Abstract
Polymorphisms in genes encoding enzymes of folate metabolism are a focus of breast cancer risk studies due of the role of these enzymes in DNA methylation, synthesis, and repair. MTHFR, encoding for 5,10-methylenetetrahydrofolate reductase, is one of the most studied genes in this regard, but findings are controversial, and the majority of studies have analyzed polymorphisms individually. In this case control study, we examined the combination of the polymorphisms MTHFR C677T and A1298C with MTR A2756G, where MTR, methionine synthase, is an important enzyme of the folate cycle in the methylation pathway. One hundred and forty-two patients with breast cancer and controls were included and the genotypes were determined using PCR-RFLP. In the population studied, individuals carrying the polymorphic allele in the heterozygous state for both enzymes, MTHFR C677T and MTR A2756G, had an increased risk [odds ratio, OR=2.77 (95% confidence interval, CI=1.19-6.52)] for disease, compared to those with the wild genotype. In addition, individuals carrying the MTR 2756 genotype AG had an increased risk when this was combined with the MTHFR 1298 genotype CC [OR=5.13 (95% CI=0.87-38.82)]. No significant results were found from the analyses associating the MTHFR C677T and A1298C genotypes. However, when stratifying the patients by age (50 years old as the cut-off), patients over 50 years old had greater risk, with the presence of both MTHFR polymorphisms in the heterozygous state [OR=5.33 (95% CI=1.42-21.03)]. This study points out the importance of the interactions between the MTHFR C677T, MTHFR A1298C and MTR A2756G polymorphisms, and also highlights the relevance of the MTR A2756G polymorphism and age in breast cancer risk.
Abbreviations: CDC: Centers for Disease Control and Prevention; CE: ceara; CI: confidence interval; CRIO: Integrated Regional Oncology Center Clinic; DNA: deoxyribonucleic acid; LBGEM: Molecular Genetics Laboratory; MTR: methionine synthase; MTHFR: 5,10-Methylenetetrahydrofolate reductase; PCR: polymerase chain reaction; OR: odds ratio; Ala: alanine; Val: valine; Glu: glutamine; SNP: single-nucleotide polymorphisms.
Breast cancer is the leading cause of death in women with malignancies worldwide, including Brazil (1). Epidemiological evidence suggests that folate metabolism imbalance may be involved in predisposition to cancer. The relationship of folate metabolism with carcinogenesis is based on its involvement in both nucleotide synthesis and DNA methylation. Several genes controlling folate metabolism are polymorphic; however, two enzymes, 5,10-methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR), play an important role in both functions. MTHFR irreversibly converts 5,10-methyle-netetrahyrdofolate to 5-methylenetetrahydrofolate, the primary circulating form of folate (2), which is the methyl-donor for remethylation of homocysteine to methionine, mediated by MTR (3). Methionine is the precursor of S-adenosylmethionine, the universal methyl donor for biological methylation reactions, including DNA methylation (4, 5).
Two common allelic variants of the MTHFR gene have been described, C677T (rs1801133) and A1298C (rs1801131), which lead to the amino acid substitutions Ala222Val and Glu429Ala, respectively, and to decreased enzyme activity (6-8). Heterozygous and homozygous carriers of the MTHFR 677T allele variant have 30-40% and 60-70% reduced enzyme activity, respectively, as determined by in vitro analysis of MTHFR activity (6, 8, 9).
A great number of studies have examined how MTHFR polymorphisms influence the risk for breast cancer development (10-31), but in the majority, the polymorphisms had been analyzed independently, with the results being controversial. Only a few studies have analyzed both MTHFR variations, considering the interactions between the two non-synonymous single-nucleotide polymorphisms (SNP) (2,32-38).
The common polymorphism MTR A2756G (rs1805087) results in an amino acid change of Asp919Gly, and has been demonstrated to contribute to alterations in the plasma levels of homocysteine and folate (39,40). Although there are some studies that evaluate this polymorphism and breast cancer risk (12, 41, 42), there are only two studies that considered the combination of this polymorphism with those of MTHFR polymorphism in breast cancer risk (18, 43, 44).
