A genome-wide association study identified PTPN2 as a population-specific susceptibility gene locus for primary biliary cholangitis.

BACKGROUND AIMS: Previous genome-wide association studies (GWAS) have indicated the involvement of shared (population-non-specific) and non-shared (population-specific) susceptibility genes in the pathogenesis of primary biliary cholangitis (PBC) among European and East-Asian populations. Although a meta-analysis of these distinct populations has recently identified more than 20 novel PBC susceptibility loci, analyses of population-specific genetic architecture are still needed for a more comprehensive search for genetic factors in PBC. APPROACH RESULTS: Protein tyrosine phosphatase non-receptor type 2 (PTPN2) was identified as a novel PBC susceptibility gene locus through a GWAS and subsequent genome-wide meta-analysis involving 2,181 cases and 2,699 controls from the Japanese population (GWAS-lead variant: rs8098858, p=2.6×10-8). In-silico and in-vitro functional analyses indicated that the risk allele of rs2292758, which is a primary functional variant, decreases PTPN2 expression by disrupting Sp1 binding to the PTPN2 promoter in T follicular helper cells (Tfh) and plasmacytoid dendritic cells (pDCs). Infiltration of PTPN2-positive T-cells and pDCs were confirmed in the portal area of the PBC-liver by immunohistochemistry. Furthermore, transcriptomic analysis of PBC-liver samples indicated the presence of a compromised negative feedback loop in-vivo between PTPN2 and IFNG in patients carrying the risk allele of rs2292758. CONCLUSIONS: PTPN2, a novel susceptibility gene for PBC in the Japanese population, may be involved in the pathogenesis of PBC via an insufficient negative feedback loop caused by the PTPN2 risk allele of rs2292758 in IFN signaling. This suggests that PTPN2 could be a potential molecular target for PBC treatment.

rs10924104 in the expression enhancer motif of CD58 confers susceptibility to human autoimmune diseases.

CD58 plays roles in cell adhesion and co-stimulation with antigen presentation from major histocompatibility complex class II on antigen-presenting cells to T-cell antigen receptors on naïve T cells. CD58 reportedly contributes to the development of various human autoimmune diseases. Recently, genome-wide association studies (GWASs) identified CD58 as a susceptibility locus for autoimmune diseases such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), and primary biliary cholangitis (PBC). However, the primary functional variant and molecular mechanisms of susceptibility to autoimmune diseases in the CD58 locus were not clarified. Here, rs10924104, located in the ZNF35-binding motif within the gene expression regulatory motif, was identified as the primary functional variant for SLE, MS, and PBC among genetic variants showing stronger linkage disequilibrium (LD) with GWAS-lead variants in the CD58 locus. Expression-quantitative trait locus (e-QTL) data for each distinct blood cell type and in vitro functional analysis using the CRISPR/Cas9 system corroborated the functional role of rs10924104 in the upregulation of CD58 transcription by the disease-risk allele. Additionally, the strength of disease susceptibility observed in the CD58 locus could be accounted for by the strength of LD between rs10924104 and each GWAS-lead variant. In conclusion, the present study demonstrated for the first time the existence of a shared autoimmune disease-related primary functional variant (i.e., rs10924104) that regulates the expression of CD58. Clarifying the molecular mechanism of disease susceptibility derived from such a shared genetic background is important for understanding human autoimmune diseases and human immunology.

rs2013278 in the multiple immunological-trait susceptibility locus CD28 regulates the production of non-functional splicing isoforms.

