Mutation and methylation of hMLH1 in gastric carcinomas with microsatellite instability

Abstract

Aim: To appraise the correlation of mutation and methylation of hMSH1 with microsatellite instability (MSI) in gastric cancers.

Methods: Mutation of hMLH1 was detected by Two-dimensional electrophoresis (Two-D) and DNA sequencing; Methylation of hMLH1 promoter was measured with methylation-specific PCR; MSI was analyzed by PCR-based methods.

Results: Sixty-eight cases of sporadic gastric carcinoma were studied for mutation and methylation of hMLH1 promoter and MSI. Three mutations were found, two of them were caused by a single bp substitution and one was caused by a 2 bp substitution, which displayed similar Two-D band pattern. Methylation of hMLH1 promoter was detected in 11(16.2 %) gastric cancer. By using five MSI markers, MSI in at least one locus was detected in 17/68(25 %) of the tumors analyzed. Three hMLH1 mutations were all detected in MSI-H (>=2 loci, n=8), but no mutation was found in MSI-L (only one locus, n=9) or MSS (tumor lacking MSI or stable, n=51). Methylation frequency of hMLH1 in MSI-H (87.5 %, 7/8) was significantly higher than that in MSI-L (11.1 %, 1/9) or MSS (5.9 %, 3/51) (P<0.01-0.001), but no difference was found between MSI-L and MSS (P>0.05).

Conclusion: Both mutation and methylation of hMLH1 are involved in the MSI pathway but not related to the LOH pathway in gastric carcinogenesis.

Figure 1

MSI in gastric cancer using 5 microsatellite loci (BAT-25, BAT-26, BAT-40, D2S123, and D5S346). Arrows indicate variant conformers. N: normal DNA pattern; T: tumor speci-mens containing variant conformers representing MSI.

Figure 2

Detection of mismatching repair gene hMLH1 muta-tion in gastric cancer by two-dimentional DNA electrophoresis. Four-band pattern was observed at exon 8.

Figure 3

Methylation of hMLH1 in gastric cancer. A: No me-thylation was found in normal mucosa; B: Methylation of hMLH1 in MSI-H gastric cancer. Cases 1, 3 showed both hypermethylation and unmethylation; C: Methylation of hMLH1 in MSI-L gastric cancer. Case 3 showed both hypermethylation and unmethylation; U: Unmethylation; M: methylation.