High-throughput quantification of hMLH1 promoter methylation using Pyrosequencing™ technology

Abstract

Promoter hypermethylation of hMLH1 is believed to account for the majority of hMLH1 down-regulation in sporadic gastric and colorectal cancers. As a key DNA mismatch repair gene, hMLH1 silencing leads to the manifestation of microsatellite instability (MSI), which is recognized as a specific tumorigenic pathway in both cancer types. Although biallelic methylation of the hMLH1 promoter has been demonstrated in colon cancer cell lines, such information is rarely available in primary tumors, neither can it be implied without quantitative data on methylation level. Currently, few techniques offer simple, high-throughput quantitative measurement of methylation at multiple CpG sites. Pyrosequencing technology has recently emerged for quantifying allele frequency at polymorphic sites. Its application has been extended to the measurement of methylation levels, where the cytosine methylation at individual CpG sites is viewed as a C/T polymorphism after bisulphite modification and PCR amplification. We report here a Pyrosequencing protocol for the accurate measurement of hMLH1 promoter methylation level. Assessment of methylation levels at five individual CpG sites showed a good correlation with the expected percentages of methylated population generated by the mixing of DNA from a methylated and an unmethylated cell lines in variable proportions (R2=0.97). These assessed sites were previously shown by our group and others to have a strong positive correlation with gene expression level. The specific primers chosen here showed minimal PCR bias in amplification of the methylated and unmethylated alleles. Upon measurement in gastric adenocarcinoma, a clear cut-off of below 5% methylated population (mean of five positions) was found in all non-neoplastic gastric mucosa (n=20) as well as in those tumors without hMLH1 protein loss (n=31). For those tumors with protein loss (n=19, all manifested MSI), methylation levels of 20-80% were noted. In parallel, methylation status of all tumors were determined by direct bisulphite genomic sequencing, from which data was in complete concordance with those from Pyrosequencing. Interestingly, among the methylated tumors, 42% (8/19) presented with a methylation level of less than 50%. Upon histological review, this was unlikely attributed solely to normal cells contamination, and thus may suggest additional genetic events for hMLH1 inactivation in these cases. In conclusion, Pyrosequencing was validated as a simple and reliable method for the quantification of methylation. Its application in hMLH1 methylation study, together with mutation analysis, offer a comprehensive assessment of the underlying mechanisms for hMLH1 silencing. This information will be especially useful in genetic counseling and risk prediction in patients with cancers involving hMLH1 inactivation, such as the Hereditary Non-Polyposis Colorectal Cancer.