Identification of novel methylation/expression markers and genes associated with breast cancer

A96 Purpose: to identify cancer related genes undergoing altered epigenetic regulation in breast cancer; to evaluate methylation and expression alterations of the identified genes in breast cancer.Methods. We have elaborated a novel modification of arbitrary-primed methylation sensitive PCR to identify CpG islands differentially methylated in breast cancer and adjacent morphologically intact tissues. Genomic locations of the identified fragments were investigated by direct sequencing and BLAST search. Methylation frequencies in 108 breast cancer samples were evaluated by methylation sensitive PCR and direct sequencing of bisulphite-treated DNA. Fine methylation maps for the identified genes9 promoter CpG islands were constructed by means of direct bisulphite sequencing. Expression patterns were evaluated for each of the genes under study by real-time RT PCR.Results. CpG islands identified as differentially methylated in breast cancer and control samples in this study appeared to belong to SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, RPSA, CACNG4 and PSMF1 genes. The frequencies of abnormal methylation in breast cancer varied from 2% in RPSA to 38% in SEMA6B. Significant frequencies of abnormal methylation were detected for SEMA6B, BIN1, and LAMC3 (38%, 18%, and 8%, correspondingly). Methylation of the genes under study was not characteristic for morphologically intact breast tissues. Methylation mapping revealed nonrandom distribution of methylated cytosines across the CpG islands with certain regions within the same island being frequently and some rarely/never methylated in breast cancer. Abnotmal methylation was not necessarily restricted to the core regions of the CpG islands. Significant expression distortions were observed for all the genes under study with a pronounced shift towards underexpression. Downregulation of SEMA6B, BIN1, VCPIP1, LAMC3, KCNH2, RPSA, CACNG4 and PSMF1 in breast cancer was as frequent as 44% (SEMA6B) - 94% (LAMC3). Genes with higher methylation scores demonstrate more pronounced downregulation (by quantitative expression analysis, RT PCR).Conclusions. We have identified a number of candidate cancer related genes abnormal methylation of any of which has not been reported previously. Expression studies demonstrate their frequent downregulation in breast cancer. Further research will reveal their roles in carcinogenesis and may promote the development of novel cancer molecular markers.The study was partially supported by Applied Biosystems, USA.