Densely methylated MLH1 promoter correlates with decreased mRNA expression in sporadic colorectal cancers

It has been reported that MLH1 is silenced by promoter methylation, and that this phenomenon is associated with microsatellite instability (MSI) in sporadic colorectal cancer (CRC). To clarify the significance of MLH1 promoter methylation in sporadic CRC, we examined the correlation between methylation status over the entire promoter region and mRNA expression in cases showing high-frequency MSI (MSI-H). MLH1 promoter methylation was analyzed using the bisulfite modification sequencing in 48 MSI-H cases. We also screened for somatic mutation, loss of heterozygosity, and immunohistochemical staining of MLH1. The results showed that methylation patterns could be subdivided into three types: methylation of more than 80% of the CpG sites analyzed (type 1 methylation), methylation of less than 20% (type 2 methylation), and methylation mainly in the region 500 to 921 bases upstream from the translation start site (type 3 methylation). Of the three types, only type 1 methylation correlated with decreased mRNA expression. The frequency of type 1 methylation was significantly higher in cases involving the proximal colon (66.7%, 18/27) compared to that of the distal colon and rectum (23.8%, 5/21, P = 0.004). Immunohistochemical staining of MSI-H cases showed that decreased MLH1 was found in 77.1% (37/48). Of the cases with decreased MLH1, type 1 methylation was present in 59.5% (22/37). Overall, our data suggested that the type 1 methylation pattern may affect MLH1 mRNA expression, such that the majority of MSI-H cases in sporadic CRC, especially proximal colon cancer, exhibited type 1 methylation.