It has recently been recognized that cancer is a manifestation of both abnormal genetic and epigenetic events 1. Dysregulated epigenetic controls, which usually are represented by abnormal DNA methylation patterns such as global hypomethylation and region specific hypermethylation, are a hallmark of most cancers. Although the precise mechanisms underlying methylation alterations are far from being fully understood, the overall methylation process is mainly regulated by several groups of regulatory proteins 234.

The methyl-CpG-binding domain (MBD) proteins are among these protein families that bind specifically to a methylated gene and mediate transcriptional repression via effects on chromatin structure. Thus far, five MBD genes have been identified in mammalian cells that encode putative MBDs, namely MeCP2, MBD1, MBD2, MBD3, and MBD4 567. Human MBD genes are considered housekeeping genes because they are widely expressed in somatic tissues 6. Given the epigenetic role of MBD proteins in regulating gene expression, MBDs may be involved in cancer development by affecting the expression of cancer related genes. In fact, there is growing evidence that aberrant expression of MBD proteins is associated with human cancers 89.

The MBD2 gene is mapped to the conserved region within human chromosome 18q21 10. Genomic sequence analysis determined that the MBD2 gene contains six exons and one noncoding exon spanning more than 50 kb in the genome. The MBD2 gene encodes two potential forms of protein MBD2 that correspond to the initiation of translation starting at either the first (MBD2a; 43.5 kDa) or second (MBD2b; 29.1 kDa) methionine codons. The signal functions of MBD2 are to bind specifically to methylated gene promoters and recruit histone deacetylases and chromatin remodeling proteins. The altered chromatin structure resulting from the binding of these factors may be resistant to the transcriptional machinery and, as a result, repress gene expression 1112. A recent finding also suggests that MBD2 has potential DNA demethylase activity 13, implying that it might mediate gene activation in addition to transcriptional repression. However, two subsequent studies could not demonstrate any demethylase activity of MBD2 1415, and this inconsistency in the functions of MBD2 remains to be resolved.

Although our understanding of the exact function of MBD2 in epigenetics is still in its early stages, several studies in human cancer research have demonstrated that the MBD2 protein plays a role in tumorigenesis. For example, a recent study 16 showed that breast carcinomas exhibit alterations in MBD2 expression. One interesting finding from that study was that breast carcinomas can be divided into two groups, with one expressing very high levels of MBD2 and the other expressing a much lower level. MBD2 has also been reported to be involved in the repression of GSTP1 transcription in breast cancer cells 17. Moreover, a significant reduction in MBD2 mRNA expression was found in human colorectal and gastric cancerous tissues 18 and peripheral blood lymphocytes 19 in bladder cancer patients, implying a protective role for MBD2 in tumorigenesis. MBD2 protein expression and its demethylase activity were detected in normal human prostate tissue but not in cancerous tissue 20. These differences between types of cancers in the abundance of MBD2 levels may reflect different roles for MBD2 either in transcriptional repression or in the demethylation process.

Given that there is a potential role for MBD2 in tumorigenesis, we hypothesized that genetic polymorphisms in the MBD2 gene may modify an individual's susceptibility to human cancers. In this molecular epidemiologic study, we genotyped two single nucleotide polymorphisms (SNPs; rs1259938 and rs609791) in the MBD2 gene to investigate whether genetic variations in the MBD2 gene are associated with breast cancer risk and whether the potential associations are modified by menopausal status.

This study, which included 393 breast cancer cases and 436 controls, was composed entirely of Caucasians. Table 1 presents the distribution of selected baseline characteristics for cases and controls. There were significantly more postmenopausal women in the case population (77.6%) than among controls (66.5%), indicating an increased risk for breast cancer associated with menopausal status. In addition, data showed that more controls had higher family income (31%) compared to the incidence of high family income in breast cancer cases (23.5%). No other baseline factors exhibited a material difference between cases and controls.

The genotype distributions of both SNP loci for cases and controls (Table 2) were in Hardy–Weinberg equilibrium (χ² = 0.35, P = 0.56 for rs1259938; χ² = 0.31, P = 0.57 for rs609791).

Among all women, we found no overall associations between genotypes at these two loci and breast cancer risk after adjustment for age, menopausal status, family history of breast cancer in first-degree relatives, family income, body mass index, lifetime months of breastfeeding, and study site. Among premenopausal women, a reduced breast cancer risk was significantly associated with variant genotypes (homozygous minor allele + heterozygote) at both SNP loci. Specifically, women with G/A and A/A at rs1259938 had 59% reduced breast cancer risk (OR = 0.41, 95% CI = 0.23–0.72) and women with C/G and G/G at rs609791 had 46% reduced breast cancer risk (OR = 0.54, 95% CI = 0.30–0.96). However, no significant associations were detected among postmenopausal women.

These two SNP loci in the MBD2 gene may generate four possible haplotypes, and their frequency distributions among cases and controls are shown in Table 3. G-C was the most common haplotype, with a frequency of 66.90% in our control group. The frequencies of the other three haplotypes were 5.56% (G-G), 11.70% (A-C), and 15.84% (A-G) in controls. Among all female participants, none of these haplotypes was associated with breast cancer risk. However, in premenopausal women the two rare haplotypes halved breast cancer risk, with ORs of 0.40 (95% CI = 0.20–0.83, P = 0.013) for A-C and 0.47 (95% CI = 0.26–0.84, P = 0.011) for A-G. Similar associations were not observed in postmenopausal women.

It is becoming clear that carcinogenesis is a stepwise process of accumulation of both genetic and epigenetic abnormalities that can lead to cellular dysfunction. A large body of evidence has demonstrated that the epigenetic process is involved in breast carcinogenesis by influencing several broad gene categories, including cell cycle regulation, cell growth, steroid receptors, tumor susceptibility, carcinogen detoxification, cell adhesion, and inhibitors of matrix metalloproteinase genes. For example, methylation of p16 promoter and exon 1 regions are observed in both human breast cancer cell lines and 20–30% of primary breast cancers 2627. Methylation of the promoter region of GSTP1, a member of the glutathione S-transferases, which are a supergene family involved in the detoxification of carcinogens, is associated with gene inactivation in about 30% of primary breast carcinomas 28. DNA methylation has also been found to be an alternative mechanism of inactivation of BRCA1 2930, a gene that accounts for one half of inherited breast carcinomas 31. Moreover, three members of the steroid hormone superfamily, including estrogen receptor, progesterone receptor and retinoic acid receptor, have long been linked to mammary carcinogenesis 32 and recent studies have shown that epigenetic alterations appear to play a role in silencing estrogen receptors and retinoic acid receptors in breast malignancy 333435. Given the increasing evidence for a role of the epigenetic process, especially DNA methylation, in breast carcinogenesis, it is speculated that some genetic variations in methylation related genes may affect the expression and function of these genes and consequently contribute to breast cancer development.

Findings from the present study show associations between genotypes and haplotypes of the MBD2 gene and breast cancer, which have not previously been examined. These results support a potential role for methylation related genes in breast tumorigenesis. Interestingly, MBD2 polymorphisms have different effects in women depending on menopausal status. Our results demonstrate significant associations between MBD2 genotypes and haplotypes and breast cancer risk in premenopausal women but not in postmenopausal women. In fact, menopausal effects on breast cancer risk have also been observed in a previous study investigating genetic polymorphisms in catechol-O-methyltransferase 36. That study found that the low-activity allele of catechol-O-methyltransferase was associated with increased risk among premenopausal women (OR = 2.1, 95% CI = 1.4–4.3) but was inversely associated with postmenopausal risk (OR = 0.4, 95% CI = 0.2–0.7). Our findings support arguments from previous studies suggesting that different etiologies may be involved in breast carcinogenesis between premenopausal and postmenopausal women 3637.

Although the mechanisms are not elucidated, menopausal effects on the role of MBD2 in breast cancer development may be related to changes in sex hormone levels. One of the phases of breast cancer pathogenesis is exposure of breast tissue to ovarian hormones that drive the kinetics of breast tissue stem cells, resulting in carcinogenesis 38. Dividing cells are particularly susceptible to alterations in DNA synthesis, DNA repair, and DNA methylation.

These biologic and physiologic effects of sex hormones are controlled by hormone receptors, the expression of which is regulated by the methylation status of their promoter regions 333435.

On the other hand, steroid hormones may influence the epigenetic blue print of methylation of certain genes and consequently activate or inactivate gene expression 39. Even though MBD2 might be involved in the epigenetic regulation of steroid hormone receptor gene expression, MBD2 itself could also be affected by steroid hormones. It is possible that MBD2 plays different roles in breast carcinogenesis when hormone levels dramatically change. Our findings support this speculation in that we found a significantly protective role of MBD2 variants in premenopausal women but no significant associations in postmenopausal women.

There are limitations to the present study. Only Caucasian women were included in the study, and so hypotheses must be further examined in multi-ethnic groups. In addition, the sample sizes of our study limit the analyses to explore other potential risk factors. Traditional risk factors such as parity and family history of cancer did not differ between cases and controls, which could also be due to the sample sizes. Inaccurate recall may affect our assessment of family history of cancer as well.

These findings imply a potential link between DNA methylation processes and hormonal expression. Although large molecular epidemiologic studies are warranted to further examine associations between MBD2 polymorphisms and breast cancer in multi-ethnic groups, this study suggests that genetic variations in methylation related genes may serve as a promising biomarker in risk estimate of breast cancer.

bp = base pairs; CI = confidence interval; MBD = methyl-CpG-binding domain; OR = odds ratio; PCR = polymerase chain reaction; SNP = single nucleotide polymorphism.

The authors declare that they have no competing interests.

YZ designed the study and drafted the manuscript. HNB carried out the genotyping analysis. YZ and TRH performed the statistical analysis. TZ is the principal investigator for the parent case–control study and participated in the design of the present project and manuscript preparation. All authors read and approved the final manuscript.