DNA methylation variations in familial female and male breast cancer

Abstract

In total, ~25% of familial breast cancer (BC) is attributed to germline mutations of the BRCA1 and BRCA2 genes, while the rest of the cases are included in the BRCAX group. BC is also known to affect men, with a worldwide incidence of 1%. Epigenetic alterations, including DNA methylation, have been rarely studied in male breast cancer (MBC) on a genome-wide level. The aim of the present study was to examine the global DNA methylation profiles of patients with BC to identify differences between familial female breast cancer (FBC) and MBC, and according to BRCA1, BRCA2 or BRCAX mutation status. The genomic DNA of formalin-fixed paraffin-embedded tissues from 17 women and 7 men with BC was subjected to methylated DNA immunoprecipitation and hybridized on human promoter microarrays. The comparison between FBC and MBC revealed 2,846 significant differentially methylated regions corresponding to 2,486 annotated genes. Gene Ontology enrichment analysis revealed molecular function terms, such as the GTPase superfamily genes (particularly the GTPase Rho GAP/GEF and GTPase RAB), and cellular component terms associated with cytoskeletal architecture, such as 'cytoskeletal part', 'keratin filament' and 'intermediate filament'. When only FBC was considered, several cancer-associated pathways were among the most enriched KEGG pathways of differentially methylated genes when the BRCA2 group was compared with the BRCAX or BRCA1+BRCAX groups. The comparison between the BRCA1 and BRCA2+BRCAX groups comprised the molecular function term 'cytoskeletal protein binding'. Finally, the functional annotation of differentially methylated genes between the BRCAX and BRCA1+BRCA2 groups indicated that the most enriched molecular function terms were associated with GTPase activity. In conclusion, to the best of our knowledge, the present study was the first to compare the global DNA methylation profile of familial FBC and MBC. The results may provide useful insights into the epigenomic subtyping of BC and shed light on a possible novel molecular mechanism underlying BC carcinogenesis.

Keywords: DNA methylation; Gene Ontology; epigenomics; female breast cancer; male breast cancer; methylated DNA immunoprecipitation on chip.

Figure 1.

Evaluation of MeDip enrichment. The Ct values of H19 gene and CTRL regions were evaluated by qPCR in MeDip DNA and input DNA fractions from 3 randomly selected breast cancer cases. The enrichment levels for the methylated regions were calculated by the qPCR threshold cycle using the formula 2^{−[(H19me-H19in)-(CTRLme-CTRLin)]}, where, in case of an undetected Ct value, a value of 40 was used. Each sample was tested in triplicate. Error bars represent Ct standard error. Ct, threshold cycle; qPCR, quantitative PCR; MeDip, methylated DNA immunoprecipitation; Undet., undetermined; CTRL, control.

Figure 2.

Hierarchical clustering of the Human Promoter array methylation data. Hierarchical clustering of microarray methylation data of input and MeDip DNA from the examined BC cases. Two arrays per BC case (1 input DNA and 1 MeDip DNA) were performed. BC, breast cancer; FBC, female BC; MBC, male BC; MeDip, methylated DNA immunoprecipitation.

Figure 3.

PCA of the Human Promoter array methylation data. PCA of microarray methylation data of input (yellow) and MeDip (green) DNA from the examined BC cases. Two arrays per BC case (1 input DNA and 1 MeDip DNA) were performed. BC, breast cancer; MeDip, methylated DNA immunoprecipitation; PCA, principal component analysis.

Figure 4.

Genomic distribution of identified DMRs. Pie chart showing the percentage of total DMRs according to their functional genomic distribution. AnnotatePeak (ChIPSeeker) was used to assign the DMRs according to their genomic positions, considering the promoter regions (in 1 kb region upstream from TSS-in the region between 1 kb and 5 kb upstream from TSS-in the region between 5 kb and 8 kb upstream from TSS), 3′ and 5′UTR regions, exons, introns, the downstream regions defined as the downstream of gene end, and distal intergenic regions. DMRs, differentially methylated regions; TSS, transcriptional start sites; UTR, untranslated region.

Figure 5.

Methylation levels of keratin filament genes in FBC compared with MBC. Dot plots were generated by Partek Genomics Suite and represent the DMRs associated with GO term ‘GO: 0045095: Keratin filament’ in FBC compared with MBC. Each graph refers to a specific KRT gene and KRTAP genes. Within a graph a point represents the Δ methylation value of a DMR for a given breast cancer case. FBC cases are shown in red and MBC cases are shown in blue. A total of two DMRs were detected for the KRT78 gene, indicated as (i) and (ii). The numbers on the y-axis are Δ methylation values. DMR, differentially methylated region; FBC, female breast cancer; GO, Gene Ontology; KRT, keratin; KRTAP, keratin-associated protein; MBC, male breast cancer.