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AIM:To evaluate the association between Helicobacter pylori(H.pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability(MSI).METHODS:The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction(MSPPCR) in gastric biopsy samples from uninfected or H.pylori-infected children(n = 50),from adults with chronic gastritis(n = 97) and from adults with gastric cancer(n = 92).MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene(β actin).MSI analysis was performed by screening MSI markers at 4 loci(Bat-25,Bat-26,D17S250 and D2S123) with PCR;PCR products were analysed by single strand conformation polymorphism followed by silver staining.Statistical analyses were performed with either the χ 2 test with Yates continuity correction or Fisher’s exact test,and statistical significance for expression analysis was assessed using an unpaired Student’s t-test.RESULTS:Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children,regardless of H.pylori infection status.The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H.pylori infection(P < 0.05);this region was methylated in 66% of gastric cancer patients,and the difference in the percentage of methylated samples between these patients and those from H.pylori-infected chronic gastritis patients was statistically significant(P < 0.05).MLH1 methylation frequencies among H.pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%,respectively.We observed methylation of the MLH1 promoter(39%) and increased MSI levels(68%) in samples from gastric cancer patients in comparison to samples from H.pylori-infected adult chronic gastritis patients(P < 0.001 and P < 0.01,respectively).The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H.pylori-positive chronic gastritis samples(P < 0.05).The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated(P < 0.01).CONCLUSION:In summary,MGMT and MLH1 methylation did not occur in earlier-stage H.pylori infections and thus might depend on the duration of infection.
AIM: To evaluate the association between Helicobacter pylori (H.pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI). METHODS: The methylation status of the MLH1 and MGMT promoter region was was analyzed by methylation specific methylation-polymerase chain reactions (MSPPCR) in gastric biopsy samples from uninfected or H. pylori-infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer expression were measured by real-time PCR and normalized to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyzes were performed with either the χ 2 test with Yates continuity correction or Fisher’s exact test, and statistical significance for expression analysis was assessed using an unpaired Student’s t-test. RESULTS: Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P <0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori-infected chronic gastritis patients was significantly ( P <0.05) .MLH1 methylation weights among H.pylori-infected and non-infected chronic gastritis adult patients were 13% and 7% respectively. Observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori-infected adult chronic gastritis patients (P <0.001 and P <0.01, respectively). The frequency of promoter methylation for both genes was higher in gastric cancer s ampEL than those in H. pylori-positive chronic gastritis samples (P <0.05) .CONCLUSION: In summary, MGMT and MLH1 methylation did not occur in earlier-stage H. pylori infections and therefore might depend on the duration of infection.