Epigenetic Analysis of Colorectal Carcinoma (CRC) Related Genes by Means of Methylation-Sensitive High-Resolution Melting (MS-HRM) in CRC Patients

Tumorigenesis in the human colorectum is now regarded as based on multistage genetic and epigenetic alterations in the epithelial cells. Particularly, within the multistep process leading to colorectal carcinoma (CRC), loss of function of critical genes can result from either point mutations, chromosome rearrangements or epigenetic modifications. One of the most studied epigenetic signatures of CRC cells is DNA methylation of CpG islands within the promoter region of critical genes, usually related to gene silencing. In the present study we analysed the methylation status of CRC related genes including RASSF1A, MGMT, APC, hMLH1, and CDKN2A in the DNA obtained from epithelial cancerous cells isolated from either early or late stage CRC tissues through immunomagnetic method, in order to evaluate the association between methylation and clinicopathological features. Promoter methylation profiles have been evaluated by means of Methylation-Sensitive High- Resolution Melting (MS-HRM) technique. When possible, we also measured plasma homocysteine concentrations and serum folate and vitamin B12 values, all required for the synthesis of DNA methylation precursors, thereby affecting the intracellular methylation potential. Preliminary results on 30 CRC patients suggest that APC is the most frequently methylated gene in our cohort, showing promoter methylation in almost one-half of the patients. All the other genes are found to be methylated in approximately 20-25% of the cases. Particularly, MGMT and hMLH1 genes showed promoter methylation only in late stages of CRC. Interestingly, several CRC patients had high plasma Hcy levels (above the normal range). Present results are part of an ongoing project aimed at recruiting a large cohort of CRC subjects in order to correlate Hcy, folate, vit B12 values and tumour stadiation with the levels of methylation of selected genes