Studies examining the interaction of these polymorphisms are important, since these enzymes interact through their substrates, and therefore, an imbalance due to the presence of polymorphisms, in combination, can be more substantial than that of single polymorphisms and may determine the routes of DNA synthesis and repair or methylation. This case control study of breast cancer risk associations with MTHFR polymorphism plus genetic polymorphism of MTR, one of the key enzymes in DNA methylation, was conducted to better-understand the relevance of these polymorphisms in the studied population.
Materials and Methods
Study population. Breast cancer cases were ascertained from 142 women being seen at the Integrated Regional Oncology Center Clinic (Fortaleza, CE, Brazil) in the years from 2004 to 2010. All cases had been histologically-proven to be breast cancer. Further clinicopathological information was obtained from hospital records, and detailed family histories were obtained through a previously validated questionnaire. Controls included healthy volunteers who were blood bank donors and university staff, without any history of malignancy at the time of ascertainment. All participants were genetically unrelated and from the same geographical region (Ceará State, Northeast Brazil), and they were matched by age (±2 years). The study was performed with informed consent from the participants and after approval from the Ethical Review Board of the Hospital Complex of the Federal University of Ceará, under protocol No. 702/04, according to the Resolution 306/04 of the National Council of Health, Ministry of Health/Brazil.
Genotyping. Genomic DNA was extracted soon after blood sampling, using a standard salting-out extraction procedure. DNA quality was determined by 1% agarose gel electrophoresis, and DNA quantified using a NanoDropTM® 3300 fluorospectrometer (Wilmington, DE, USA). The MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131) and MTR A2756G (rs1805087) genotypes were determined by PCR-RFLP. All PCR procedures were carried out according to the references cited in Table I.
To ensure quality of genotyping, random samples (10% of cases and control samples for each enzyme) were re-analyzed with the identities unknown to the laboratory staff. The concordance of the analysis was 99.5% for all studied polymorphisms. For the discordant samples, the genotype assay was repeated by two independent researchers to achieve a concordance of 100%.
Statistical analysis. Deviations of genotype frequencies were assessed for each polymorphism, comparing Hardy-Weinberg equilibrium values with control values by the standard chi-square test. Genotype frequencies in the study cases and the controls were compared by conditional logistic regression. Genotype-specific risks were estimated as an odds ratio (OR) with associated 95% confidence intervals (CI). A value of p<0.05 was considered significant. All the statistical analyses were performed using the software EpiInfo TM (v3.5.1, Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA)
Results
Genotyping was successfully performed for the polymorphisms studied for all 142 individuals. All genotype frequencies were in Hardy-Weinberg equilibrium. The frequencies of MTHFR 677CC, 677CT and 677TT were 45.8%, 46.5% and 7.7% in the cases and 52.1%, 37.3% and 10.6% in the controls, respectively. The frequencies of MTHFR 1298AA, AC and CC were 38.7%, 50.0% and 11.3% in the cases and 43.7%, 50.0% and 11.3% in the controls, respectively. A very low frequency was observed for the MTR A2756G homozygous polymorphic genotype, where the frequencies for 2756AA, 2756AG and 2756GG were 50.7%, 49.3% and 0.0% in the cases and 64.1%, 35.2% and 0.7% in the controls, respectively.
Combinations of the polymorphisms were analyzed to determine the influence on breast cancer risk. The distribution of the genotype frequencies is shown in Tables II, III and IV. It was observed that individuals carrying the polymorphic allele in the heterozygous condition for both enzymes, MTHFR C677T and MTR A2756G, had an increased risk [OD=2.77 (95% CI=1.19-6.52)] for disease compared to those with the wild-type genotype. (Table II). Regarding the combination of MTHFR A1298C and MTR A2756G polymorphisms (Table III), a lower frequency for the combination of wild-type genotypes was observed in patients compared to controls, and individuals carrying the MTR 2756AG genotype had an increased risk of disease when this was combined with the MTHFR 1298CC genotype [OR=5.13 (95% CI=0.87-38.82)].
