Methylation Profiles of hMLH1 Proximal Promoter in Colorectal Tumor Progression

AIM: Even though hereditary colorectal cancer (CRC) risk could be predicted in healthy siblings when a mismatch-repair germline mutation is found, no biologic test is available to predict sporadic CRCs. In these tumors, methylation of the whole hMLH1 promoter leading to the lack of hMLH1 protein expression has been related to carcinogenesis. The aim of this study was to identify a region in hMLH1 promoter which methylation may confer to benign lesion a high risk of carcinogenesis.METHODS: To test the methylation status of the proximal hMLH1 promoter, we analyzed tumor samples obtained from 18 patients [6 showing benign polyps with microsatellite stability (MSS), 6 showing MSS CRCs and 6 showing CRCs with microsatellite instability (MSI)] by bisulfite sequencing.RESULTS: All tissues tested showed methylated CpG(s) 43 and 44. Among MSI CRCs, 3 showed a methylated status and no hMLH1 staining, and 3 showed an unmethylated status and hMLH1 staining. One benign polyp showed CpG(s) 1 to 37 methylated and 1 CRC sample with MSS status showed CpG(s) 1 to 23 methylated. Immunohistochemistry showed large areas with loss of hMLH1 expression in the benign polyp even though hMLH1 was detected in the CRC tissue sample with MSS status.CONCLUSION: The methylation of CpG(s) located in the 5' end of the hMLH1 promoter until CpG 37 may be an early event of colorectal carcinogenesis. The hMLH1 promoter region containing CpG(s) 23 to 37 might be a target zone to optimize a predictive bisulfite sequencing test to identify polyps with high risk of carcinogenesis.