Association of MHTFR Ala222Val (rs1801133) polymorphism and breast cancer susceptibility: An update meta-analysis based on 51 research studies

Background The association between MHTFR Ala222Val polymorphism and breast cancer (BC) risk are inconclusive. To derive a more precise estimation of the relationship, a systematic review and meta-analysis was performed. Methods A comprehensive search was conducted through researching MEDLINE, EMBASE, PubMed, Web of Science, Chinese Biomedical Literature database (CBM) and China National Knowledge Infrastructure (CNKI) databases before August 2012. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate the strength of the association. Results A total of 51 studies including 20,907 cases and 23,905 controls were involved in this meta-analysis. Overall, significant associations were found between MTHFR Ala222Val polymorphism and BC risk when all studies pooled into the meta-analysis (Ala/Ala vs Val/Val: OR=0.870, 95%CI=0.789–0.958,P=0.005; Ala/Val vs Val/Val: OR=0.895, 95%CI=0.821–0.976, P=0.012; dominant model: OR=0.882, 95%CI=0.808–0.963, P=0.005; and recessive model: OR = 0.944, 95%CI=0.898–0.993, P=0.026; Ala allele vs Val allele: OR = 0.935, 95%CI=0.887–0.986, P=0.013). In the subgroup analysis by ethnicity, the same results were found in Asian populations, while no significant associations were found for all comparison models in other Ethnicity populations. Conclusion In conclusion, our meta-analysis provides the evidence that MTHFR Ala222Val gene polymorphisms contributed to the breast cancer development. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1966146911851976


Introduction
Breast cancer is the most common cancer and the main cause of cancer mortality in women. The etiology towards to this disease is poorly understood, some risk factors including familial history of the disease, age of menarche and of menopause, diet, reproductive history, high estrogen exposure as well as genetic factors may contribute to its development [1,2]. Studies suggest that the effect determined by low-penetrance genes, may provide a plausible explanation for BC susceptibility.
Polymorphisms in genes are associated with a risk or protection against the disease. 5,10-methylenetetrahydrofolate reductase (MTHFR) is one important genes located at 1p36.3 [3]. MTHFR Ala222Val polymorphism has become the most commonly studied one, which has been considered to influence the enzyme activity of MTHFR [4]. The MTHFR 222Val/Val (homozygote) genotype results in 30% enzyme activity in vitro compared with the Ala/Ala wild-type [5]. Numerous epidemiological studies have evaluated the association between the MTHFR Ala222Val polymorphisms and BC risk. However, these studies have yielded conflicting results, partially because of the possible small effect of the polymorphism on BC risk and the relatively small sample size in each of published studies. The aim of this study is to derive a more precise estimation of these associations by performing this meta-analysis.

Literature search
All studies that examined the association between the MFTHR Ala222Val polymorphism and BC were identified. A comprehensive search was conducted through researching MEDLINE, EMBASE, PubMed, Web of Science, China Biomedical Literature database (CBM) and China National Knowledge Infrastructure (CNKI) databases before August 2012. The search strategy included the combination of "breast cancer," "breast neoplasm," "methylene-tetrahydrofolate reductase," "MTHFR," "Ala222Val", "rs1801133", "variant," and "polymorphism." References of the retrieved articles were also screened. Non-familial case-control studies were eligible if they determined the distribution for this polymorphism in unrelated patients with breast cancer and in a concurrent control group of healthy subjects using molecular methods for genotyping. Of the studies with the same or overlapping data by the same investigators, we selected the most recent ones with the most subjects. We evaluated all associated publications to retrieve the most eligible literatures. The reference lists of reviews and retrieved articles were hand searched at the same time. We did not include abstracts or unpublished reports. When overlapping data of the same patient population were included in more than one publication, only the most recent or complete study was used in this meta-analysis.

Inclusion and exclusion criteria
The following inclusion criteria were used to select literatures for the meta-analysis: (1) information on the evaluation of MFTHR Ala222Val polymorphism and BC susceptibility; (2)Only the cohort and case-control studies were considered;(3) sufficient genotype data were presented to calculate the OR with 95% CI. Major reasons for exclusion of studies were: (1) none-case-control studies; (2) reviews and duplication of the previous publication; (3) control population including malignant tumor patients; (4) no usable data reported.

Data extraction
Two investigators reviewed and extracted information from all eligible publications independently, according to the inclusion and exclusion criteria listed above. An agreement was reached by discussion between the two reviewers whenever there was a conflict. The following items were collected from each study: first author's surname, year of publication, ethnicity, total number of cases and controls with Ala/Ala, Ala/Val, and Val/Val genotypes, respectively. Different descents were categorized as Caucasians, Asians, and Mixed populations which included more than one ethnic descent. For casecontrol studies, data were extracted separately for each group whenever possible.

Statistical analysis
The strength of the association between MFHTR Ala222Val polymorphism and BC risk was measured by ORs, whereas a sense of the precision of the estimate was given by 95% Cls. Two models including the fixed-effects model and the random-effects model of meta-analysis were applied for dichotomous outcomes. The fixed-effects model assumes that studies are sampled from populations with the same effect size, making an adjustment to the study weights according to the in-study variance. The randomeffects model assumes that studies are taken from populations with varying effect sizes, calculating the study weights both from in-study and between-study variances, considering the extent of variation, or heterogeneity. A P-value ≥0.10 for the Q-test indicated lack of heterogeneity among the studies, and so the summary OR estimate of each study was calculated by the fixedeffects modelm [6]. Otherwise, the random-effects model (DerSimonian and Laird method) was used [7]. I 2 statistic can be used to quantify heterogeneity irrespective of the number of studies. The significance of the pooled OR was determined by the Z-test and P<0.05 was considered as statistically significant. Subgroup analyses were performed by ethnicity to explore the reasons of heterogeneity. Sensitivity analyses were performed to assess the stability of the results. To investigate whether publication bias might affect the validity of the estimates, funnel plot were constructed. An asymmetric plot suggests a possible publication bias. Funnel plot asymmetry was assessed by the method of Egger's linear regression test, a linear regression approach to measure funnel plot asymmetry on the natural logarithm scale of OR. The significance of the intercept was determined by the t-test suggested by Egger (P<0.05 was considered  Figure 1 Forest plot of overall breast cancer risk associated with the MTHFR Ala222Val polymorphism (Ala/Ala versus Val/Val).   representative of statistically significant publication bias). All statistical tests were performed with Stata (Version 12.0, Stata Corporation, College Station, TX), using twosided P-values.

