of publication, ethnicity, source of the controls, total number of cases and controls, and genotype distributions in cases and controls.

Quality assessment
Quality assessment of case-control studies in this metaanalysis was performed using the Newcastle Ottawa scale (NOS) as recommended by the Cochrane Non-Randomized Studies Methods Working Group [10]. This instrument was developed to assess the quality of nonrandomized studies, specifically cohort and casecontrol studies. Based on the NOS, case-control studies were judged based on three broad perspectives: selection of study groups (1 criterion), comparability of study groups (4 criteria), and ascertainment of outcome of interest (3 criteria). Given the variability in quality of observational studies found on our initial literature search, we considered studies that met 5 or more of the NOS criteria as high quality [10].

Statistical analysis
We examined the association between MTHFR Ala222Val polymorphism and the bladder cancer risk by calculating pooled odds ratio (ORs) and 95% confidence intervals (95%CI). We explored the association between (Alaallele vs Val-allele) comparison and bladder cancer development, as well as homozygote comparison (Ala/Ala versus Val/Val), dominant model [(Ala/Ala + Ala/Val) versus Val/Val)] and recessive model [(Ala/Ala versus Ala/Val + Val/Val)]. The significance of pooled OR was tested by Z test. The χ2-based Q-test was also used to examine the heterogeneity assumption [11]. If studies' findings only differ by the sampling (P≥0.05), a fixedeffects model could be used to calculate the combined OR. By contrast, if the P value of the Q tests is below 0.05, which suggested that the study results statistically differ by heterogeneous case and sampling, a randomeffects model could be more suitable. The significance of the pooled OR was determined by the Z-test, and P < 0.05 was considered as statistically significant.
The leave-one-out sensitivity analysis was conducted to determine whether our assumptions or decisions have a major effect on the results of the review by omitting each study [12]. Furthermore, subgroup analyses were performed to test whether the effect size varied by the ethnicity and the source of control population. To evaluate the published bias, we used Begg's [13] and Egger's [14] formal statistical test and by visual inspection of the funnel plot. All statistical analyses were performed with Stata software (version 12.0; Stata Corp LP, College Station, TX), using two-sided P values.

Study characteristics
In total, 13 publications including 15 case-control studies with 3,570 cases and 3,926 controls met the selection criteria [15][16][17][18][19][20][21][22][23][24][25][26][27]. The characteristics of the studies included in this meta-analysis are listed in Table 1. Among the 15 studies, 5 studies of Caucasians, 5 studies of Asians, 4 studies of Africans and 1 study of mixed population, 8 studies of population-based controls, 7 studies of hospital-based controls. The distribution of the MTHFR Ala222Val genotype in the control groups of these 15 studies was all consistent with HWE (All P HWE values were more than 0.05, Table 1). According to the quality criteria, all the 15 studies were high quality.

Quantitative synthesis
Overall, there was no association between bladder cancer risk and the variant genotypes in different genetic models when all the eligible studies were pooled into the meta-analysis. As show in Table 2, compared with the wild-type homozygote genotype, the homozygote variant genotype Val/Val was not associated with a statistically significant increase risk of bladder cancer (OR=0.96, 95%CI=0.76-1.21, P=0.73, Figure 1). Simultaneously, no significant effects were observed in dominant (OR=1.00, 95%CI=0.87-1.47, P=0.96, Figure 2), recessive models (OR=0.92, 95%CI=0.79-1.07, P=0.28, Figure 3), and Ala allele vs Val allele comparison model (OR=0.96, 95% CI=0.86-1.07, P=0.43, Figure 4). When we analyzed the relationship of MTHFR Ala222Val polymorphism and bladder cancer risk in different ethnicity subgroup, our data showed that this polymorphism did not contribute to the bladder cancer risk among different descent populations. Subgroup analyses were also performed according to the source of control population. We didn't observe any significant association both populationbased study and hospital-based study in any genetic model using random effect model (Table 2).

Sensitivity analysis
In order to compare the sensitivity of this meta-analysis, we conducted a leave-one-out sensitivity analysis. A  Ethnicity single study involved in this meta-analysis was evaluated each time to reflect the influence of the individual data set to pooled ORs. The results pattern was not impacted by single study in all genetic models, for example, under the allele contrast model (Ala versus Val) was shown in Figure 5. The P for Q test and the I 2 value also showed that none of single study affected the heterogeneity of this meta-analysis.

Publication bias
Begg's funnel plot and Egger's test were used to assess the publication bias in this meta-analysis. The Table 2 Meta-analysis on the association between MTHFR Ala222Val polymorphism and bladder cancer risk (Continued)     Begg's test for the allele contrast model was shown in Figure 6, and Egger's test for the allele contrast model was shown in Figure 7. Thus, the above results suggest that publication bias was not evident in this meta-analysis.

Discussion
It is wide acknowledged that genetics play an important role in determining cancer risk and association studies have been identified to evaluate cancer susceptibility [28]. However, many associated studies failed to provide convincing evidence of linkage and resulted in contradicting findings. Meta-analysis provided a popular method for combining world literatures across studies to resolve the statistical power and discrepancy problem in associate studies [29]. It was more systematic for its statistical methods than single case-control studies and cohort studies [30][31][32]. Previous epidemiological studies have evaluated the association between bladder cancer risk and MTHFR Ala222Val polymorphism, but with inconclusive results. An initial cohort study suggested a 5.5 fold risk of bladder and kidney cancer combined among 222 Ala>Val variant homozygotes [33]. In contrast, three case-control studies provided the evidence that there was no overall association of MTHFR Ala222Val genotype with bladder cancer [15][16][17], one study observed a reduced risk among heterozygotes for MTHFR Ala222val [18].
Based on 15 studies providing data on MTHFR Ala222Val polymorphism and bladder cancer risk, we conduced a meta-analysis involving in 3,570 cases and 3,926 controls to indicate if this polymorphism was significantly associated with bladder cancer risk. Moreover, we also evaluated the publication bias. The MTHFR Ala222Val genotypes funnel plot was approximately symmetrical. Furthermore, Begg's and Eggle's test showed that there was no publication bias in this study.  We found that there were no significant associations between this polymorphism and bladder cancer risk in the all genetic models. These results suggested that the MTHFR Ala222Val polymorphism might be not contributed to the development of bladder cancer. It has been well known that cancer occurrence and mortality varied by ethnicity and geographic location [34]. In this metaanalysis, all subjects were sub-grouped into four groups (Caucasian, Asian, African and mixed populations). No association of Ala222Val polymorphism with bladder cancer risk was detected in different descent populations. When stratified by source of controls, the similar results were found both in population-based and hospital-based studies.
Some limitations of our meta-analysis should be addressed. Firstly, the numbers of published studies collected in our analysis were not large enough for the comprehensive analysis. Secondly, lacking the original data of the included studies limited our study to further evaluate the potential interactions, since geneenvironment and gene-gene interactions may modulate bladder cancer risk. So, a more precise analysis needs to be conducted if individual data such as age and sex are available. Nevertheless, advantages in our metaanalysis should also be acknowledged. A systematic review of the association of MTHFR Ala222Val polymorphism with bladder cancer risk is statistically more powerful than any single study. Furthermore, the studies included in our meta-analysis strictly and satisfactory met our selection criteria.

Conclusions
In conclusion, our meta-analysis provided the evidence that the MTHFR Ala222Val polymorphism maybe not contributed to the development of bladder cancer. In addition, further studies with larger sample sizes and careful design are needed to identify this association more comprehensively.