BACKGROUND: Ligation of CD28 with ligands such as CD80 or CD86 provides a critical second signal alongside antigen presentation by class II major histocompatibility complex expressed on antigen-presenting cells through the T cell antigen receptor for naïve T cell activation. A number of studies suggested that CD28 plays an important role in the pathogenesis of various human diseases. Recent genome-wide association studies (GWASs) identified CD28 as a susceptibility locus for lymphocyte and eosinophil counts, multiple sclerosis, ulcerative colitis, celiac disease, rheumatoid arthritis, asthma, and primary biliary cholangitis. However, the primary functional variant and molecular mechanisms of disease susceptibility in this locus remain to be elucidated. This study aimed to identify the primary functional variant from thousands of genetic variants in the CD28 locus and elucidate its functional effect on the CD28 molecule. RESULTS: Among the genetic variants exhibiting stronger linkage disequilibrium (LD) with all GWAS-lead variants in the CD28 locus, rs2013278, located in the Rbfox binding motif related to splicing regulation, was identified as a primary functional variant related to multiple immunological traits. Relative endogenous expression levels of CD28 splicing isoforms (CD28i and CD28Δex2) compared with full-length CD28 in allele knock-in cell lines generated using CRISPR/Cas9 were directly regulated by rs2013278 (P < 0.05). Although full-length CD28 protein expressed on Jurkat T cells showed higher binding affinity for CD80/CD86, both CD28i and CD28Δex2 encoded loss-of-function isoforms. CONCLUSION: The present study demonstrated for the first time that CD28 has a shared disease-related primary functional variant (i.e., rs2013278) that regulates the CD28 alternative splicing that generates loss-of-function isoforms. They reduce disease risk by inducing anergy of effector T cells that over-react to autoantigens and allergens.

X Chromosome Contribution to the Genetic Architecture of Primary Biliary Cholangitis.

BACKGROUND & AIMS: Genome-wide association studies in primary biliary cholangitis (PBC) have failed to find X chromosome (chrX) variants associated with the disease. Here, we specifically explore the chrX contribution to PBC, a sexually dimorphic complex autoimmune disease. METHODS: We performed a chrX-wide association study, including genotype data from 5 genome-wide association studies (from Italy, United Kingdom, Canada, China, and Japan; 5244 case patients and 11,875 control individuals). RESULTS: Single-marker association analyses found approximately 100 loci displaying P < 5 × 10-4, with the most significant being a signal within the OTUD5 gene (rs3027490; P = 4.80 × 10-6; odds ratio [OR], 1.39; 95% confidence interval [CI], 1.028-1.88; Japanese cohort). Although the transethnic meta-analysis evidenced only a suggestive signal (rs2239452, mapping within the PIM2 gene; OR, 1.17; 95% CI, 1.09-1.26; P = 9.93 × 10-8), the population-specific meta-analysis showed a genome-wide significant locus in East Asian individuals pointing to the same region (rs7059064, mapping within the GRIPAP1 gene; P = 6.2 × 10-9; OR, 1.33; 95% CI, 1.21-1.46). Indeed, rs7059064 tags a unique linkage disequilibrium block including 7 genes: TIMM17B, PQBP1, PIM2, SLC35A2, OTUD5, KCND1, and GRIPAP1, as well as a superenhancer (GH0XJ048933 within OTUD5) targeting all these genes. GH0XJ048933 is also predicted to target FOXP3, the main T-regulatory cell lineage specification factor. Consistently, OTUD5 and FOXP3 RNA levels were up-regulated in PBC case patients (1.75- and 1.64-fold, respectively). CONCLUSIONS: This work represents the first comprehensive study, to our knowledge, of the chrX contribution to the genetics of an autoimmune liver disease and shows a novel PBC-related genome-wide significant locus.

rs9459874 and rs1012656 in CCR6/FGFR1OP confer susceptibility to primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic cholestatic autoimmune liver disease that appears to be strongly influenced by genetic factors. Recently, an international meta-analysis of genome-wide association studies (GWAS) identified CC-Motif Chemokine Receptor-6 (CCR6) and FGFR1 Oncogene-Partner (FGFR1OP) as PBC-susceptibility genes. However, the lead single nucleotide polymorphisms (SNPs) of CCR6/FGFR1OP showed low linkage disequilibrium with each other in East Asian and European populations. Additionally, the primary functional variants and the molecular mechanisms responsible for PBC-susceptibility remain unclear. Here, among the PBC-susceptibility SNPs identified by high-density association mapping in our previous meta-GWAS (Patients: n = 10,516; healthy controls: n = 20,772) within the CCR6/FGFR1OP locus, rs9459874 and rs1012656 were identified as primary functional variants. These functional variants accounted for the effects of GWAS-identified lead SNPs in CCR6/FGFR1OP. Additionally, the roles of rs9459874 and rs1012656 in regulating FGFR1OP transcription and CCR6 translation, respectively, were supported by expression quantitative trait loci (eQTL) analysis and gene editing technology using the CRISPR/Cas9 system. Immunohistochemistry showed higher expression of CCR6 protein in the livers of patients with PBC than in those of a non-diseased control. In conclusion, we identified primary functional variants in CCR6/FGFR1OP and revealed the molecular mechanisms by which these variants confer PBC-susceptibility in an eQTL-dependent or -independent manner. The approach in this study is applicable for the elucidation of the pathogenesis of other autoimmune disorders in which CCR6/FGFR1OP is known as a susceptibility locus, as well as PBC.

