[Methylation Chip Screening and Verification of Differential Genes Related to Tuberculosis Infection]

To screen the genes with significant changes in DNA methylation level in active tuberculosis patients, we used the methylation chips and expanded the sample size to verify candidate genes.

① This study enrolled 9 cases of active tuberculosis patients, 3 cases of latent tuberculosis patients and 3 cases of healthy controls whose age and gender were all matched. Genome DNA was extracted from peripheral blood mononuclear cell in blood samples collected from these candidates, and bisulfite conversion treatment was then conducted. After hybridization with the Illumina HD 450K Infinium Mehtylation BeadChip, the results were compared between patients group and control group, and GO and KEGG pathway analyses 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 (age-and gender-matched), DNA was extracted from their peripheral blood and also followed bisulfite conversion treatment. Pyrosequencing method was used to detect the methylation levels of candidate genes (IFNGR2, PTPN6, CRK1, ATP6V0B, WIF1, DKK1 and SFRP1) screened by gene chip.

Compared with healthy controls, the fragments in the patients that showed low methylation change accounted for the vast majority. Most of the methylation differential fragments (DMRs) were located in the main body region, followed by the upstream region of transcription initiation site, and the lowest DMRs distribution area was 3´UTR area. GO and Pathway analysis showed that the functions of the differentially methylated regions related genes are mainly enriched in the biological processes of the regulation of leukocyte differentiation, apoptosis, cytokine regulation and inflammatory response which are closely related to tuberculosis. There were 32 CpG sites involved in the verified 7 tuberculosis related genes, and 16 CpG locus showed significant difference (P<0.05), they were distributed in 6 genes: PTPN6, WIF1, CRK1, SFRP1, DKK1 and IFNGR2.Of these genes with significant difference, PTPN6 genes showed hypermethylation status and WIF1, CRK1, SFRP1, DKK1 and IFNGR2 genes exhibited demethylation status in the patients group compared to the health controls. SFRP1 and CRK-1 mRNA up-regulated in the patients group compared with health controls.

In the course of MTB infection, the methylation status of genomic DNA is altered, and most of the differentially methylated regions (DMRs) are showed status of demethylation. The expressions ofSFRP1and CRK-1gene up-regulate in tuberculosis infection.