Associations of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) Polymorphisms with Susceptibility to Bladder Cancer: A Systematic Review and Meta-Analysis

The effects of the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms on bladder cancer risk have been evaluated in some studies. However, the results were conflicting and ambiguous. Therefore, we aimed to perform a comprehensive meta-analysis to investigate the association of these polymorphisms with risk of bladder cancer from all eligible case-control studies. PubMed, Web of science, Scopus, SID, CNKI and SciELO databases were searched to identify all relevant studies published up to 1 January, 2021. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the strength of associations. A total of 20 case-control studies including 11 studies with 3463 cases and 3927 controls on MTHFR rs1801133 (677C>T) and 9 studies with 3177 cases and 3502 controls on rs180113 (1298A>C) polymorphism were selected. Pooled data revealed that the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms were not associated with risk bladder cancer in overall. Stratified analysis by ethnicity revealed that the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms were associated with bladder cancer risk in Asians, but not in Caucasians. There was no publication bias. The current meta-analysis revealed that the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms were not risk factor for development of bladder cancer globally. However, large sample size, well-designed, and population-based studies should be performed to verify the association of the MTHFR polymorphisms with bladder cancer risk.


Introduction
Bladder cancer is the second most common genitourinary malignancy, after prostate cancer in human (Oeyen et al., 2019;Lenis et al., 2020). It is the sixth most prevalent malignancy in the United States and causes more than 16,000 deaths annually (Degeorge et al., 2017), which represents 4.4% of all new cancer diagnoses in the USA (Wong et al., 2018). More than 60% of all bladder cancer cases and half of all the 165,000 bladder cancer deaths occur in the less developed regions of the world (Antoni et al., 2017). Bladder cancer is more common in men than women, with a respective incidence of 9.6 among men and 2.4 among women per 100,000 person-year globally,

Abstract
The effects of the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms on bladder cancer risk have been evaluated in some studies. However, the results were conflicting and ambiguous. Therefore, we aimed to perform a comprehensive meta-analysis to investigate the association of these polymorphisms with risk of bladder cancer from all eligible case-control studies. PubMed, Web of science, Scopus, SID, CNKI and SciELO databases were searched to identify all relevant studies published up to 1 January, 2021. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the strength of associations. A total of 20 case-control studies including 11 studies with 3463 cases and 3927 controls on MTHFR rs1801133 (677C>T) and 9 studies with 3177 cases and 3502 controls on rs180113 (1298A>C) polymorphism were selected. Pooled data revealed that the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms were not associated with risk bladder cancer in overall. Stratified analysis by ethnicity revealed that the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms were associated with bladder cancer risk in Asians, but not in Caucasians. There was no publication bias. The current meta-analysis revealed that the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms were not risk factor for development of bladder cancer globally. However, large sample size, well-designed, and population-based studies should be performed to verify the association of the MTHFR polymorphisms with bladder cancer risk.
Keywords: Bladder cancer-urinary neoplasms-folate-MTHFR-polymorphism-meta-analysis REVIEW Associations of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) Polymorphisms with Susceptibility to Bladder Cancer: A Systematic Review and Meta-Analysis respectively (Bouffioux, 1984). Bladder cancer is one of the most expensive cancer to care for from diagnosis to death due to the frequent procedures required for this malignancy monitoring and treatment (Andreassen et al., 2016).
The leading risk factor for development of bladder cancer is tobacco use, which actually accounts for more 50 percent of the cases and increases the chance of development of the disease by three times compared to not smoking individuals (Freedman et al., 2011;Mobley and Baum, 2015). Moreover, studies have found that occupational exposures (such as paint, textiles, rubber, leather, and dyes) and pollutants in drinking water (such as arsenic and chlorinated byproducts) constitute the second most important risk factor for development of bladder cancer (Gu and Wu, 2011;Aminian et al., 2014). Therefore, bladder cancer is an excellent model for studying genetic susceptibility and gene-environment interaction in cancer etiology (Gu and Wu, 2011). Since the major environmental risk factors for development of bladder cancer have been identified (Letaiová et al., 2012;Al-Zalabani et al., 2016) some efforts were made in the last few years to identify genetic variations in the pathways involved in the carcinogenesis processes, including metabolism of carcinogens, DNA repair, cell cycle checkpoints, apoptosis and inflammatory response (Grotenhuis et al., 2010;Gu and Wu, 2011). It is wellknown that folate metabolism may be has an important role in development of several tumours through its involvement in both DNA methylation and nucleotide synthesis . It has been shown that cigarette smoke exposure is associated with decreased serum levels of folate and vitamin B12 antioxidants (Tungtrongchitr et al., 2003;Wu et al., 2007). Folate and other B vitamins play important roles in the one-carbon metabolism pathway, which is associated with DNA methylation, synthesis and impaired DNA repair (Pan et al., 2019). The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) is involved in the circulation form of folate as it catalyzes the irreversible reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (Maruti et al., 2009;Soleimani-Jadidi et al., 2020;Tabatabaei et al., 2020).
The candidate gene approaches revealed that functional polymorphisms at the MTHFR gene may be play an important role in development of bladder cancer (Mannino et al., 2003;Tungtrongchitr et al., 2003;Baghestani et al., 2018;Ahmadi et al., 2021). The human MTHFR gene is located on chromosome 1 at 1p36.3, composed of 11 exons and consists of 17 kb (Rosenberg et al., 2002). It was found that the rs1801133 (677C>T) in exon 4 and rs180113 (1298A>C) in exon 7 of the MTHFR gene resulted in amino acid substitution and a reduction of MTHFR activity ( Liu et al., 2020). To date, several epidemiological studies have evaluated the association of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms with susceptibility to bladder cancer (You et al., 2013;Xu and Zuo, 2020). However, these associations were still inconclusive. Although two meta-analyses have reported the association of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms and bladder cancer risk (Wang et al., 2009;Xu et al., 2013;Akbari et al., 2015), they did not perform subgroup analysis by country of origin and source of controls. Thus, to comprehensively estimate the association of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms with susceptibility to bladder cancer, we carried out this systematic review and meta-analysis.