An international genome-wide meta-analysis of primary biliary cholangitis: Novel risk loci and candidate drugs.

BACKGROUNDS & AIMS: Primary biliary cholangitis (PBC) is a chronic liver disease in which autoimmune destruction of the small intrahepatic bile ducts eventually leads to cirrhosis. Many patients have inadequate response to licensed medications, motivating the search for novel therapies. Previous genome-wide association studies (GWAS) and meta-analyses (GWMA) of PBC have identified numerous risk loci for this condition, providing insight into its aetiology. We undertook the largest GWMA of PBC to date, aiming to identify additional risk loci and prioritise candidate genes for in silico drug efficacy screening. METHODS: We combined new and existing genotype data for 10,516 cases and 20,772 controls from 5 European and 2 East Asian cohorts. RESULTS: We identified 56 genome-wide significant loci (20 novel) including 46 in European, 13 in Asian, and 41 in combined cohorts; and a 57th genome-wide significant locus (also novel) in conditional analysis of the European cohorts. Candidate genes at newly identified loci include FCRL3, INAVA, PRDM1, IRF7, CCR6, CD226, and IL12RB1, which each play key roles in immunity. Pathway analysis reiterated the likely importance of pattern recognition receptor and TNF signalling, JAK-STAT signalling, and differentiation of T helper (TH)1 and TH17 cells in the pathogenesis of this disease. Drug efficacy screening identified several medications predicted to be therapeutic in PBC, some of which are well-established in the treatment of other autoimmune disorders. CONCLUSIONS: This study has identified additional risk loci for PBC, provided a hierarchy of agents that could be trialled in this condition, and emphasised the value of genetic and genomic approaches to drug discovery in complex disorders. LAY SUMMARY: Primary biliary cholangitis (PBC) is a chronic liver disease that eventually leads to cirrhosis. In this study, we analysed genetic information from 10,516 people with PBC and 20,772 healthy individuals recruited in Canada, China, Italy, Japan, the UK, or the USA. We identified several genetic regions associated with PBC. Each of these regions contains several genes. For each region, we used diverse sources of evidence to help us choose the gene most likely to be involved in causing PBC. We used these 'candidate genes' to help us identify medications that are currently used for treatment of other conditions, which might also be useful for treatment of PBC.

Genome-wide association studies identify PRKCB as a novel genetic susceptibility locus for primary biliary cholangitis in the Japanese population.

A previous genome-wide association study (GWAS) performed in 963 Japanese individuals (487 primary biliary cholangitis [PBC] cases and 476 healthy controls) identified TNFSF15 (rs4979462) and POU2AF1 (rs4938534) as strong susceptibility loci for PBC. In this study, we performed GWAS in additional 1,923 Japanese individuals (894 PBC cases and 1,029 healthy controls), and combined the results with the previous data. This GWAS, together with a subsequent replication study in an independent set of 7,024 Japanese individuals (512 PBC cases and 6,512 healthy controls), identified PRKCB (rs7404928) as a novel susceptibility locus for PBC (odds ratio [OR] = 1.26, P = 4.13 × 10-9). Furthermore, a primary functional variant of PRKCB (rs35015313) was identified by genotype imputation using a phased panel of 1,070 Japanese individuals from a prospective, general population cohort study and subsequent in vitro functional analyses. These results may lead to improved understanding of the disease pathways involved in PBC, forming a basis for prevention of PBC and development of novel therapeutics.

