Promoter hypermethylation of MMR genes.
(a) Diagram showing the in silico mapping of putative CpG islands in the promoter region of hMLH1, hMSH6 and hMSH2 genes with position of forward and reverse primers demarcated as half arrows which were used in methylation assays. TSS indicates the transcriptional start site.(b) Gel photograph illustrating the methylation status of hMLH1 and hMSH2 promoter region CpG islands in prostate cancer and BPH tissues as determined by methylation-specific PCR in the left and right panels respectively. Primer sets for amplification were designated as unmethylated (U) and methylated (M).The presence of PCR product in lanes marked U indicates unmethylated hMLH1 and hMSH2; product in lanes marked M indicates hypermethylated hMLH1 and hMSH2. L indicates 100 bp plus ladder. (c) Distribution of methylation quotients [MQ = (logMLH1/ACTB) X1000] obtained from analyzing prostate cancer and BPH tissue DNAs using qMSP. The calibration curve was generated to determine quantitative accuracy of qMSP with five different dilutions of in vitro fully methylated DNA from normal healthy human lymphocytes.(d) Plot showing correlation between transcript levels of hMLH1 (∆Cq) in prostate cancer tissue samples with methylation quotients corresponding hMLH1 promoter. Pearson’s correlation coefficient (r) and P values are indicated for each test. * indicates P <0.05.
