Meta-analysis of the association of MTHFR polymorphisms with multiple myeloma risk

The association of methylenetetrahydrofolate reductase (MTHFR) polymorphisms with multiple myeloma (MM) risk has been explored, but the results remain controversial. Thus, a meta-analysis was performed to provide a comprehensively estimate. The case-control studies about MTHFR C677T and A1298C polymorphisms with MM risk were collected by searching PubMed, Elsevier, China National Knowledge Infrastructure and Wanfang Databases. Odds ratios (ORs) with 95% confidence intervals (CIs) were applied to assess the strength of association. Overall, no significant association was found between MTHFR A1298C polymorphism and MM risk under all four genetic models (AC vs. AA, OR = 0.99, 95%CI = 0.82-1.20; CC vs. AA, OR = 1.14, 95%CI = 0.77-1.68; recessive model, OR = 1.10, 95%CI = 0.76-1.59; dominant model, OR = 1.01, 95%CI = 0.84-1.22). The risk was also not significantly altered for C677T polymorphism and MM in overall comparisons (CT vs. CC, OR = 1.04, 95%CI = 0.93-1.17; TT vs. CC, OR = 1.16, 95%CI = 0.98-1.37; recessive model, OR = 1.13, 95%CI = 0.98-1.32; dominant model, OR = 1.07, 95%CI = 0.96-1.20). In subgroup analyses by ethnicity, no significant association was observed in both Caucasians and Asians. This meta-analysis suggested that MTHFR polymorphisms were not associated with MM risk.

folate and significantly reduced activity of the MTHFR enzyme [18][19][20] . Previous studies have demonstrated that folate deficiency might lead to misincorporation of uracil in place of thymidine during DNA replication, resulting in DNA strand breaks and chromosomal translocation and deletion 21,22 . In addition, the hypomethylation of DNA may also result in activation and increased expression of proto-oncogenes, contributing to an increased prevalence of cancer 23 . Hence, individual susceptibility to cancer may be modified by some functional polymorphisms of MTHFR gene through the alteration of DNA synthesis and methylation. There have been several studies investigating the relationship between MTHFR C677T and A1298C genetic polymorphisms and MM susceptibility, but the published results are inconsistent, which may be attributed to the relatively small sample size and different ethnic background in each study 24,25 . Therefore, a meta-analysis was carried out to comprehensively evaluate the association of MTHFR C677T or A1298C polymorphisms with MM risk.

Results
Study characteristics. The process of study selection was showed in Fig. 1. A total of 15 potentially relevant publications were obtained through the literature search. After screening the titles, abstracts, and full-texts, five articles were excluded due to irrelevant research, review, commentary, and data duplication. Finally, a total of nine studies including 2,092 cases and 4,954 controls were included for the C677T polymorphism [24][25][26][27][28][29][30][31][32] , seven studies bearing 732 cases and 2,841 controls for the A1298C polymorphism 24,[26][27][28][29][30]32,33 . Of these publications, there were seven studies for Caucasians [24][25][26][29][30][31]33 , and three studies for Asians 27,28,32 . When divided by the source of controls, seven studies were population-based 24,26,[28][29][30]32,33 , and one was hospital-based designed 27 , respectively. The controls in the study by Martino et al. were selected among the general population and hospitalized subjects with diagnoses excluding cancer 31 . Seven studies with a quality score 7 or greater were considered as high quality, and three were classified into intermediate quality with score of 4-6 points. Genotypes distribution in the controls of all included studies were in consistent with HWE, except for A1298C polymorphism in Lima et al. 29 Table 1 showed the detailed characteristics of included studies and the genotypes distribution of MTHFR C677T and A1298C polymorphisms in cases and controls was listed in Table 2.
Results of meta-analysis. The main results of meta-analysis and heterogeneity test were summarized in Table 3. Overall, no significant association was found between MTHFR A1298C polymorphism and MM risk under all four genetic models (AC vs. AA, OR = 0.99, 95%CI = 0.82-1.20, P = 0.92; CC vs. AA, OR = 1.14, 95%CI = 0.77-1.68, P = 0.51; CC vs. AA + AC, OR = 1.10, 95%CI = 0.76-1.59, P = 0.62; AC + CC vs. AA, OR = 1.01, 95%CI = 0.84-1.22, P = 0.89). The similar results were obtained in the stratified analyses by ethnicity, source of controls (population-based), and quality score of studies (Fig. 2, Table 3). The risk was also not significantly altered for MTHFR  Publication bias and sensitivity analysis. The publication bias was detected using funnel plot and the results showed that there was no obvious asymmetry in the funnel plots, suggesting the absence of publication bias in the overall meta-analysis. We also assessed the stability of the overall results by sequential omission of individual studies. The result of sensitive analysis showed that no individual study could significantly influence the combined results, indicating the reliability and stability of our results.

