甲基化芯片筛选结核感染相关基因及后续验证

Methylation Analysis and Validation of Whole Genome DNA in Active Tuberculosis

目的筛选活动性结核患者DNA甲基化水平显著变化的基因并进行验证。方法 (1)纳入年龄与性别匹配的9例活动性结核患者、3例潜伏性结核患者和3例健康对照,将人血中单个核细胞DNA与芯片杂交,比较结核组与健康组的甲基化差异基因,并对差异基因进行GO功能和Pathway功能分析,以及与表达芯片进行联合分析;(2)收集年龄与性别匹配的活动性结核与健康对照各60例,分别提取全血DNA,使用焦磷酸测序方法对甲基化芯片预测出的结核病相关基因(TLR1、TLR2、TLR4)进行甲基化程度检测。结果 (1)活动性结核患者与健康对照对比,大部分呈现甲基化下调状态,GO分析和Pathway分析结果显示,甲基化差异基因的功能主要富集在白细胞分化、凋亡、自噬、细胞因子调节及炎症反应等与结核密切相关的生物过程中;(2)待验证的3个基因TLR1、TLR2、TLR4包含10个CpG位点,其中TLR1基因的CpG1位点(P<0.001)呈现高甲基化状态,TLR4基因的CpG2位点(P=0.012)呈现低甲基化状态。结论在结核分枝杆菌感染过程中存在基因组DNA的甲基化状态改变,以低甲基化变化为主。其中TLR1基因呈现高甲基化状态,TLR4呈现低甲基化状态,提示上述基因可能在结核病的发生发展过程中具有一定的作用。

Objective To screen and identify the gene of DNA methylation in patients with active tuberculosis. Methods ① This study enrolled 9 cases of active tuberculosis patients （including 3 newly diagnosed tuberculosis patients and 6 cases of retreatment of active tuberculosis patients）, 3 cases of latent tuberculosis patients and 3 cases of healthy controls. Genome DNA was extracted from Peripheral Blood Mononuclear Cell and following bisulfite conversion treatment. After hybridization with the Illumina HD 450K Infinium Mehtylation BeadChip, the results were compared between patients group and control group, GO and Pathway analysis were performed to evaluate the function of differentially expressed genes; ② We further enrolled 60 cases of active tuberculosis patients and 60 cases of health controls （their age and gender were matched）. By using pyrosequencing method to detect the methylation levels of candidate genes （TLR1, TLR2, TLR4） screened by gene chip. Results① Compared with healthy controls, we found that most of them were showed demethylation status. GO and Pathway analysis showed that the functions of the differentially methylated regions related genes were mainly enriched in the biological processes of the regulation of leukocyte apoptosis, cytokine regulation and inflammatory response which were closely related to tuberculosis. ②There were 10 CpG sites involved in the verified tuberculosis related genes （TLR1, TLR2, TLR4）, the CpG sites of TLR1 gene showed the hypermethylation status （P〈0. 001）, the CpG sites of TLR4 gene showed demethylation status （P= 0. 012）. Conclusions The present study demonstrated that in the course of MTB infection, the methylation status of genomic DNA was altered, and most of the Differentially Methylated Regions （DMRs） were showed status of demethylation. TLR1 gene and TLR4 gene may play an important role in the occurrence and development of tuberculosis.