The first genome-wide association study identifying new susceptibility loci for obstetric antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is the most important treatable cause of recurrent pregnancy loss. The live birth rate is limited to only 70-80% in patients with APS undergoing established anticoagulant therapy. Lupus anticoagulant (LA), but not anticardiolipin antibody (aCL), was found to predict adverse pregnancy outcome. Recent genome-wide association studies (GWAS) of APS focusing on aCL have shown that several molecules may be involved. This is the first GWAS for obstetric APS focusing on LA. A GWAS was performed to compare 115 Japanese patients with obstetric APS, diagnosed according to criteria of the International Congress on APS, and 419 healthy individuals. Allele or genotype frequencies were compared in a total of 426 344 single-nucleotide polymorphisms (SNPs). Imputation analyses were also performed for the candidate regions detected by the GWAS. One SNP (rs2288493) located on the 3'-UTR of TSHR showed an experiment-wide significant APS association (P=7.85E-08, OR=6.18) under a recessive model after Bonferroni correction considering the number of analyzed SNPs. Another SNP (rs79154414) located around the C1D showed a genome-wide significant APS association (P=4.84E-08, OR=6.20) under an allelic model after applying the SNP imputation. Our findings demonstrate that a specific genotype of TSHR and C1D genes can be a risk factor for obstetric APS.

Human primary biliary cirrhosis-susceptible allele of rs4979462 enhances TNFSF15 expression by binding NF-1.

A genome-wide association study (GWAS) identified tumor necrosis factor superfamily member 15 (TNFSF15) as the strongest associated gene with susceptibility to primary biliary cirrhosis (PBC) outside the HLA loci in the Japanese population. However, causal functional variants of the TNFSF15 locus and the molecular mechanism underlying disease susceptibility have not been clarified. Here, to identify the functional causal variants of the TNFSF15 locus, integrated analysis comprising in silico analysis, a case-control association study and in vitro functional analysis was performed. Initially, 32 functional candidate single-nucleotide polymorphisms (SNPs) in the expression regulatory motifs, the coding region, or the untranslated regions (UTRs) of the TNFSF15 locus were selected by in silico analysis. By the case-control association studies using PBC patients (n = 1279) and healthy controls (n = 1091) in the Japanese population, rs4979462 [P = 1.85 × 10(-14) (our previous study)], rs56211063 (P = 2.21 × 10(-14)), and rs55768522 (r(2) = 1 with rs4979462) were likely candidates for causal variants. Among these SNPs, rs4979462 was identified as the causal variant by in vitro functional analysis using luciferase assay and electrophoretic mobility shift assay (EMSA). Super-shift assay clarified that PBC-susceptible allele of rs4979462 generated a novel NF-1 binding site. Moreover, higher endogenous TNFSF15 protein and mRNA expression levels were observed in individuals with the PBC-susceptible allele of rs4979462. This study identified the causal variant for PBC susceptibility in the TNFSF15 locus and clarified its underlying molecular mechanism. TNFSF15 and NF-1 are considered to be potential targets for the treatment of PBC.

Genome-wide association study identifies TNFSF15 and POU2AF1 as susceptibility loci for primary biliary cirrhosis in the Japanese population.

For the identification of susceptibility loci for primary biliary cirrhosis (PBC), a genome-wide association study (GWAS) was performed in 963 Japanese individuals (487 PBC cases and 476 healthy controls) and in a subsequent replication study that included 1,402 other Japanese individuals (787 cases and 615 controls). In addition to the most significant susceptibility region, human leukocyte antigen (HLA), we identified two significant susceptibility loci, TNFSF15 (rs4979462) and POU2AF1 (rs4938534) (combined odds ratio [OR] = 1.56, p = 2.84 × 10(-14) for rs4979462, and combined OR = 1.39, p = 2.38 × 10(-8) for rs4938534). Among 21 non-HLA susceptibility loci for PBC identified in GWASs of individuals of European descent, three loci (IL7R, IKZF3, and CD80) showed significant associations (combined p = 3.66 × 10(-8), 3.66 × 10(-9), and 3.04 × 10(-9), respectively) and STAT4 and NFKB1 loci showed suggestive association with PBC (combined p = 1.11 × 10(-6) and 1.42 × 10(-7), respectively) in the Japanese population. These observations indicated the existence of ethnic differences in genetic susceptibility loci to PBC and the importance of TNF signaling and B cell differentiation for the development of PBC in individuals of European descent and Japanese individuals.

Disease susceptibility genes shared by primary biliary cirrhosis and Crohn's disease in the Japanese population.