Results
Eligible studies 51 eligible studies on MTHFR Ala222Val genotypes and colorectal cancer were identified through literature search and selection based on the inclusion and exclusion criteria .  Table 1.

Meta-analysis results
Overall, there was statistically significant difference in BC risk between the patients with Ala/Ala genotype and those with Val/Val genotype (OR=0.870, 95%CI=0.789-0.958, P=0.005; Figure 1). Similarly, significant associations were Platek   Table 2). Moreover, we found significant association between Ala222Val polymorphism and BC when examining the contrast of Ala versus Val (OR=0.935, 95% CI=0.887-0.986, P=0.013; Figure 2). In the stratified analysis by ethnicity, there was significant association between Ala222Val polymorphism and BC risk for Ala/Ala vs   Figure 4) among Asian populations. For Caucasian and Mixed populations, there was no significant association between Ala222Val polymorphism and breast cancer risk (Table 2).   Figure 4 Forest plot of a meta-analysis of the association between the MTHFR Ala222Val polymorphism and breast cancer susceptibility in Asians (Ala-allele versus Ala-allele).

Sensitivity analysis
In order to compare the difference and evaluate the sensitivity of the meta-analyses, we conducted one-way sensitivity analysis to evaluate the stability of the meta-analysis. The statistical significance of the results was not altered when any single study was omitted, confirming the stability of the results. Hence, results of the sensitivity analysis suggest that the data in this meta-analysis are relatively stable and credible.

Publication bias
Begg's funnel plot and Egger's test were performed to assess the publication bias. The shape of funnel plots did not reveal any evidence of obvious asymmetry in all comparison models, and the Egger's test was used to provide statistical evidence of funnel plot symmetry. The results of Begg's test did not show any evidence of publication bias.

Discussion
Breast cancer is currently the most frequently occurring cancer and the leading causes of cancer-related death among women in the world. Single nucleotide polymorphism (SNP) is the most common form of human genetic variation, and may contribute to individual's susceptibility to cancer, however, the underlying molecular mechanism is unknown. Previous study suggested that some variants, especially those in the promoter regions of genes, may affect either the expression or activity levels of enzymes [59][60][61] and therefore may be mechanistically associated with cancer risk. Previous studies on the relationship between MTHFR Ala222Val polymorphisms and BC risk were contradictory. These inconsistent results are possibly because of a small effect of the polymorphism on BC risk or the relatively low statistical power of the published studies. Hence, the meta-analysis was needed to provide a quantitative approach for combining the results of various studies with the same topic, and for estimating and explaining their diversity. Meta analysis has great power for elucidating genetic factors in cancer. On the bases of the character of cancer, the effect of one genetic component on the development of the disease can be easily masked by other genetic and environmental factors. A meta-analysis potentially investigates a large number of individuals and can estimate the effect of a genetic factor on the risk of the disease [62,63]. The present study included data from 51 association studies that had investigated the relationship between the MTHFR Ala222Val polymorphism and BC.
This present meta-analysis, including 20,907 cases and 23,905 controls, concerned the Ala222Val polymorphism of MTHFR gene and BC risk. In the meta-analysis, we found that the variant genotypes of the MTHFR Ala222Val polymorphisms were significantly associated with BC risk. Simultaneously, the same results presented in stratified analysis by ethnicity. We found that the variant genotype of the MTHFR Ala222Val polymorphism, in Asian populations, was associated with significant increase in BC risk. Although the MTHFR Ala222Val polymorphism may be associated with DNA repair activity, no significant association of the variant genotype with BC risk was found in Caucasian and Mixed populations, suggesting the influence of the genetic variant may be masked by the presence of other as-yet unidentified causal genes involved in colorectal cancer.
Some limitations of this meta-analysis should be acknowledged. First, our result was based on unadjusted estimates, while a more precise analysis should be conducted adjusted by other factors like diet habit, smoking, drinking status, environmental factors and so on. Second, in the subgroup analyses by ethnicity, relatively limited study numbers to perform ethnic subgroup analysis of mixed populations. Moreover, there are no American and African-American descent populations. Thus, additional studies are warranted to evaluate the effect of this functional polymorphism on BC risk in different ethnicities, especially in American, African-American and Mixed populations. In addition, our analysis did not consider the possibility of gene-gene or SNP-SNP interactions or the possibility of linkage disequilibrium between polymorphisms.
Despite of some limitations, this meta-analysis provided evidence of the association between the MTHFR Ala222Val polymorphisms and BC risk, supporting the hypothesis that MTHFR Ala222Val polymorphism contributes to overall BC risk. In subgroup analysis, the same results were found in Asian populations. In order to verify our findings, welldesigned studies including different ethnic groups with a careful matching between cases and controls should be considered in future association studies to confirm the results from our meta-analysis. Moreover, further evaluating the effect of gene-gene and gene-environment interactions on the Ala222Val polymorphism and BC risk are necessary.