Unidade Integrada de Farmacologia e Gastroenterologia, Universidade São Francisco, Bragança Paulista 12916-900, SP, Brazil.
World J Gastroenterol. 2013 May 28;19(20):3043-51. doi: 10.3748/wjg.v19.i20.3043.
To evaluate the association between Helicobacter pylori (H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI).
The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSP-PCR) 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.
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).
In summary, MGMT and MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection.
评估幽门螺杆菌（H. pylori）感染与 MLH1 和 MGMT 甲基化的相关性及其与微卫星不稳定性（MSI）的关系。
采用甲基化特异性聚合酶链反应（MSP-PCR）分析未感染或感染 H. pylori 的儿童（n=50）、慢性胃炎成人（n=97）和胃癌成人（n=92）胃活检样本中 MLH1 和 MGMT 启动子区域的甲基化状态。采用实时 PCR 测量 MLH1 和 MGMT mRNA 表达，并标准化为组成型基因（β肌动蛋白）。通过 PCR 筛选 4 个微卫星标记物（Bat-25、Bat-26、D17S250 和 D2S123）进行 MSI 分析；PCR 产物通过单链构象多态性分析，然后用银染色。采用 χ(2)检验和 Yates 连续性校正或 Fisher 确切检验进行统计学分析，采用未配对学生 t 检验评估表达分析的统计学意义。
儿童胃活检样本中 MLH1 和 MGMT 启动子区域未检测到甲基化，无论 H. pylori 感染状态如何。51%的慢性胃炎成人患者 MGMT 启动子发生甲基化，与 H. pylori 感染相关（P<0.05）；66%的胃癌患者该区域发生甲基化，胃癌患者与 H. pylori 感染慢性胃炎患者相比，甲基化样本的百分比差异有统计学意义（P<0.05）。H. pylori 感染和未感染的慢性胃炎成人患者中 MLH1 甲基化频率分别为 13%和 7%。与 H. pylori 感染的成人慢性胃炎患者相比，胃癌患者的 MLH1 启动子甲基化（39%）和 MSI 水平升高（68%）（P<0.001 和 P<0.01）。与 H. pylori 阳性慢性胃炎样本相比，胃癌样本中两个基因的启动子甲基化频率更高（P<0.05）。MLH1 和 MGMT mRNA 水平在同时发生高甲基化的慢性胃炎样本中显著降低（P<0.01）。
总之，MGMT 和 MLH1 甲基化在早期 H. pylori 感染中并未发生，因此可能取决于感染时间。
