Detection of MLH1 Promoter Methylation in Colorectal Cancer using Methylation-Sensitive High-Resolution Melting (MS-HRM) Analysis

Abstract

Colorectal cancer (CRC) is one of the most prevalent types of cancer in both genders worldwide. In Indonesia alone, CRC ranks second in men and third in women for its prevalence. Microsatellite instability (MSI) occurs in 15% of all CRC cases from hereditary and sporadic origins. In sporadic cases, more than 90% of MSI cases results from mismatch repair (MMR) MLH1 gene promoter hypermethylation. The analysis of MLH1 methylation plays a crucial role in the decision-making algorithm towards having genetic testing for CRC with MSI phenotype. Methylation-sensitive high-resolution melting (MS-HRM) analysis has shown a future potential as a tool for detecting DNA methylation and determining methylation status. This study aims to optimize the detection of methylation of MLH1 promoter in CRC samples using MS-HRM analysis. There were 14 CRC patient samples used (8 MSS samples and 6 MSI samples). The methods included bisulfite conversion of isolated DNA samples of CRC, quality control using MYOD1 PCR, MS-HRM analysis for MLH1, and data analysis to determine the MLH1 methylation status of each sample. This study showed that 50% of the samples with loss of MLH1 expression and MSI scoring was able to be detected for partial methylation of MLH1. Meanwhile, the other 50% of the MSI samples were determined as MLH1-unmethylated. The results of this study suggested the importance of MLH1 methylation analysis in the genetic testing algorithm of CRC, as well as the potential of MS-HRM as the analysis method.