This is the first case-control study to investigate the associations between the methylation of WT1, CA10 in leukocyte DNA and breast cancer risk, and the risk of different molecular types of breast cancer in a Chinese female population. After PS adjustment, we found that methylation of WT1 was significantly elevated breast cancer risk by 2.069-fold, CA10 methylation was marginally associated with breast cancer risk with OR of 1.354. Women with WT1 methylation presented a 1.620 higher risk of Luminal A and 2.231 higher risk of Luminal B subtype of breast cancer than those without methylation. CA10 methylation was significantly associated with the risk of Luminal B subtype with OR of 1.798. We further used GEO-GSE51032, a nested case control study with clear temporal relationship between methylation changes and breast cancer, as an external dataset to validate our retrospective study’s results. The nested case control study’s results showed a lower but still significant association between WT1 methylation and breast cancer risk, but the association between CA10 methylation and breast cancer risk was not statistically significant. Furthermore, we found that low level of dietary zinc intake combined with WT1, CA10 methylation was strongly associated with breast cancer risk.
Breast cancer is a heterogeneous disease with different molecular subtypes, which may present different genetic and epigenetic susceptibilities. Previous studies predominantly focused on the aberrant methylation in tissue samples and its association with the risk of different molecular types of breast cancer [30, 31], few studies have focused on the gene-specific methylation in leukocyte DNA. The methylation alternation in leukocyte DNA presented a response of the hematopoietic system [32]. Leukocyte DNA methylation could represent germline methylation, which can be applied to analyze the association with cancer risk [33]. It was further reported that BRCA1 hypermethylation in peripheral blood DNA was associated with TNBC with an OR of 5.0 [34]. Our study indicated that, after adjustment of PS, WT1 methylation was associated with the risk of Luminal A and Luminal B subtype of breast cancer with ORs of 2.620 and 3.231, and CA10 methylation was significantly associated with Luminal B subtype of breast cancer with OR of 1.798.
WT1 is a zinc finger transcription factor located on 11p13, which was first identified as a tumor suppressor gene. WT1 exon displayed significantly increased methylation in cancer tissue compared to nonmalignant breast tissue [17]. WT1 methylation in the promoter and first exon region was associated with silencing WT1 mRNA expression in MCF-7 and MDA-MB-231 breast cancer cells [9]. Our investigated sequence was 160 bp downstream of the Laux et al. sequence position. Here, we reported the methylation of the CpG island in the first exon of WT1 in blood leukocyte DNA, which contains 11 CpGs in CpG island. Furthermore, we used external data of IPEC- Italy (GEO-GSE51032) with nested case control study design and found the significant association between WT1 methylation and breast cancer risk, with two CpG probes inside our sequence, with OR of 1.879.
Previous study has shown CA10 could undergo methylation during breast carcinogenesis in tumor tissue [17]. CA10 was reported to be hypermethylated among a panel of genes in urine, which may contribute to highly accurate early detection of bladder cancer [35]. Our study suggested that CA10 methylation in leukocyte DNA was marginally associated with an elevated breast cancer risk after the adjustment of PS. The amplified sequence contained 7 CpGs and located at the second exon of CA10. The external validation dataset of GEO-GSE51032 only included 2 CpG probes and did not observe statistically significant association between CA10 hypermethylation and breast cancer risk.
To further investigate the functional relevance of the observed associations, it would be important to test whether methylation in the CpGs associated with the alteration of the expression of WT1 and CA10. Therefore, we investigated the correlations between methylation probes and expression using TCGA (http://cancergenome.nih.gov/) and Mexpress (https://mexpress.be/) The result showed that WT1 hypermethylation was negatively correlated with its expression (Cg14657517, r=-0.204, P < 0.001; Cg19074340, r=-0.201, P < 0.001), and CA10 hypermethylation was negatively related to its mRNA expression as well (Cg14054928, r=-0.182, P < 0.001; Cg20405017, r=-0.162, P < 0.001). Although discounted by different sample-derived DNA, the significant negative correlations between WT1, CA10 methylation and gene expression were consistent with our study and indicated promising potential in breast cancer risk assessment.
Recently, more researchers have focused on nutri-epigenetics, which refers to the study of nutrient with any genetic or epigenetic interaction that leads to phenotypic changes [36]. Zinc is a trace mineral that is vital for numerous cellular processes and may play an important role in cancer etiology [37]. Zinc is also an essential component of DNA-binding proteins with zinc fingers, such as WT1 [23], and carbonic anhydrase function might be influenced by the concentration of erythrocyte Zn [38]. Studies on dietary zinc intake showed that high zinc intake could reduce the risk of various cancers[20, 39, 40], including breast cancer [40]. Previous studies also reported that zinc deficiency in dietary might be associated with increasing level of specific gene methylation [25, 41]. In this study, we first investigated if dietary zinc intake interacted with DNA methylation to affect the risk of breast cancer. Although we did not observe statistically significant interactions between lower dietary zinc intake and WT1, CA10 methylation on breast cancer risk, we did find that lower zinc intake combined with methylation of WT1, CA10 significantly increased the breast cancer risk by 10.220-fold and 3.145-fold, respectively.
Our previous study have tested the accuracy of MS-HRM by detecting the WT1 methylation level with both MS-HRM and pyrosequencing, and the results were highly correlated between these two methods [42]. However, the methylation level of leukocyte DNA is relatively low and the limitation of pyrosequencing is 2%. As a reliable and high sensitive technique, MS-HRM can assess the methylation level of targeted CpGs as low as 0.1% [43]. The high consistency of our test for different runs which making the non-misclassification of methylation level between case and control and the probability of higher sensitivity of MS-HRM comparing pyrosequencing can make our study result more conserved [44].
Limitations should be taken into consideration before drawing a conclusion: first, as in all retrospective analyses, our study may have some recall bias when collecting information on environmental factors including the intake of zinc. Second, the sample size of our study is not large enough for subgroup analysis, including the subgroup analyses of low frequency environmental factors, like smoking behavior, therefore their associations with DNA methylation of WT1, CA10 could not be analyzed.
In summary, our study suggested that methylation of WT1 and CA10 in blood leukocytes may associate with the risk of breast cancer. There were also associations between WT1 methylation and risk of Luminal subtypes of breast cancer, CA10 methylation and risk of Luminal B subtype of breast cancer. Lower level of dietary zinc intake combined with methylation of WT1, CA10 may be associated with breast cancer risk.