No significant results were found from the analyses associating the MTHFR C677T and A1298C genotypes (Table IV). However, stratifying the patients by age, using 50 years of age as cut-off (Table V), showed that in patients more than 50 years old, the presence of both MTHFR polymorphisms in the heterozygous state was associated with risk [OR=5.33(95% CI=1.42-21.03)]. On the other hand, no significant results were found for patients 50 years old and younger.
Discussion
Genetic variations in enzymes involved in one-carbon metabolism have been the focus of many studies on breast cancer risk. As MTHFR is a critical gene in the one-carbon metabolism pathway, the two non-synonymous polymorphisms, C677T and A1298C, located in the coding region, have been extensively studied, but their associations with breast cancer risk are conflicting. Folate metabolism works as a cycle, where enzymes interact with each other through their substrates. Consequently, genotypes with more than one polymorphism play differential and interrelated roles in the pathophysiology of cancer. Therefore, in this study we analyzed the interaction of these two polymorphisms of MTHFR with that of MTR, in order to determine the impact of the presence of the combined polymorphisms on breast cancer risk, since these alterations in both enzymes may affect the metabolism of folate in different ways.
In this study, an increased risk was associated with the presence of the MTHFR C677T CT and MTR AG genotypes. This result is in contrast to the findings of Naushad et al. (43) and Shrubsole et al. (44), in which no association was found. In addition, we found association for the combination of the MTR 2756AG and MTHFR 1298CC genotypes with risk, and to the best of our knowledge there are no other studies reported in the literature, correlating these polymorphisms in breast cancer development. These data demonstrate the relevance of the polymorphism of MTR, in the studied population, in combination with the MTHFR polymorphisms. In our previous study with the same patients (12), in which these polymorphisms were individually analyzed, we identified breast cancer risk as being associated only with the MTR A2756G polymorphism and no risk was observed related to either MTHFR polymorphism in the same population. In contrast, other authors (30, 43) have found risk only for MTHFR polymorphisms and not MTR A2756G. The significant associations found here could explain the controversial results found in the literature, in which the polymorphisms were analyzed independently. The interaction of these polymorphisms, characterized by reduction in enzyme activity, may result in reduced methylation, thereby allowing for the activation of oncogenes, which promotes susceptibility to cancer development.
On the other hand, no association with breast cancer risk was observed in the analysis considering the two MTHFR polymorphisms. Some studies have analyzed this interaction but have yielded controversial results. The studies by Forsti et al. (34), Vainer et al. (37) and Zivo et al. (38) found no association with breast cancer risk. On the contrary, Chou et al. (45) and Sharp et al. (2) found a protective effect against breast cancer with the presence of the polymorphic allele of both enzymes, while Chen et al. (32), Ergul et al. (33) and Gao et al. (35) found an increased risk.
However, when the MTHFR polymorphisms were stratified by patient age, an increased risk was observed in women aged >50 years when carrying both MTHFR polymorphisms in the heterozygous state. We used a cut-off age of 50 because a previous study with the same patients (12) showed that this cutoff is suitable as a discriminator to stratify risk in these two age groups.
In conclusion, an association of breast cancer risk in the studied population was observed with the interaction of the polymorphic allele of MTR and both MTHFR polymorphic alleles. Moreover, we observed an age-dependent risk related to the interaction of the two MTHFR polymorphisms. In addition, we emphasized on the importance of studies that take into account the interaction between polymorphisms involved in the same metabolic cycle.
Acknowledgements
Authors are grateful to all the doctors, nursing and technical staff and staff of the Integrated Regional Oncology Center Clinic (CRIO). We are greatly indebted to the staff of the Molecular Genetics Laboratory (LABGEM) for their support and helpful discussions. Financial support was provided by the National Council for Scientific and Technological Development (CNPq). Dr. A. Leyva helped with the English language editing of the manuscript.
- Received August 7, 2012.
- Revision received October 5, 2012.
- Accepted October 8, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved