MLH1 Methylation Status and Microsatellite Instability in Patients with Colorectal Cancer

Abstract

Background/Objectives: The purpose of the current study was to compare the methylation of five regions of the CpG island of MLH1 with the presence of microsatellite instability (MSI) in colorectal cancer (CRC) patients. Methods: The study analyzed 138 CRC tumor samples. DNA extraction was performed, followed by bisulfite conversion. MLH1 gene methylation was assessed by methylation-specific PCR (MS-PCR), and the resulting fragments were analyzed using polyacrylamide gels. MSI was evaluated using multiplex PCR, and the fragments were run through capillary electrophoresis. R studio (v4.4.1) and SPSS (v29.0) software were used for the statistical analysis, and values of p < 0.05 were considered statistically significant. Results: The study showed 75.4% unmethylated, 21% partially methylated, and 3.6% fully methylated samples, with region A frequently methylated. MSI was observed in 7.2% of cases (MSI-H: 5.8%, MSI-L: 1.4%). BAT-26 was the most unstable marker. A significant difference between MLH1 methylation and MSI-H (p < 0.01) was identified, but there was no relationship with specific MLH1 regions. Conclusions: No differences were identified when analyzing specific methylation regions in relation to MSI. This study is the first to describe MSI frequency in Mexican patients regardless of age.

Keywords: MLH1; colorectal cancer; methylation; microsatellite instability.

Figure 1

MLH1 gene promoter region. The image represents the promoter of the MLH1 gene with a length of 1781 bp and some key elements. At the bottom, the five analyzed regions of the CpG island are shown in colored boxes, along with the fragment size in base pairs for each one. At the top, above the black line, the locations of regulatory elements in the MLH1 promoter are outlined, such as the initiator indicated by the red triangle (−620 bp), the GC and CCAAT boxes represented by the purple (−385, −244, −174, −90, −58 bp) and green dots (−250, −113 bp), respectively. The transcription start site (TSS) is marked by the black arrow, and exon 1 in the blue box. Taken from the Eukaryotic Promoter Database [19]. Created by BioRender.

Figure 2

MLH1 methylation across MSI categories. Percentages represent the distribution of each methylation category within the MSI categories. The X-axis represents the three MSA categories (MSI-H: Microsatellite Instability High, MSI-L: Microsatellite Instability Low, and MSS: Microsatellite Stability), and the Y-axis is represented by colors corresponding to the three methylation categories: orange (UM: Unmethylated), green (PM: Partially methylated), and blue (M: methylated).