DNA methylation variations in familial female and male breast cancer

About 25% of familial breast cancer (BC) is attributed to germline mutations of BRCA1 and BRCA2 genes while the rest of patients are included in the BRCAX group. BC also affects men with a worldwide incidence of 1%. The epigenetic alterations, including those DNA methylation, have been rarely studied in the male breast cancer (MBC) on a genome-wide level. The aim of the current work was to study the global DNA methylation profiles of BC patients to look for differences between familial female breast cancer (FBC) and MBC and according to BRCA1, BRCA2 and BRCAX mutation status. The genomic DNA from FFPE tissues of 17 female and 7 male patients with BC was subjected to methylated DNA immunoprecipitation (MeDIP) and hybridized on human promoter microarrays. The comparison between FBC and MBC showed 2846 differentially methylated regions (DMRs) corresponding to 2486 distinct annotated genes. The gene ontology enrichment analysis revealdrelevant molecular function terms such as the GTPase superfamily genes (in particular the GTPase Rho GAP/GEF and GTPase RAB) and cellular component terms associated to cytoskeletal architecture such as “cytoskeletal part”, “keratin filament”, “intermediate filament". By considering only FBC, several cancer-associated pathways were the most enriched KEGG pathways of differentially methylated genes between BRCA2 and BRCAX or BRCA1+BRCAX groups. The comparison between BRCA1 group vs BRCA2+BRCAX group displayed the enriched molecular function term “cytoskeletal protein binding”. Finally, the functional annotation of differentially methylated genes between BRCAX and BRCA1+BRCA2 groups indicated that the most enriched molecular function terms were related to GTPase activity. In summary, this is the first study that compares the global DNA methylation profile of familial FBC and MBC and the results may provide useful insights into the epigenomic subtyping of breast cancer and shed light on a possible new molecular mechanisms underlying BC carcinogenesis. The global DNA methylation profiles of 24 breast cancer cases were obtained by methylated DNA immunoprecipitation coupled with Affymetrix Human Promoter 1.0R Tiling Arrays (MeDip-chip). The genomic DNA from 24 breast cancer FFPE tissues was extracted using QIAamp DNA FFPE Tissue Kit supplied by Qiagen- The genomic DNA was digested using micrococcal nuclease (New England Biolabs) to obtain DNA fragments ranging from 200 to 500 nucleotides and used them as input (IN) DNA. Agilent Bioanalyzer with the RNA 6000 Nano LabChip Kit was used to check the size of fragmented DNA. Part of IN DNA (4 µg) was immunoprecipitated (IP) with 10 µl of anti-5-MethylCytosine Antibody (Eurogentec, BI-MECY-0100) using the MeDip protocol. The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol. A total of 200 ng of IN and IP DNA was amplified with the Affymetrix Chromatin Immunoprecipitation Assay Protocol. Totalling 2 arrays per each breast cancer case were performed (1 IP DNA and 1 IN DNA). The CEL files were imported into Partek® Genomics Suite® (PGS). We performed 9 different comparisons according to gender and mutation status (BRCA1, BRCA2 and BRCAX). For each comparison performed the significantly differentially methylated regions (DMR) were obtained using ANOVA followed by MAT algorithm (model-based analysis of tiling arrays algorithm). Using PGS function, each DMR was associated to its nearest gene of human genome. The genomic coordinates of each DMR were calculated based on UCSC human genomic assembly version 18.