Aheavy methyl real-time pcr assay for detection of aberrant methylated sept9 in sporadic colorectal cancer

Epigenetic markers based on differential methylation of DNA sequences are used in cancer screening and diagnostics. The SEPT9 gene methylation assay is the first FDA-approved blood Epi proColon(Epigenomics AG, Berlin, Germany) assay for colorectal cancer (CRC) screening. It was shown recently major changes in the methylation pattern of SEPT9_V2 transcript in colon adenoma and cancer tissues are confined to only one of the CpG islands, the CGI3. Hypermethylation at CGI3 may suppress the normal expression of SEPT9_V2, which, in turn, disturbs the formation of structured filaments and the key cellular functions. In this study, we aimed to analyze promoter methylation status of SEPT9 genes in CRC patients.Genomic DNA was extracted from 30 CRC tumor tissues and pairedadjacent healthy tissues. Extracted DNA underwent bisulfite conversion and real-time analysis. The real-time assay for SEPT9 was developed based on Heavy Methyl technology in which oligonucleotideprimers were designed for bisulfite converted sequences using methylation non-specific primers (containing no CpG sites), methylation-specific probes and blocker sequences specific for un- methylated DNA to inhibit amplification of undesired sequence. This study demonstrated that there was a significantly higher (p-value< 0.05) frequency of SEPT9 methylated DNA in tumor tissue versus paired adjacent healthy tissue with a sensitivity and specificity of 87% and 93%, respectively. In conclusion, our results demonstrated that SEPT9 promoter methylation in tumor tissue samples had a high sensitivity and specificity for the detection of CRC

کلیدواژه ها:

Colorectal Cancer ، Cancer Diagnosis ، Cancer Prevention ، Cell and Cancer ، Cancer Genetics