Search Strategy
We carried out a comprehensive online literature search on electronic databases including PubMed, Scopus, EMBASE, Web of Knowledge, Cochrane Library, Google Scholar, Scientific Information Database (SID), WanFang, VIP, Chinese Biomedical Database (CBD), Scientific Electronic Library Online (SciELO) and China National Knowledge Infrastructure (CNKI) database to identify all relevant studies on the association of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms with susceptibility to bladder cancer up to 1 January, 2021. We used the combination of following keywords and terms: (''Bladder Cancer'' OR ''Urinary Cancer'' OR ''Urinary Bladder Neoplasm'') AND ("Methylenetetrahydrofolate Reductase" OR ''MTFR'' OR "Methionine Synthase Reductase" OR "Folate Pathway'') and Languages were limited to English, Portuguese, Farsi and Chinese. Moreover, the reference lists of retrieved studies including case-control studies, previous meta-analyses and reviews were manually searched to find other relevant publications.

Selection Criteria
Studies meeting the following criteria were included: 1) studies with case-control or cohort design; 2) studies evaluated the association of the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms with bladder cancer risk; 3) genotype distributions in cases and healthy controls were available for calculating an odds ratio (OR) with 95% confidence interval (CI). The following were exclusion criteria: 1) animal studies or in vitro studies; 2) studies evaluated other polymorphisms at MTHFR gene; 3) case only studies; 4) linkage studies and family based studies (sibling, twins and trios-parents studies); 5) studies did not report genotype frequencies; 6) abstracts, posters, case reports, reviews, meta-analyses, commentaries, editorials, conference articles, and proceedings; 7) duplicates of previous published studies or studies with overlapping data. If more than one study was published by the same author(s) using repeated or overlapped data, the most complete one or more recently published study was selected.

