Association Of CCR6 Polymorphism With Genetic Susceptibility To Primary Biliary Cholangitis In Han Chinese Population

Primary biliary cholangitis（PBC）,formerly named primary biliary cirrhosis,is a chronic cholestasis disease characterized by the small and medium bile ducts injury in liver.The disease is more common in middle-aged and older women,the male-female ratio is about 1:9.The incidence of PBC has increased in recent years.The pathogenesis of PBC is not yet clear,and the high degree of familial aggregation,in particular,between identical twins supports the role of genetic factors in PBC pathogenesis.Recently,multiple genome-wide association studies（GWAS）further confirmed that PBC has a strong genetic susceptibility and identified a series of genetic loci shared with or distinct to different populations.In order to identify specific genetic susceptibility loci associated with Chinese Han PBC population,our group used Human Omni Zhong Hua-8 chip to conduct large-scale genome-wide association analysis in1126 Han PBC patients and 4036 healthy controls.Based on our GWAS results,multiple SNPs in the CCR6 region showed significance association with P value less than 1×10^-5.These sites can be divided into susceptible SNPs and protective SNPs.This current study will focus on susceptibility loci and verify the potential genetic association of these SNPs with Han Chinese PBC.Objective:Based on our GWAS results,we intended to verify genetic association of SNPs in CCR6 locus with PBC susceptibility in Chinese Han PBC,and perform linear regression analysis to identify the major SNPs representing the genetic susceptibility for CCR6 locus.Methods:1.According to the PBC diagnostic criteria,a total of 1456 PBC patients and 3316healthy controls were collected for case-control association analysis.2.Taq Man-MGB method and Sanger sequencing were used for genotyping in replication samples.3.Case-control association analysis on the genotyping results of the SNPs were performed using IBM SPSS Statistics 23.0;meta-analysis on the data combination of the GWAS stage and the replication stage was analyzed with Stata SE 12.0;linkage disequilibrium analysis and conditional logistic regression analysis was conducted with Haploview 4.1 and PLINK 1.9.Results:The linkage disequilibrium（LD）analysis of the potential susceptibility SNPs in CCR6locus in the GWAS stage revealed two LD blocks.The tag SNPs were selected from both blocks,with rs12529876 from the first block,and rs4710181 and rs6905911 selected from the second block.In addition,according to linkage disequilibrium analysis,the first SNP rs968334 in the CCR6 dinucleotide polymorphism site（CCR6DNP）was found to be linked to rs2301436 from the first block with r²=0.70 in Han Chinese population.These SNPs were genotyped in 1456cases and 3316 controls.The statistical significance of allele frequency between case and control was calculated with P=2.51×10^-5（OR=1.22,95%CI=1.11-1.33）for rs12529876,P=4.41×10^-3（OR=1.14,95%CI=1.04-1.25）for rs4710181,and P=2.66×10^-4（OR=1.18,95%CI=1.08-1.29）for rs6905911.Meta analysis results with 2582 cases and 7352 controls showed that rs12529876(P=5.59×10^-9,OR=1.22,95%CI=1.14-1.30)and rs6905911(P=2.68×10^-9,OR=1.22,95%CI=1.14-1.30)reached the significant level of GWAS.Case-control association analysis of CCR6DNP and its 2 SNPs rs968334 and rs117912866 showed that rs968334(P=4.80×10^-4,OR=1.25,95%CI=1.10-1.42)was significantly related to PBC,but rs117912866（P=0.93,OR=1.01,95%CI=0.79-1.29）did not differ significantly between the two groups.The linkage disequilibrium analysis showed that the degree of linkage between rs4710181 and rs6905911 was consistent with the results of the GWAS stage,r²=0.76,located in the same group.rs12529876 and rs968334 have a certain linkage,r²=0.66.Conditional logistic regression analysis showed that only rs12529876 and rs968334 can eliminate the significance of all other loci,which indicate that rs12529876 or rs968334 can represent the genetic susceptibility observed in CCR6 locus.Conclusion:This study confirmed CCR6 locus is associated with PBC susceptibility in Han Chinese population and rs12529876 or rs968334 can represent the genetic susceptibility observed in CCR6 locus.Further linkage analysis and functional verification are required to identify the causative variants associated with PBC susceptibility in CCR6 locus.