Association Study Of Single Nucleotide Polymorphisms Of TNFSF15 Gene And Its Expression With Primary Biliary Cholangitis

Objective: This study is to explore the association of the level of human tumor necrosis factor-like ligand 1A(TL1A)and single nucleotide polymorphisms(SNP)of tumor necrosis factor superfamily 15(TNFSF15)with primary biliary cholangitis(PBC),and further to conduct functional prediction of the positive SNP loci in association analysis,aiming to screen the high-risk PBC population and provide clues for finding new therapeutic targets.Methods:1.Peripheral venous blood was collected from PBC patients and healthy individuals.Plasma and blood cells were separated,and peripheral blood mononuclear cells(PBMC)were isolated by density gradient centrifugation to extract the genomic DNA and RNA.2.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of TL1 A in the peripheral blood plasma of the PBC patients and healthy controls.3.Real-time quantitative PCR was used to detect the relative expression levels of TNFSF15 mRNA in PBMCs from the PBC patients and healthy controls.4.The target SNPs were screened.The genotyping of SNPs in the study population was performed by Sanger sequencing technology,and then their correlations with plasma TL1 A levels were analyzed.5.The function of the positive SNPs in the association analysis was predicted using the bioinformatics software JASPAR.Results:1.The plasma TL1 A level in PBC patients was significantly higher than that in the healthy group(31.45±17.03 ng/L VS 19.64±5.74 ng/L)(P<0.05).Plasma TL1 A level predicted that the area under the ROC curve of PBC was 0.838,which is greater than the area under the opportunity reference(P <0.05).2.The results of Real-time quantitative PCR showed that the relative expression of TNFSF15 mRNA in PBC group was 2.975 times higher than that in healthy control group with the statistical difference(P<0.05).3.Six target SNPs of TNFSF15 gene were selected including rs55717217,rs6478108,rs4979462,rs10114470,rs1857335 and rs12235514.there were statistical differences in the genotypes and allele frequency distributions of rs4979462,rs55717217,rs6478108,rs1857335 loci between PBC group and control group((P<0.05).However,There was no statistical difference of rs10114470 and rs12235514 loci.4.Association analysis results showed that plasma TL1 A levels were positively correlated with AST,ALT,ALP,GGT,and TBA(r=0.202,P=0.002;r=0.252,P<0.001;r=0.313,P<0.001;r= 0.328,P<0.001;r = 0.129,P=0.046),but there is no linear correlation with TBIL,DBIL and ALB.There was no statistical difference in the distribution of TL1 A levels in PBC group with a positive autoantibody(P>0.05).Secondary alleles at rs55717217,rs4979462 and rs1857335 loci were associated with the elevated plasma TL1 A levels(P<0.001,P<0.001,P=0.002).5.SNP function prediction results show that the rs4979462 for retinoid X receptor alpha(RXRA),rs55717217 for forkhead box L1(FOXL1),rs1857335 for specificity protein 1(SP1)were potential binding sites.Conclusions:1.The level of TL1 A and the expression of TNFSF15 gene mRNA in peripheral blood of the PBC patients are significantly higher than those in healthy controls.The TL1 A level in plasma is related to the severity of hepatobiliary system injury.The TL1 A level in plasma may be a biomarker for PBC.2.Polymorphisms of rs4979462,rs55717217,rs6478108 and rs1857335 in TNFSF15 gene are related to susceptibility to PBC,whereas polymorphisms of rs10114470 and rs12235514 are not related to PBC.Among them,this study found that rs55717217 and rs1857335 polymorphisms are associated with PBC susceptibility for the first time.TNFSF15 gene rs4979462,rs55717217 and rs1857335 locus polymorphisms are correlated with high expression of TL1 A in plasma.3.The binding of rs4979462 site with the transcription factor RXRA,rs55717217 site with the transcription factor FOXL1 and rs1857335 site with the transcription factor SP1 may be existed,thereby promoting the transcriptional expression of TNFSF15 gene.