Data Extraction
Two authors carefully reviewed and extracted data from all eligible studies according to the inclusion criteria. If any disagreement appeared, a third author was consulted to resolve the dispute and the final consensus was made by the majority of the votes. The following data were extracted from each study: the name of first author, year of publication, country of origin, ethnicity (Caucasian, Asian, African, Mixed populations), source of controls (hospital based or population based), genotyping methods, sample size, alleles and genotypes frequencies for MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms in cases and controls, Minor Allele Frequency (MAFs) and Hardy-Weinberg equilibrium (HWE) in healthy controls. The ''mixed'' group means mixed or unknown populations. conducted using Cochran's Q test, in which P ≤ 0.01 indicated a significant heterogeneity. In addition, I 2 statistic was used to quantify the proportion of the between-study heterogeneity (range of 0 to 100%: I 2 ≤50%, no heterogeneity; I 2 ≥50%, presence of heterogeneity). Thus, when the heterogeneity was absent the fixed-effect model (Mantel-Haenszel method) was used to calculate the overall or pooled OR; otherwise, the random-effects model (DerSimonian and Laird method) was applied. To explore sources of between-study heterogeneity, we have performed subgroup analysis by ethnicity, country, source of controls and HWE status. We used the Chi-squared test to evaluate Hardy-Weinberg equilibrium (HWE) in controls, and we considered p < 0.05 as a significant deviation from HWE (HWE-violating) (Bahrami et al., 2020). Sensitivity analyses were performed to assess the stability of the results by sequential removing of each study. The Begg's visual inspection of funnel plot and the Egger's regression tests were used to evaluate publication bias in the literature, in which P<0.05 was considered statistically significant. All of the statistical calculations were performed using Comprehensive Meta-Analysis (CMA) software version 2.0 (Biostat, USA). Two-sided P-values < 0.05 were considered statistically significant.

Characteristics of Selected Studies
As shown in Figure 1, our initial search yielded 315 Quality score assessment The Newcastle-Ottawa Score (NOS) were performed to assess the quality of included studies in the meta-analysis and to assess the various aspects of the methodology used by the observational research, which are relevant to the quality of the study. This standard assessed 3 sections (selection of cases, comparability of groups, and determination of exposure) and 8 items. In the selection and exposure categories, a quality research item received 1 star, and a comparable category could receive at most 2 stars. The quality assessment values ranged from 0 stars (worst) to 9 stars (best), and studies with a score ≥7 were defined as high quality. Generally, the study which scored at least 5 points was considered to be included in meta-analysis and any discrepant opinions were resolved by discussion and consensus.

Statistical Analysis
Crude odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to evaluate the strength of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms with susceptibility to bladder cancer. The statistical significance of pooled ORs was assessed by the Z test, in which p-value less than 0.05 was considered as statistically significant. The associations was estimated under all five genetic models, i.e., allele (B vs. A), homozygote (BB vs. AA), heterozygote (BA vs. AA), dominant (BB+BA vs. AA), and the recessive (BB vs. BA+AA). A test of between-studies heterogeneity was   studies on MTHFR polymorphisms and bladder cancer, with duplicate studies removed resulting in 181 studies remaining. Among them, 83 studies were excluded based on titles and abstracts. Following the inclusion exclusion criteria 78 studies were excluded to case reports, review, previous meta-analyses, and other polymorphisms of MTHFR gene or lack of the relevant data. Finally, a total of 20 case-control studies from eleven independent papers (Kimura et al., 2001;Sanyal et al., 2004;Moore et al., 2004Moore et al., , 2007Lin et al., 2004;Karagas et al., 2005;Cai et al., 2009;Wang et al., 2009;Rouissi et al., 2009;Chung et al., 2010;Safarinejad et al., 2011;Amooee et al., 2019) were selected. The main characteristics of each study identified are listed in Table 1. Of them, eleven case-control studies with 3,463 cases and 3,927 controls were on MTHFR rs1801133 (677C>T) and nine case-control studies with 3,177 cases and 3,502 controls were on MTHFR rs180113 (1298A>C). For the MTHFR rs1801133 (677C>T), five studies were conducted on Caucasians, four on Asians and one study on African and mixed population. For the MTHFR rs180113 (1298A>C), four studies were conducted on Caucasians, three on Asians and one study on African and mixed population. The eligible studies were published between 2001 and 2011. In term of study design, there were 14 hospital-based (HB) and six population-based (PB). Genotyping methods were conducted using restrictive fragment length polymorphism (PCR-RFLP) and TaqMan. The alleles, genotypes and minor allele frequencies (MAFs) distributions for both MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms in the cases and controls are present in Table 1. The distribution of genotypes in the controls was in agreement with Hardy-Weinberg equilibrium (HWE) for all selected studies (Table 1). The NOS score of eligible articles ranged from 7 to 9, which indicated that all included studies were of high quality (Table 1).
There was no significant association between MTHFR rs1801133 (677C>T) polymorphism and risk of bladder cancer by source of controls and in Chinese population.

