Introduction

Multiple myeloma (MM), the second most common hematological cancer, is a kind of plasma cells cancer characterized by bone marrow plasmacytosis and presence of monoclonal immunoglobulin1,2. MM constitutes nearly one-fifth of all hematological malignancies and its prevalence is expected to rise in Western countries due to the population aging3,4. It has been estimated that every year, there are about 86,000 newly diagnosed patients with MM, accounting for approximately 0.8% of all new cancer cases and 63,000 related deaths, which constitute 0.9% of all cancer deaths3,5. However, the etiology of MM remains largely unknown. In general, MM is considered as a multifactorial disease, facilitated by the interaction between various environmental and promoter factors3,6. Risk factors such as increased age, positive family history, tobacco smoking, alcohol consumption, ionizing radiation, industrial occupation and obesity have been reported to influence the development of MM7,8. Recently, some evidence have demonstrated that genetic predisposition is also involved in MM carcinogenesis and genetic polymorphisms in candidate genes, including the immune response, DNA repair and folate metabolism, have been found to be associated with the susceptibility to MM9,10,11. Moreover, increasing epidemiological studies have suggested that the promoter methylation of some candidate genes may be connected with MM pathogenesis12,13.

Methylenetetrahydrofolate reductase (MTHFR), the most critical enzyme in folate-metabolizing pathway, catalyzes the irreversible reduction of 5.10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which acts as a methyl donor for the remethylation of homocysteine into methionine14. Therefore, MTHFR plays an important role in the folate metabolism network and is a critical metabolic juncture in the regulation of DNA synthesis, methylation and repair15,16. The MTHFR gene locates in chromosome 1p36.317. Two common functional polymorphisms in the MTHFR gene, C677T (rs1801133) and A1298C (rs1801131), have been identified and the variants are associated with low levels of plasma folate and significantly reduced activity of the MTHFR enzyme18,19,20. 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 deletion21,22. In addition, the hypomethylation of DNA may also result in activation and increased expression of proto-oncogenes, contributing to an increased prevalence of cancer23. 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 study24,25. 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 polymorphism24,25,26,27,28,29,30,31,32, seven studies bearing 732 cases and 2,841 controls for the A1298C polymorphism24,26,27,28,29,30,32,33. Of these publications, there were seven studies for Caucasians24,25,26,29,30,31,33 and three studies for Asians27,28,32. When divided by the source of controls, seven studies were population-based24,26,28,29,30,32,33 and one was hospital-based designed28, respectively. The controls in the study by Martino et al. were selected among the general population and hospitalized subjects with diagnoses excluding cancer32. 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.30 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.

Table 1 Main characteristics of studies included in the meta-analysis.
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Table 2 The genotypes distribution of MTHFR C677T and A1298C polymorphisms in cases and controls.
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Figure 1
figure 1

Flow diagram of study selection.

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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 C677T polymorphism and MM in overall comparisons (CT vs. CC, OR = 1.04, 95%CI = 0.93-1.17, P = 0.49; TT vs. CC, OR = 1.16, 95%CI = 0.98-1.37, P = 0.08; TT vs. CC + CT, OR = 1.13, 95%CI = 0.98-1.32, P = 0.10; CT + TT vs. CC, OR = 1.07, 95%CI = 0.96-1.20, P = 0.22). In the subgroup analyses according to ethnicity, no significant association was observed in both Caucasians and Asians (Fig. 3, Table 3). However, when stratified by the quality score of studies (high and intermediate), a significantly increased risk for MTHFR C677T polymorphism and MM was detected in studies with intermediate quality (CT vs. CC, OR = 1.53, 95%CI = 1.03-2.27, P = 0.04; TT vs. CC, OR = 2.45, 95%CI = 1.36-4.43, P = 0.003; TT vs. CC + CT, OR = 1.94, 95%CI = 1.13-3.31, P = 0.02; CT + TT vs. CC, OR = 1.67, 95%CI = 1.15-2.44, P = 0.007) (Table 3).

Table 3 Results of meta-analysis for MTHFR C677T and A1298C polymorphisms with MM risk.
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Figure 2
figure 2

Meta-analysis for association of MTHFR A1298C polymorphism with MM risk (AC + CC vs. AA; stratified by ethnicity).

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Figure 3
figure 3

Meta-analysis for association of MTHFR C677T polymorphism with MM risk (TT vs. CC + CT; stratified by ethnicity).

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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 malignancies18,34. Previous studies have confirmed that these common functional polymorphisms of MTHFR give rise to a thermolabile enzyme with significantly reduced enzyme activity19,20. 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 cancer35 and an increased risk for non-Hodgkin lymphoma36. 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.27 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.32 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 polymorphism32. However, the study carried out by Martino et al.32 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 reviewers37. 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.

Statistical analysis

The strength of association of MTHFR C677T and A1298C polymorphisms with MM risk was assessed by odds ratios (ORs) with 95% confidence intervals (CIs) under the heterozygote model (C677T: CT vs. CC; A1298C: AC vs. AA), homozygote model (C677T: TT vs. CC; A1298C: CC vs. AA), recessive model (C677T: TT vs. CC + CT; A1298C: CC vs. AA + AC) and dominant model (C677T: CT + TT vs. CC; A1298C: AC + CC vs. AA). 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 χ2-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).

Additional Information

How to cite this article: Ma, L.-M. et al. Meta-analysis of the association of MTHFR polymorphisms with multiple myeloma risk. Sci. Rep. 5, 10735; doi: 10.1038/srep10735 (2015).