Methylation Analysis and Validation of Whole Genome DNA in Active Tuberculosis

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. Results ① 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.