Between-Study Heterogeneity
As shown in Tables 2 and 3, there was a significant between-study heterogeneity in overall population under most genetic models for both MTHFR rs1801133 (677C>T) and rs180113 (1298A>C). Thus, we utilized a random-effects model (DerSimonian and Laird method) for those genetic models. To explore the potential sources of between-study heterogeneity, we conducted subgroup analyses by ethnicity, country of origin and source of controls. Results revealed a significant heterogeneity in Asians studies for both MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms. However, the heterogeneity was reduced or disappeared in Caucasian studies. Subgroup analysis revealed that ethnicity and source of controls (for rs180113) might be source of heterogeneity in the current meta-analysis.

Sensitivity Analysis
We conducted a sensitivity analysis to evaluate the stability of the results by sequentially removing each study from our meta-analysis. As shown in Figures 4 and 5, the data from sensitivity analysis revealed that none of the studies changed the pooled OR for MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) under all five genetic models, and it shows that the meta-analysis is stable.

Publication Bias
We performed potential publication bias with the Begg's test and the Egger's test. As Figure 6 indicated, the symmetrical funnel plot indicated that there is no significant publication bias for both for MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms under all five genetic models. Moreover, the Egger's test was performed to provide the statistical evidence of funnel plot (Tables 2 and 3).

