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目的探讨人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)基因启动子区CpG岛甲基化及甲基化位点与儿童白血病临床特征的相关性。方法收集儿童白血病患者173例(Leu组),根据白血病亚型不同分为:急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)94例、急性髓细胞白血病(acute myelocytic leukemia,AML)52例、慢性粒细胞白血病(chronic granulocytic leukemia,CML)19例及复发难治的白血病(acute leukemia,AL)8例(ALL 5例,AML 3例),以非恶性血液病(NL组)42例作为对照。分离患者外周血单核细胞,提取基因组DNA,经甲基化修饰试剂盒修饰后,采用甲基化特异性-PCR(methylation specific-PCR,MS-PCR)法分别检测Leu组、NL组患者外周血标本单个核细胞中hTERT基因启动子区两个CpG岛不同位点甲基化情况,并分析其与儿童白血病临床特征的相关性。结果 hTERT基因启动子区CpG岛在NL组呈完全非甲基化,在复发难治的白血病组呈甲基化状态。NL组外周血中hTERT基因启动子区2号CpG岛(-2016～-1532和-1415～-1151)呈完全非甲基化状态,而Leu组呈甲基化状态,且甲基化更易发生在1号和2号(-2016～-1532)位点,但hTERT基因启动子区1号CpG岛甲基化在NL组和Leu组中差异无统计学意义(P>0.05)。hTERT基因启动子区甲基化与患者年龄、外周血白细胞数、免疫分型、细胞遗传标志及危险分级相关(P<0.05),与患者性别、融合基因无关(P>0.05)。结论儿童白血病患者hTERT基因启动子区CpG岛易发生甲基化,与甲基化位点分布相关,对儿童白血病的诊断具有重要意义。
Objective To investigate the relationship between methylation and methylation of CpG island in human telomerase reverse transcriptase (hTERT) promoter and clinical features of childhood leukemia. Methods A total of 173 childhood leukemia patients (Leu group) were collected and divided into four groups according to leukemia subtypes: 94 cases of acute lymphoblastic leukemia (ALL), 52 cases of acute myelocytic leukemia (AML) Nineteen patients with chronic granulocytic leukemia (CML) and 8 patients with refractory leukemia (ALL) (ALL 5, AML 3) and non-hematologic malignancies (NL) 42 patients served as controls. Peripheral blood mononuclear cells were isolated and genomic DNA was extracted. After modification by methylation modification kit, the peripheral The methylation status of two CpG islands in the hTERT gene promoter region in blood samples of mononuclear cells was analyzed and its correlation with the clinical features of childhood leukemia was analyzed. Results CpG island of hTERT gene promoter region was completely unmethylated in NL group and methylated in leukemia group with refractory relapse. NLG group hTERT gene promoter region No. 2 CpG island (-2016 ~ -1532 and -1415 ~ -1151) was completely unmethylated state, while the Leu group was methylated, and methylation was more likely to occur There was no significant difference in CpG island methylation between the 1st and 2nd (-2016 ~ -1532) sites in the NL group and the Leu group (P> 0.05). The promoter methylation of hTERT gene was correlated with age, peripheral blood leukocyte count, immunophenotyping, cytogenetic marker and risk grade (P <0.05), but not with sex and fusion gene (P> 0.05). Conclusions Methylation of CpG island in promoter region of hTERT gene in childhood leukemia is easily correlated with the distribution of methylation sites, which is of great significance in the diagnosis of childhood leukemia.