Takano Hikaru, Shibata Tomoyuki, Nakamura Masakatsu, Sakurai Naoko, Hayashi Tasuku, Ota Masafumi, Nomura-Horita Tomoe, Hayashi Ranji, Shimasaki Takeo, Otsuka Toshimi, Tahara Tomomitsu, Arisawa Tomiyasu
Department of Gastroenterology, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Ishikawa, 920-0293, Japan.
Department of Gastroenterology, Fujita Health University, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, 470-1192, Japan.
BMC Med Genet. 2020 Oct 16;21(1):205. doi: 10.1186/s12881-020-01142-7.
CpG methylation of tumor suppressor genes occurs in the early stage of carcinogenesis. Detecting risk factors for aberrant CpG methylation is clinically important for predicting cancer development. DNA methyltransferase (DNMT) 3a is considered to play critical roles in the DNA methylation process during pathogenesis. In this study, we evaluated the association between DNMT3A polymorphisms (rs6733868 and rs13428812) and CpG methylation status in non-cancerous gastric mucosa.
We determined the DNMT3A genotype and CpG methylation status of 4 genes (p14, p16, DAPK, and CDH1) in 510 subjects without gastric cancer. Helicobacter pylori (HP) infection status was determined by the rapid urease test, urea breath test, speculum examination, or serum antibody test. We determined the DNMT3A genotype using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). CpG methylation status was determined by methylation-specific polymerase chain reaction (MSP). When the methylated band was stronger than 10 ng/μL according to the DNA marker, we judged CpG island hypermethylation (CIHM) to be present. Associations between genotypes and susceptibilities were assessed by logistic regression analysis.
The minor allele frequencies of both polymorphisms (rs6733868 and rs13428812) were lower in the CpG methylated groups of each of the 4 genes (p14, p16, DAPK, and CDH1). Using a dominant genetic model, rs6733868 was significantly associated with the hypermethylation of each gene, whereas rs13428812 was associated with the methylation of 3 genes (all except p14). When low-CIHM was defined as 1 or 2 CpG islands methylated and high-CIHM was defined as 3 or more CpG islands methylated, carrying the minor allele of rs6733868 was associated with both decreased low- and high-CIHM, and that of rs13428812 also was associated with a decrease. Comparing low-CIHM with high-CIHM, carrying the minor alleles of rs6733868 or rs13428812 was related to decreased susceptibility to high-CIHM. In HP-infected subjects, carrying the minor alleles of rs6733868 or rs13428812 had a significantly greater association with decreased susceptibility to high-CIHM.
Our study indicates that polymorphisms of DNMT3A are associated with the accumulation of gene methylation in gastric mucosa. Carrying the minor alleles of rs6733868 or rs13428812 inhibits aberrant gene methylations, which are typically enhanced by HP infection.
肿瘤抑制基因的CpG甲基化发生在癌变的早期阶段。检测异常CpG甲基化的危险因素对于预测癌症发展具有重要的临床意义。DNA甲基转移酶（DNMT）3a被认为在发病过程中的DNA甲基化过程中起关键作用。在本研究中，我们评估了DNMT3A基因多态性（rs6733868和rs13428812）与非癌性胃黏膜中CpG甲基化状态之间的关联。
我们测定了510名无胃癌受试者中4个基因（p14、p16、DAPK和CDH1）的DNMT3A基因型和CpG甲基化状态。幽门螺杆菌（HP）感染状态通过快速尿素酶试验、尿素呼气试验、窥镜检查或血清抗体试验确定。我们使用聚合酶链反应单链构象多态性（PCR-SSCP）测定DNMT3A基因型。通过甲基化特异性聚合酶链反应（MSP）确定CpG甲基化状态。当根据DNA标志物甲基化条带强于10 ng/μL时，我们判断存在CpG岛高甲基化（CIHM）。通过逻辑回归分析评估基因型与易感性之间的关联。
在4个基因（p14、p16、DAPK和CDH1）各自的CpG甲基化组中，两种多态性（rs6733868和rs13428812）的次要等位基因频率均较低。使用显性遗传模型，rs6733868与每个基因的高甲基化显著相关，而rs13428812与3个基因（除p14外的所有基因）的甲基化相关。当低CIHM定义为1个或2个CpG岛甲基化，高CIHM定义为3个或更多CpG岛甲基化时，携带rs6733868的次要等位基因与低CIHM和高CIHM的降低均相关，rs13428812的次要等位基因也与降低相关。将低CIHM与高CIHM进行比较，携带rs6733868或rs13428812的次要等位基因与高CIHM易感性降低相关。在HP感染的受试者中，携带rs6733868或rs13428812的次要等位基因与高CIHM易感性降低的关联显著更大。
我们的研究表明，DNMT3A基因多态性与胃黏膜中基因甲基化的积累有关。携带rs6733868或rs13428812的次要等位基因可抑制异常基因甲基化，而这种甲基化通常会因HP感染而增强。
