骨髓增生异常综合征GSTM5基因启动子区甲基化的状态及意义

Study on promoter methylation status of glutathione transferase mu5 gene in MDS and its significance

目的探讨谷胱甘肽转移酶mu5(GSTM5)启动子区甲基化状态在骨髓增生异常综合征(MDS)发生发展中的作用,为MDS的早期诊断提供新的潜在分子标记物。方法收集2018年10月至2021年6月在解放军总医院血液科初诊的40例MDS[MDS伴单系发育异常(MDS-SLD)5例,MDS伴多系发育异常(MDS-MLD)7例,MDS伴环形铁粒幼红细胞(MDS-RS)6例,MDS伴原始细胞增多Ⅰ型(RAEB1)13例,MDS伴原始细胞增多Ⅱ型(RAEB2)9例]患者、8例MDS转化为急性髓系白血病(sAML)患者和6例对照(4例缺铁性贫血、2例营养性巨幼细胞性贫血)患者的骨髓血标本。采用Agena MassARRAY核酸质谱技术对3组标本进行GSTM5基因启动子区甲基化的定量检测。采用Wilcoxon非参数检验比较不同组GSTM5基因甲基化水平。采用受试者工作特征(ROC)曲线评价试验的特异度、敏感度和准确度。结果聚类分析结果显示,MDS组GSTM5甲基化率高于对照组[0.50(0.27,0.79)比0.29(0.10,0.45),P<0.05];sAML组GSTM5甲基化率明显高于MDS组[0.67(0.36,0.86)比0.50(0.27,0.79),P<0.05]。分析各CpG位点甲基化率的差异,在CpG_1、CpG_4,5、CpG_6,7,8、CpG_11、CpG_13,14、CpG_15、CpG_16、CpG_22和CpG_24位点,MDS组与对照组比较,差异有统计学意义(P<0.05)。ROC曲线分析结果显示,在CpG_6,7,8单位曲线下面积最大,AUC=0.861(95%CI 0.717~1.000,P<0.05),敏感度和特异度分别为85%和83%。分析GSTM5甲基化与MDS疾病发展的关系,在骨髓原始细胞较高组和高危亚组(RAEB)中GSTM5基因甲基化水平较高。结论GSTM5基因启动子区甲基化在MDS中较常见,并且在MDS发生发展过程中可能起重要作用,可作为诊断MDS的潜在肿瘤标志物。

Objective To investigate the role of methylation status of glutathione transferase mu5(GSTM5)promoter region in the occurrence and development of myelodysplastic syndrome(MDS)and provide a new potential molecular marker for the early diagnosis of MDS.Method Bone marrow blood samples were collected from 40 patients with initial diagnosis of MDS[5 cases of MDS with single dysplasia(MDS-SLD),7 cases of MDS with multilineage dysplasia(MDS-MLD),6 cases of MDS with ringed sideroblasts(MDS-RS),13 patients with refractory with excess blast-1(RAEB1),9 patients with refractory with excess blast-2(RAEB2)],8 patients with AML secondary to MDS and 6 patients with non-malignant blood diseases(4 patients with iron deficiency anemia and 2 patients with nutritional megaloblastic anemia)in PLA General Hospital from October 2018 to June 2021.Methylation status of the promoter region of GSTM5 gene in three groups were detected by the Agena MassArray nucleic acid mass spectrometry.The Wilcoxon nonparametric test[non-normally distributed data,median(IQR)]was used to compare the methylation levels of GSTM5 gene in different groups.Receiver operating characteristic(ROC)curve was used to evaluate the specificity,sensitivity and accuracy of the test.Results Cluster analysis showed that the methylation status of GSTM5 in MDS group was higher than that in control group[0.50(0.27,0.79)vs.0.29(0.10,0.45),P<0.05];The methylation status of GSTM5 in sAML group was significantly higher than that in MDS group[0.67(0.36,0.86)vs.0.50(0.27,0.79),P<0.05].The differences in the methylation status of each CpG site were analyzed,and there were statistically significant differences between the MDS group and the control group at CpG_1,CpG_4,5,CpG_6,7,8,CpG_11,CpG_13,14,CpG_15,CpG_16,CpG_22 and CpG_24 sites(P<0.05).The results of ROC curve analysis showed that the area under the CpG_6,7,8 site curves was the largest,with AUC=0.861(95%CI 0.717-1.000;P<0.05),and the sensitivity and specificity were 85%and 83%,respectively.By analyzing the relationship between GSTM5 methylation and MDS disease development,GSTM5 methylation levels were significantly increased in the higher bone marrow blast group and the high-risk subgroup(RAEB).Conclusion Aberrant DNA promoter methylation of GSTM5 was a frequent event in MDS and may play an important role in the occurrence and development of MDS.It might be served as a promising biomarker in the diagnosis of MDS.