Colorectal cancer risk: role of one carbon metabolism on gene specific methylation

Colorectal cancer (CRC) is characterized by the accumulation of genetic and epigenetic alterations affecting oncogenes and tumour suppressor genes. This thesis is aimed to search for a possible correlation among genetic, epigenetic and environmental factors in colorectal cancer, focusing on the following items: 1) the detection of methylation levels by means of MS-HRM in promoters of APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in CRC and healthy adjacent tissue; 2) the analysis of the correlation among the methylation status of the chosen genes and both physical and clinicopathological features of the patients; 3) the analysis of one-carbon metabolism gene polymorphisms RFC1 80G>A, MTHFR 677C>T, MTHFR 1298A>C, MTRR 66A>G, MTR 2756A>G, TYMS 28 bp repeats, TYMS 1494 6 bp ins/del, DNMT3B -149C>T, DNMT3B -579G>T by means of PCR-RFLP technique and the correlation among the studied polymorphisms and the methylation levels of APC, MGMT, hMLH1, RASSF1A, CDKN2A gene promoters; 4) the analysis of folate and homocysteine values in CRC patients and their correlation with gene promoter methylation. For all the studied genes, with the exception of APC that showed a similar frequency of methylated samples in both CRC and healthy tissue, we observed a statistically significant increased number of methylated samples in CRC respect to adjacent healthy tissues. APC and MGMT resulted frequently methylated in healthy tissues but the level of promoter methylation was relatively low and not higher than 10%. Similar results were obtained also for the three other genes, that however resulted methylated only in very few healthy tissues. We found statistically a significant association between MGMT promoter methylation and stage, particularly higher methylation levels were found in adenomas vs. all other stages (stage I, II, III, IV) (P=0.036), and between RASSF1A promoter methylation and stages with stage III showing higher methylation levels than other stages (P=0.02); for about tumour size a statistically significant association between APC promoter methylation and tumour size was found, particularly T3 vs. T4 (P=0.006). Further we found an interesting positive association between age and both hMLH1 (P=0.0006) and MGMT (P=0.0023) methylation levels and a statistically significant association between hMLH1 promoter methylation and gender, particularly females showed higher methylation levels with respect to males (P=0.04). A significant correlation between age and APC promoter methylation in healthy tissue was found (P=0.01). Finally statistically significant correlations were found between the MTR 2756AG genotype (vs. AA) and higher methylation levels of both RASSF1A (P=0.0013) and MGMT (P=0.0179) in CRC samples and of APC (P=0.03) in the adjacent healthy mucosa samples, between the TYMS 1494 6bp ins/del polymorphism and both CDKN2A promoter methylation levels in CRC tissues (P=0.028) and MGMT promoter methylation in the adjacent healthy tissues (P=0.043), between the MTHFR 677TT genotype (vs. CC+CT) and CDKN2A promoter methylation in CRC tissues (P=0.018) and between the DNMT3B -579TT genotype (vs. GG+GT) and MGMT promoter methylation in CRC tissues (P=0.043). RASSF1A hypomethylation correlated with high hcy level (P=0.01) and hMLH1 hypemethylation with low folate levels, even if this last correlation was borderline (P=0.06)