Discussion
MTHFR plays an important role in the regulation of DNA synthesis, methylation, and repair. Thus, DNA methylation and synthesis may be affected by alterations in the enzyme activity of MTHFR, which subsequently increases the incidence of malignancies 18,34 . Previous studies have confirmed that these common functional polymorphisms of MTHFR give rise to a thermolabile enzyme with significantly reduced enzyme activity 19,20 . Numerous studies have been conducted to investigate the relationship between MTHFR C677T or A1298C polymorphisms and the cancers risk, and these polymorphisms were associated with a low risk of colorectal cancer 35 , and an increased risk for non-Hodgkin lymphoma 36 . However, there are controversial findings about the role of MTHFR C677T and A1298C polymorphisms in the development of MM. González Ordóñez et al. 25 showed that the 677CC genotype of MTHFR gene could be an effective protective factor against MM. Hatzimichael et al. 33 did not observe significant difference in genotype distribution of MTHFR A1298C polymorphism between MM patients and controls. No significant association between MTHFR C677T or A1298C polymorphisms and MM susceptibility was found in Chiusolo et al. 24 , suggesting that variant alleles might not play a vital role in the development risk of MM. To quantitatively and comprehensively evaluate the effect of MTHFR C677T and A1298C polymorphisms on MM risk, a meta-analysis including 10 case-control studies was performed. The present meta-analysis suggested that there was no significant association between MTHFR C677T or A1298C   polymorphism and MM risk in overall comparisons and subgroup analyses by ethnicity and source of controls. Therefore, the extensively investigated C677T and A1298C functional polymorphisms in MTHFR may not play a crucial role in the etiology of MM, which was consistent with the study reported by Martino et al. 31 The results of large-scale study with high statistical power clarified that none of the previously reported single-nucleotide polymorphisms, which were identified to be associated with genetic susceptibility to MM in the last years, were significantly associated with MM risk with the exception of one polymorphism in women, and none of the meta-analyses showed any significant association with MM risk including MTHFR C677T polymorphism 31 . However, the study carried out by Martino et al. 31 did not provide any data about the meta-analysis and did not synthetically evaluate the association of MTHFR A1298C polymorphism with MM risk. In addition, the stratification analyses by quality score of studies found that C677T polymorphism in MTHFR was significantly associated with an increased risk for MM under all four genetic models in studies with intermediate quality, but not in high-quality studies, which suggested that the methodological quality of the included studies might be a critical effect factor on the association. However, this meta-analysis has some limitations which need to be addressed. Our analyses were based on unadjusted OR values without adjustment for other covariates such as age, gender, folate intake status, and exposures, which may result in relatively low power to estimate the real association. Some stratification analyses might have insufficient statistical power to detect the effect because of the limited number of included studies. Folate status may influence the association of MTHFR polymorphisms with MM risk through gene-nutrition interaction. However, the potential gene-environment effect was not evaluated in this study due to the unavailability of original data.
In summary, the current meta-analysis found that MTHFR C677T and A1298C polymorphisms were not associated with the altered risk for MM. However, well-designed studies based on larger sample sizes are needed to validate the present findings.

Materials and Methods
Studies identification. Two authors independently conducted a systematic literature search in the PubMed, Elsevier, China National Knowledge Infrastructure platform and Wanfang databases to identify studies about the relationship between MTHFR C677T or A1298C polymorphisms and MM risk (up to December 20, 2014). The search terms and keywords used were as follows: "methylenetetrahydrofolate reductase" or "MTHFR", "polymorphism" or "variation" or "variant" or "mutant", and "multiple myeloma" or "MM" or "plasma cell myeloma" or "myeloma" or "myelomatosis", without any restriction on the language. A manual search for references cited in the eligible articles was also performed to look for additional studies.
Inclusion criteria. Studies included in this meta-analysis had to meet the following criteria: (a) case-control studies about the association of MTHFR C677T or A1298C polymorphisms with MM risk; (b) the case group had confirmed diagnosis; (c) genotype frequencies for both cases and controls were available; (d) the distribution of genotypes in the control group was in consistent with Hardy-Weinberg equilibrium (HWE). If there were multiple articles from the same study, the most relevant was included. The case reports, letters, meta-analysis, and reviews were excluded.
Data extraction. The following information were extracted from each included study: first author's name, publication year, country, ethnicity of the study population, source of controls, genotyping methods, sample size of cases and controls, genotypes distribution of the MTHFR C677T and A1298C polymorphisms in cases and controls, and HWE of control group. Two authors independently extracted information and disagreement was addressed by discussion between them.
Quality assessment. The Newcastle-Ottawa Scale (NOS) was applied to assess the quality of the included studies independently by two reviewers 37 . The NOS includes three parameters of quality for case-control studies: selection of the study population, comparability of subjects, and exposure assessment. This scale, with a maximum score of 9 points, assigns 4 points for selection, 2 for comparability, and 3 for exposure. NOS scores of 7-9, 4-6, and 1-3 were considered as high, intermediate, and low-quality studies, respectively. Any discrepancies were addressed by re-evaluation of the original studies. The Z-test was used to determine the significance of combined ORs. The heterogeneity between included studies was evaluated by the Q-test. If P > 0.05, indicating that there exists no significant heterogeneity, the fixed-effects model (Mantel-Haenszel) was selected to combine the data, otherwise, the random-effects model (DerSimonian-Laird) was applied. Subgroup analyses were performed according to ethnicity (Asians and Caucasians), source of controls (population-based and hospital-based), and quality score of studies (high and intermediate). The publication bias was detected using funnel plot and sensitivity analysis was performed by sequential omission of individual studies to assess the stability of results. HWE of genotypes distribution in the control group was checked by the χ 2 -test. All the tests were two-sided and P < 0.05 was considered as statistically significant. The data analyses were performed using the software STATA v12.0 (Stata Corporation, College Station, TX) and Review Manager v5.2 (The Cochrane Collaboration, Oxford, UK).