We previously identified TNFSF15 as the most significant susceptibility gene at non-HLA loci for both primary biliary cirrhosis (PBC) and Crohn's diseases (CD) in the Japanese population. The aim of this study is to identify further disease susceptibility genes shared by PBC and CD. We selected 15 and 33 genetic variants that were significantly associated with PBC and CD, respectively, based on previously reported genome-wide association studies of the Japanese population. Next, an association study was independently performed for these genetic variants in CD (1312 CD patients and 3331 healthy controls) and PBC (1279 PBC patients and 1015 healthy controls) cohorts. Two CD susceptibility genes, ICOSLG rs2838519 and IL12B rs6556412, were also nominally associated with susceptibility to PBC (P=3.85 × 10(-2) and P=8.40 × 10(-3), respectively). Three PBC susceptibility genes, CXCR5 rs6421571, STAT4 rs7574865 and NFKB1 rs230534, were nominally associated with susceptibility to CD (P=2.82 × 10(-2), P=3.88 × 10(-2) and P=2.04 × 10(-2), respectively). The effect of ICOSLG and CXCR5 variants were concordant but the effect of STAT4, NFKB1 and IL12B variants were discordant for PBC and CD. TNFSF15 and ICOSLG-CXCR5 might constitute a shared pathogenic pathway in the development of PBC and CD in the Japanese population, whereas IL12B-STAT4-NFKB1 might constitute an opposite pathogenic pathway, reflecting the different balance between Th1 and Th17 in the two diseases.

Systemic and local expression levels of TNF-like ligand 1A and its decoy receptor 3 are increased in primary biliary cirrhosis.

BACKGROUND & AIMS: Through a genome-wide association study of a Japanese population, we recently identified TNFSF15, a gene encoding TNF-like ligand 1A (TL1A), as a susceptibility gene for primary biliary cirrhosis (PBC). We investigated the clinical significance of TL1A and one of its receptors, decoy receptor 3 (DcR3), in PBC. METHODS: We analysed the systemic and local expression of TL1A and DcR3 in 110 PBC patients and 46 healthy controls using enzyme-linked immunosorbent assay, quantitative polymerase chain reaction and immunohistochemical staining. RESULTS: Serum TL1A levels were significantly increased in PBC patients at both early and late stages as compared with healthy controls, and its levels were significantly decreased in early-stage PBC patients after ursodeoxycholic acid (UDCA) treatment. TL1A was immunohistochemically localized to biliary epithelial cells, Kupffer cells, blood vessels and infiltrating mononuclear cells in the PBC liver. In addition, TL1A messenger RNA expression was increased in the PBC liver as compared with the non-diseased liver. Serum DcR3 levels were also significantly increased in PBC patients, and were significantly decreased after UDCA treatment in early-stage PBC patients. CONCLUSIONS: These results indicate that TL1A and DcR3 may play an important role in the pathogenesis of PBC.

Systematic evaluation of personal genome services for Japanese individuals.

Disease risk prediction (DRP) is one of the most important challenges in personal genome research. Although many direct-to-consumer genetic test (DTC) companies have begun to offer personal genome services for DRP, there is still no consensus on what constitutes a gold-standard service. Here, we systematically evaluated the distributions of DRPs from three DTC companies, that is, 23andMe, Navigenics and deCODEme, for 22 diseases using three Japanese samples. We systematically quantified and analyzed the differences between each DTC company's DRPs. Our independency test showed that the overall prediction results were correlated with each other, but not perfectly matched; less than onethird mismatching of the opposite direction occurred in eight diseases. Moreover, we found that the differences could mainly be attributed to four factors: (1) single nucleotide polymorphism (SNP) selection, (2) average risk estimation, (3) the disease risk calculation algorithm and (4) ethnicity adjustment. In particular, only 7.1% of SNPs over 22 diseases were reviewed by all three companies. Therefore, development of a universal core SNPs list for non-Caucasian samples will be important for achieving better prediction capacity for Japanese samples. This systematic methodology provides useful insights for improving the capacity of DRPs in future personal genome services.

Novel HLA allele associations with susceptibility, staging, symptomatic state, autoimmune hepatitis and hepatocellular carcinoma events for primary biliary cholangitis in the Japanese population.