Discussion
MTHFR is a key enzyme in folate metabolism, which converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate that is required for the remethylation of homocysteine to methionine (Gohari et al., 2019;Karimi-Zarchi et al., 2019;Sadeghiyeh et al., 2020;Bahrami et al., 2021). The rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms are responsible for the synthesis of a thermolabile form of MTHFR enzyme with decreased enzymatic activity (Kiseljaković et al., 2008). It is suggested that the homozygote mutant genotypes of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms are linked with higher plasma homocysteine level (Azarpira et al., 2018;Niktabar et al., 2021). Some published molecular epidemiological studies have demonstrated an association between MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms and an increased risk of bladder cancer. However, Safarinejad et al., reported that MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms did not risk factor for development of bladder cancer among Iranian population (Safarinejad et al., 2011). This trend inconsistent results between the rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms at MTHFR gene and risk of bladder cancer may be caused for limited number of related studies and small sample sizes. In the current meta-analysis, we evaluated the associations between rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms at MTHFR gene and susceptibility to bladder cancer based on 20 eligible studies. Our pooled OR indicated that both rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms at MTHFR gene were not associated with susceptibility to bladder cancer globally.
In the current meta-analysis, pooled ORs showed that MTHFR rs1801133 (677C>T) polymorphism was not associated with an increased risk of bladder cancer in overall population. Stratified analysis by showed that both polymorphisms were associated with bladder cancer in Asians, but not in Caucasian. Similarly, Shi et al., (2014) in a meta-analysis of 3,463 cases and 3,927 controls revealed that the MTHFR rs1801133 (677C>T) polymorphism was not associated with risk of bladder cancer in overall population. However, their stratified analysis by ethnicity showed that the MTHFR rs1801133 (677C>T) polymorphism was significantly associated with susceptibility to bladder cancer in Middle Eastern populations. Li et al., (2013) in a meta-analysis based on 15 case-control studies with 3,570 cases and 3,926 healthy subjects demonstrated that the MTHFR rs1801133 (677C>T) polymorphism did not associate with risk of bladder cancer. Their subgroup analysis still revealed that the rs1801133 (677C>T) polymorphism was not risk factor for bladder cancer by ethnicity and sources of controls. The previous meta-analyses were not performed subgroup analyses by country of origin for MTHFR rs1801133 (677C>T) polymorphism. Thus, this polymorphism association with bladder cancer needs to be evaluated. Moreover, their conclusions reliability is considerably smaller than that needed to achieve the robust conclusions.
Our pooled data failed to show a significant association between the MTHFR rs180113 (1298A>C) and an increased risk of bladder risk in the global population. Our subgroup analysis by ethnicity showed a significant association between MTHFR rs180113 (1298A>C) and bladder cancer risk in Asians, but not in Caucasians. Moreover, some epidemiological studies revealed that the MTHFR rs180113 (1298A>C) polymorphism was associated with susceptibility to bladder risk in Chinese (Lin et al., 2004;Cai et al., 2009). However, our subgroup analysis by country of origin did not show an association between the MTHFR rs180113 (1298A>C) polymorphism and susceptibility to bladder cancer in Chinese patients. Similarly, Safarinejad et al., in case-control study revealed that the MTHFR rs180113 (1298A>C) was not risk factor for development of bladder cancer in Iranian population (Ghaemmaghami et al., 2008;Safarinejad et al., 2011;Binesh et al., 2012). According to our findings, the association of the MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms with bladder cancer may be due to differences in ethnicity, genetic background, life style, smoking habits, and etc. in a meta-analysis, Zhang et al., (2018) verified the relationship between TP53 codon 72 and bladder cancer risk in Asians, but not Caucasians. These results indicated that genetic variants might be an ethnicity related factor of susceptibility to bladder cancer. In another way, it seemed that different populations with multiple genetic backgrounds have different genetic variants risk in development of bladder cancer (McConkey et al., 2010;Meng et al., 2017). Still, further studies are needed to explore this difference between Asians and Caucasians.
Between-study heterogeneity may have affected a meta-analysis result when interpreting of the pooled ORs (Edraki et al., 2019;Sayad, Ahmadi, Nekouian, et al., 2020;Sayad, Ahmadi, Moradi, et al., 2020). In the current meta-analysis, significant between study heterogeneity existed for both MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms under most genetic models. Subgroup analysis revealed that ethnicity might be the potential source of heterogeneity in the metaanalysis (Karimi-Zarchi et al., 2013;Mojtaba Sohrevardi et al., 2016). However, heterogeneity may be due to many factors, such as differences in the characteristics of controls, life style, diverse genotyping methods, small sample size, and a mixed population from different geographic regions (Yang et al., 2014). Moreover, certain HWE deviations were revealed in the distributions of controls in some included studies, which may be due to the small sample size or other experimental technique errors in the study. However, we have selected all eligible studies even HWE-violating studies.
There are potential limitations in our meta-analysis should be considered. First, the numbers of studies as well as sample sizes for each ethnicity and some subgroup analyses were relatively limited, which Type-II error might not be dismissed. Second, the research subjects of the included studies were mostly from Asian and Caucasian origins. Thus, the bias of racial diversity could not be avoided and the results are not applicable to all populations. Third, the reliability and authenticity of our results may be influenced by the limited number of studies and small sample sizes. Fourth, moderate heterogeneity existed in some genetic models for both MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms. And the subsequent meta-regression could not identify any interfering factors contributing to heterogeneity. Selection bias, although no publication bias was observed, might be a possible major source of between-study heterogeneity in this meta-analysis. Fifth, the detailed individual data in some studies was not available; leading to failure to adjust risk of MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms with bladder cancer based on potential risk factors, such as age, gender, smoking, occupation, environmental factors, lifestyle habits, and other covariates of this disease. Finally, bladder cancer as other malignancies is a multi-factorial disease that results from complex interactions between various genetic and environmental factors. However, due to the lack of the individual original data in the selected studies, we were unable to evaluate the effect of gene-environment interactions, gene-gene interactions and also different polymorphisms within MTHFR gene on development of bladder cancer.
In conclusion, our pooled data revealed that MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms might be not risk factor for development of bladder cancer. However, stratified analysis by ethnicity revealed that both MTHFR rs1801133 (677C>T) and rs180113 (1298A>C) polymorphisms were significantly associated with an increased risk of bladder cancer in Asians. Further studies with larger sample size, well-designed, and population-based studies among different ethnicities required to validate our findings.

Author Contribution Statement
Conceived and designed the study and experiments: SAD and FA. Performed the experiments: HM, JSY and SHS. Analyzed the data: SAD and HN. Contributed reagents/materials/analysis tools: FA, SK and MZS. Wrote the paper: HN, JSY and EA. All authors reviewed the manuscript.