Vitamin D Concentration And The Methylation In Its Metabolic Pathway Genes In Association With The Risk And Prognosis Of Tuberculosis

Background and objectives:Tuberculosis is a chronic infectious disease caused by the pathogen of Mycobacterium tuberculosis(MTB),and has been a major public health threat in the world.Most infected individuals are in the latent infection state,and only 5-10%will develop the active tuberculosis.The active molecular of vitamin D can activate macrophages and anti-tuberculosis immune responses.Observational studies have shown that the deficiency of vitamin D may increase the risk of tuberculosis,and supplement with vitamin D can be an adjunctive treatment of tuberculosis,but without consistent results.Studies have suggested that host genetic factors determine the outcome of MTB-host interactions.Genetic and epigenetic changes in key the genes of vitamin D metabolic pathway influence the vitamin D activity and affect the immunity of anti-tuberculosis.Some studies have shown that high methylation in the promoter region of VDR and CYP genes can cause gene silencing and affect the level of active vitamin D,which may affect the risk and prognosis of tuberculosis.Based on the above background,we selected several key genes in vitamin D metabolic pathway and performed a case-control study together with a prospective follow-up study,by using the field investigation methods and molecular epidemiological techniques.We sequenced the promoter region of these genes to explore the methylation status.The serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D concentration were also measured.We aims to explore the epigenetic biomarkers related to the risk and prognosis of tuberculosis,and to provide a theoretical basis and population research data for further individualized vitamin D based anti-tuberculosis treatment.Methods:This study consists of two parts.The first part aims to explore the association between the methylation of vitamin D metabolic pathway genes and the risk of tuberculosis.We recruited 122 patients with pulmonary tuberculosis from Zhenjiang and Lianyungang,Jiangsu province from 2014 to 2016.We also selected 118 healthy controls from a pool of individuals who participated in the local community-based health examination programs,They were group-matched(by sex and age)with cases.Venous blood samples were collected for measuring serum 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol concentration.We selected five key genes(CYP24A1,CYP27A1,CYP27B1,CYP2R1 and VDR)in vitamin D metabolic pathway,and sequenced the CpG islands in the promoter region of these genes using the second-generation sequencing method.DNA methylation determined by combining the target region enrichment technique and bioinformatics data.Univariate and multivariate logistic regression analysis were used to calculate the odds ratio(OR)and 95%confidence interval(CI).The second part aims to explore the association between the methylation of vitamin D metabolic pathway genes and the prognosis of tuberculosis.We followed up thesetuberculosis cases recruited in the first part.The serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D concentration were measured at different time during the anti-tuberculosis treatment period.The Kaplan-Meier method was used to estimate the time to initial sputum conversion of sputum smear positive patients.Spearman correlation analysis was used to calculate the correlation between vitamin D concentration and cumulative methylation status of vitamin D metabolic pathway genes.The individual and cumulative methylation in the key genes were compared between cases and controls.The test level was set at 0.05.Results:(1)The baseline serum 25-hydroxyvitamin D concentration in the case group was 51.60 ± 27.25nmol/L,which was significantly lower than that in the control group(117.50 ± 75.50nmol/L)(Z =-8.515,P<0.001).The baseline serum 1,25-dihydroxyvitamin D concentration was 82.63 ± 51.43pmol/L,which was significantly lower than that in the control group(94.02 ± 49.26pmol/L)(Z =-2.165,P = 0.03).The 1,25-dihydroxyvitamin D concentration after the intensive treatment stage was 70.81 ± 44.50pmol/L,which was significantly lower than that at the baseline stage(Z=-2.606,P = 0.009).After the Bonferroni correction,there were 55 CpG sites with significantly different methylation frequency between cases and controls.The proportion of differentially methylated sites was 41.5%in CYP27B1 gene,31.7%in CYP24A1 gene,14.7%in VDR gene and 12.3%in CYP27A1 gene.The cumulative methylation levels were estimated using four different models.The results showed that the cumulative methylation rates of CYP24A1,CYP27A1,CYP27B1 and VDR in all models were significantly different between cases and controls(P<0.05).In model 1,the AUC was 0.747(95%CI:0.685-0.809).In model 2,the AUC was 0.805(95%CI:0.749-0.860).In model 3,the AUC was 0.838(95%CI:0.789-0.888).In model 4,the AUC was 0.810(95%CI:0.754-0.866).We observed that the methylation of CYP27A1,CYP27A1,CYP27B1 and VDR genes were positively correlated with the concentration of 25-hydroxyvitamin D(P<0.05).The interaction between CYP27A1 gene methylation and 1,25-dihydroxyvitamin D concentration in model 1 and model 4 was statistically significant(P<0.05).The interaction between VDR gene methylation and 1,25-dihydroxyvitamin D concentration was statistically significant(P<0.05).(2)Follow-up analysis revealed that 1,25-dihydroxyvitamin D concentration at the end of intensive treatment stage was associated with treatment outcomes of tuberculosis patients(P=0.008).The sputum smear negative conversion rate was significantly higher in patients with higher 1,25-dihydroxyvitamin D concentration(P=0.003).The proportion of differentially methylated sites was 14.6%in CYP24A1 gene,2.7%in CYP27A1 gene,5.7%in CYP27B1 gene,5.9%in CYP2R1 gene,and 10.7%in VDR gene.There was a positive interaction between the methylation of CYP2R1 gene and 25-hydroxyvitamin D concentration at the end of intensive treatment for the treatment outcomes of patients(P<0.05).Conclusion:The methylation of CYP24A1,CYP27A1,CYP27B1,CYP2R1 and VDR genes in the metabolic pathway of vitamin D are related to the risk and prognosis of tuberculosis.To investigate the role of abnormal methylation of vitamin D metabolic pathway in the diagnosis and prognosis of tuberculosis is of positive significance to the prevention and control of tuberculosis.