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, 16 (28), 3553-60

Promoter Methylation Status of hMLH1, MGMT, and CDKN2A/p16 in Colorectal Adenomas


Promoter Methylation Status of hMLH1, MGMT, and CDKN2A/p16 in Colorectal Adenomas

Vasiliki Psofaki et al. World J Gastroenterol.


Aim: To investigate aberrant DNA methylation of CpG islands and subsequent low- or high-level DNA microsatellite instability (MSI) which is assumed to drive colon carcinogenesis.

Methods: DNA of healthy individuals, adenoma (tubular or villous/tubulovillous) patients, and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1 (hMLH1), Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), and O-6-methylguanine DNA methyltransferase (MGMT), as well as their relation to MSI.

Results: The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma. MGMT showed the highest frequency in each group. MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas (tubular vs tubullovillous and villous adenomas). All patients with tubulovillous/villous adenomas, as well as all colorectal cancer patients, showed promoter methylation in at least one of the examined loci. These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progression in colorectal carcinogenesis. MSI and methylation seem to be interdependent, as simultaneous hMLH1, CDKN2A/p16, and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype.

Conclusion: Methylation analysis of hMLH1, CDKN2A/p16, and MGMT revealed specific methylation profiles for tubular adenomas, tubulovillous/villous adenomas, and colorectal cancers, supporting the use of these alterations in assessment of colorectal tumorigenesis.


Figure 1
Figure 1
CpG island methylation at three loci and the microsatellite instability phenotype in G I (n = 20), G II (n = 18), G III (n = 21) and G IV (n = 20). The rows represent individual cases and the dark boxes indicate methylation or microsatellite instability (MSI) phenotype; the three high-level MSI (MSI-H) cases are also indicated.
Figure 2
Figure 2
Methylation-specific polymerase chain reaction and sequence analysis. A: Methylation specific polymerase chain reaction (PCR) for hMLH1, CDKN2A/p16 and MGMT promoter methylation. In this panel, the MSI-H patients P11 and P28 show promoter hypermethylation for hMLH1, CDKN2A/p16, and MGMT. The low-level MSI (MSI-L) patient P33 shows promoter hypermethylation for MGMT. The MSI stable (MSS) patient P22 does not show promoter hypermethylation for any gene, whereas the MSS patient P43 shows promoter hypermethylation only for CDKN2A/p16.The healthy MSS patient P50 shows promoter hypermethylation only for MGMT; B: Sequence analysis of methylation-specific PCR (MSP) PCR products of hMLH1 promoter. The sequence analysis of MSP products for patients P22 (unmethylated) and P11 (methylated) reveals the complete transition of non-methylated cytosines to thymine (indicated by arrows) after bisulfite treatment. There also cytosines that are partially methylated, as indicated from the co-existence of T and C. The sequence from the bottom to top: 5’-TGG tGT TTG AtG TtG TGT TtG tGG GTA GT-3’ P22 (unmethylated); 5’-TGG t(C)GT TTG At(C)G Tt(C)G TGT TCG CGG GTA GT-3’ P11 (methylated); 5’-TGG CGT TTG ACG TCG TGT TCG CGG GTA GT-3’ (wild type). Lower case letters represent thymines derived from unmethylated cytosines, the letters in parenthesis represent partially methylated cytosines and the bolds the CpG islands. MW: Molecular weight; M: Methylated promoter; U: Unmethylated promoter.

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