DNA Methylation And Exosomes Protein Biomarkers Of Active Tuberculosis Onset From Latent Tuberculosis And Response Of Preventive Treatment

Background:Individuals with latent tuberculosis infection(LTBI)were regarded as an enormous reservoir of cases with active tuberculosis(TB).To strengthen LTBI management and achieve precise intervention,biomarkers and tools are urgently required for identifying active TB from LTBI and monitoring response to preventive treatment in a fast and effective way.Methods:On the basis of prospective cohort study,nested case-control study was used to study methylation sites related differential diagnosis of LTBI and TB and exosome proteins related to preventive treatment response.The details are as follows:1.Screening methylation sites related to active TB onset from LTBI:Based on an open-label randomized controlled trial aiming to explore short-course LTBI treatment regimens,DNA methylation profiles were retrospectively detected to explore potential biomarkers,which could discriminate active TB from LTBI.Infinium MethylationEPIC BeadChip array were used to analyze genome-wide DNA methylation levels for 15 LTBIs who later developed active TB and 15 LTBI controls who stayed healthy.The differentially methylated CpGs(dmCpGs)located in the promoter regions pre-and-post TB diagnosis were selected(p<0.05 and |Δβ|>0.10)and evaluated by receiver operating characteristic(ROC)analysis.The area under ROC curve(AUC)and the evaluation specificity under 90%sensitivity of the World Health Organization(WHO)target product benchmark(TPP)were used to evaluate the screening and diagnostic value.2.The preventive treatment response:In order to obtain a molecular marker to monitor response to preventive treatment of LTBI,based on a randomized controlled trial to explore the preventive treatment scheme in patients with previous pulmonary TB,the plasma exosomes of 80 LTBIs who participated in preventive treatment were analyzed by Label free quantitative protein mass spectrometry.Including 20 cases with continuous persistent positive IGRA after preventive treatment and 20 cases with IGRA reversion after preventive treatment(definition of reversion:IFNγ>0.70 IU/ml before intervention and IFN-γ<0.20 IU/ml within one week after intervention),two samples were taken before and after preventive treatment,and the parallel reaction monitoring(PRM)verification was carried out by enlarging the sample size.Results:1.The onset of TB from LTBI:863,159 methylated sites of 60 peripheral blood mononuclear cells were analyzed by 850K chip,eight dmCpGs were identified to be associated with TB occurrence,6 located in hypermethylated genes(cg02493602,cg02206980,cg02214623,cg12159502,cg14593639 and cg25764570)and 2 located in hypomethylated genes(cg02781074 and cg12321798).ROC analysis indicated that the area under curve(AUC)of these 8 dmCpGs ranged from 0.72 to 0.84.Given 90%sensitivity,the specificity was highest for cg14593639 of 66.67%.The combination analysis indicated that "cg02206980+cg02214623+cg12159502+cg12321798" showed the best performance with AUC of 0.88(95%CI:0.72,0.97),sensitivity of 93.33%(95%CI:70.18,99.66)and specificity of 86.67%(95%CI:62.12,97.63).2.The preventive treatment prognosis:A total of 2321 exosome proteins were analyzed by Label free quantitative protein mass spectrometry.Among them,102 exosome proteins showed significant differences before and after preventive treatment in IGRA reversion group,and there was no difference before and after treatment in persistent positive group.At the same time,there was no difference between the two groups before preventive treatment,which can be used to monitor response of preventive treatment.In the other 120 peripheral blood plasma samples used for verification(two samples before and after 30 in the persistent positive group and two samples before and after 30 in the negative conversion group),it was verified that the expression of exocrine proteins C4BPA and S100A9 was downregulated before and after treatment in the reversion group(p<0.05),which was consistent with the screening results.Conclusion:Our preliminary results provide potential value of DNA methylation level as diagnostic biomarker for discriminating active disease in case of LTBI testing.It needs further verification in independent populations with large sample size.Plasma exosome proteins C4BPA and S100A9 may be related to the host’s positive response to preventive treatment of LTBI,which provides basic data support for the construction of "precise intervention" technology system.