Primary biliary cholangitis (PBC) is a rare autoimmune disease with a clear predisposition for human leukocyte antigen (HLA)-DR/DQ-associated loss of immune tolerance for the E2 component of the pyruvate dehydrogenase complex. Three-field-resolution HLA imputation of 1,670 Japanese PBC patients and 2,328 healthy controls was conducted using Japanese population-specific HLA reference panels. Eighteen previously reported Japanese PBC-associated HLA alleles were confirmed and extended to 3-field-resolution, including HLA-DRB1*08:03 to HLA-DRB1*08:03:02, HLA-DQB1*03:01 to HLA-DQB1*03:01:01, HLA-DQB1*04:01 to HLA-DQB1*04:01:01 and HLA-DQB1*06:04 to HLA-DQB1*06:04:01. In addition, additional significant novel HLA alleles were identified, including 3 novel susceptible HLA-DQA1 alleles: HLA-DQA1*03:03:01, HLA-DQA1*04:01:01, HLA-DQA1*01:04:01 and 1 novel protective HLA-DQA1 allele, HLA-DQA1*05:05:01. In addition, PBC patients carrying HLA-DRB1*15:01:01 and HLA-DQA1*03:03:01 would have a higher predisposition toward developing concomitant autoimmune hepatitis (AIH). Further, late-stage and symptomatic PBC shared the same susceptible HLA alleles of HLA-A*26:01:01, HLA-DRB1*09:01:02 and HLA-DQB1*03:03:02. Lastly, HLA-DPB1*05:01:01 was identified as a potential risk HLA allele for development of hepatocellular carcinoma (HCC) in PBC patients. In conclusion, we have extended the current knowledge of HLA allele associations to 3-field resolution and identified novel HLA allele associations with predisposition risk, staging, symptomatic state, and AIH and HCC events for Japanese PBC patients.

TogoVar: A comprehensive Japanese genetic variation database.

TogoVar ( https://togovar.org ) is a database that integrates allele frequencies derived from Japanese populations and provides annotations for variant interpretation. First, a scheme to reanalyze individual-level genome sequence data deposited in the Japanese Genotype-phenotype Archive (JGA), a controlled-access database, was established to make allele frequencies publicly available. As more Japanese individual-level genome sequence data are deposited in JGA, the sample size employed in TogoVar is expected to increase, contributing to genetic study as reference data for Japanese populations. Second, public datasets of Japanese and non-Japanese populations were integrated into TogoVar to easily compare allele frequencies in Japanese and other populations. Each variant detected in Japanese populations was assigned a TogoVar ID as a permanent identifier. Third, these variants were annotated with molecular consequence, pathogenicity, and literature information for interpreting and prioritizing variants. Here, we introduce the newly developed TogoVar database that compares allele frequencies among Japanese and non-Japanese populations and describes the integrated annotations.

The NBDC-DDBJ imputation server facilitates the use of controlled access reference panel datasets in Japan.

Accurate genotype imputation requires large-scale reference panel datasets. When conducting genotype imputation on the Japanese population, researchers can use such datasets under collaborative studies or controlled access conditions in public databases. We developed the NBDC-DDBJ imputation server, which securely provides users with a web user interface to execute genotype imputation on the server. Our benchmarking analysis showed that the accuracy of genotype imputation was improved by leveraging controlled access datasets to increase the number of haplotypes available for analysis compared to using publicly available reference panels such as the 1000 Genomes Project. The NBDC-DDBJ imputation server facilitates the use of controlled access datasets for accurate genotype imputation.

rs1944919 on chromosome 11q23.1 and its effector genes COLCA1/COLCA2 confer susceptibility to primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic, progressive cholestatic liver disease in which intrahepatic bile ducts are destroyed by an autoimmune reaction. Our previous genome-wide association study (GWAS) identified chromosome 11q23.1 as a susceptibility gene locus for PBC in the Japanese population. Here, high-density association mapping based on single nucleotide polymorphism (SNP) imputation and in silico/in vitro functional analyses identified rs1944919 as the primary functional variant. Expression-quantitative trait loci analyses showed that the PBC susceptibility allele of rs1944919 was significantly associated with increased COLCA1/COLCA2 expression levels. Additionally, the effects of rs1944919 on COLCA1/COLCA2 expression levels were confirmed using genotype knock-in versions of cell lines constructed using the CRISPR/Cas9 system and differed between rs1944919-G/G clones and -T/T clones. To our knowledge, this is the first study to demonstrate the contribution of COLCA1/COLCA2 to PBC susceptibility.