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.

The Genetics of Primary Biliary Cholangitis: A GWAS and Post-GWAS Update.

Primary biliary cholangitis (PBC) is a chronic, progressive cholestatic liver disease in which the small intrahepatic bile ducts are destroyed by autoimmune reactions. Among autoimmune diseases, which are polygenic complex traits caused by the combined contribution of genetic and environmental factors, PBC exhibits the strongest involvement of genetic heritability in disease development. As at December 2022, genome-wide association studies (GWASs) and associated meta-analyses identified approximately 70 PBC susceptibility gene loci in various populations, including those of European and East Asian descent. However, the molecular mechanisms through which these susceptibility loci affect the pathogenesis of PBC are not fully understood. This study provides an overview of current data regarding the genetic factors of PBC as well as post-GWAS approaches to identifying primary functional variants and effector genes in disease-susceptibility loci. Possible mechanisms of these genetic factors in the development of PBC are also discussed, focusing on four major disease pathways identified by in silico gene set analyses, namely, (1) antigen presentation by human leukocyte antigens, (2) interleukin-12-related pathways, (3) cellular responses to tumor necrosis factor, and (4) B cell activation, maturation, and differentiation pathways.

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.

Crohn's Disease and Early Exposure to Thiopurines are Independent Risk Factors for Mosaic Chromosomal Alterations in Patients with Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Mosaic chromosomal alterations [mCAs] increase the risk for haematopoietic malignancies and may be risk factors for several other diseases. Inflammatory bowel diseases [IBDs], including Crohn's disease [CD] and ulcerative colitis [UC], are associated with mCAs, and patients may be at risk for haematopoietic malignancy development and/or modification of IBD phenotypes. In the present study, we screened patients with IBD for the presence of mCAs and explored the possible pathophysiological and genetic risk factors for mCAs. METHODS: We analysed mCAs in peripheral blood from 3339 patients with IBD and investigated the clinical and genetic risk factors for mCAs. RESULTS: CD and exposure to thiopurines before the age of 20 years were identified as novel independent risk factors for mCAs [odds ratio = 2.15 and 5.68, p = 1.17e-2 and 1.60e-3, respectively]. In contrast, there were no significant associations of disease duration, anti-tumour necrosis factor alpha antibodies, or other clinical factors with mCAs. Gene ontology enrichment analysis revealed that genes specifically located in the mCAs in patients with CD were significantly associated with factors related to mucosal immune responses. A genome-wide association study revealed that ERBIN, CD96, and AC068672.2 were significantly associated with mCAs in patients with CD [p = 1.56e-8, 1.65e-8, and 4.92e-8, respectively]. CONCLUSIONS: The difference in mCAs between patients with CD and UC supports the higher incidence of haematopoietic malignancies in CD. Caution should be exercised when using thiopurines in young patients with IBD, particularly CD, in light of possible chromosomal alterations.

Regional heritability mapping identifies several novel loci (STAT4, ULK4, and KCNH5) for primary biliary cholangitis in the Japanese population.

While the advent of GWAS more than a decade ago has ushered in remarkable advances in our understanding of complex traits, the limitations of single-SNP analysis have also led to the development of several other approaches. Simulation studies have shown that the regional heritability mapping (RHM) method, which makes use of multiple adjacent SNPs jointly to estimate the genetic effect of a given region of the genome, generally has higher detection power than single-SNP GWAS. However, thus far its use has been mostly limited to agricultural settings, and its potential for the discovery of new genes in human diseases is yet to be fully exploited. In this study, by applying the RHM method to primary biliary cholangitis (PBC) in the Japanese population, we identified three novel loci (STAT4, ULK4, and KCNH5) at the genome-wide significance level, two of which (ULK4 and KCNH5) have not been found associated with PBC in any population previously. Notably, these genes could not be detected by using conventional single-SNP GWAS, highlighting the potential of the RHM method for the detection of new susceptibility loci in human diseases. These findings thereby provide strong empirical evidence that RHM is an effective and practical complementary approach to GWAS in this context. Also, liver tissue mRNA microarray analysis revealed higher gene expression levels in ULK4 in PBC patients (P < 0.01). Lastly, we estimated the common SNP heritability of PBC in the Japanese population (0.210 ± 0.026).

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.

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.

Association of soluble T cell immunoglobulin domain and mucin-3 (sTIM-3) and mac-2 binding protein glycosylation isomer (M2BPGi) in patients with autoimmune hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is a disorder of unknown etiology in which immune-mediated liver injury progress to cirrhosis or hepatocellular carcinoma (HCC). The aim of the present study was to determine whether circulating soluble TIM3 (sTIM3) is elevated in patients with AIH patients and whether sTIM-3 levels are associated with clinical parameters of AIH. METHODS: We enrolled 123 Japanese patients with AIH who were identified from the National Hospital Organization-AIH-liver-network database, as well as 32 patients with chronic hepatitis C (CHC), 30 patients with primary biliary cholangitis (PBC) and healthy control subjects. Serum sTIM-3 concentrations were quantified by ELISA. RESULTS: Serum levels of sTIM-3 were significantly higher in AIH patients (median 4865 pg/ml; [interquartile range (IQR); 3122-7471]) compared to those in CHC (1026 pg/ml [IQR: 806-1283] p<0.001), PBC (2395 pg/ml [IQR: 2012-3422] p<0.001) or healthy controls (1285 pg/ml [IQR: 1098-1812] p<0.001). In AIH group, serum sTIM-3 were correlated with alanine aminotransferase (ALT), or total bilirubin (TB) and negatively correlated with serum levels of albumin (Alb). Serum levels of sTIM-3 were also strongly correlated with Mac-2 binding protein glycosylation isomer (M2BPGi) levels, but did not correlate with the histological grade of liver fibrosis. Steroid treatment of AIH patients significantly reduced serum sTIM-3 levels (2147±623pg/ml versus 1321±378pg/ml, p<0.001). CONCLUSIONS: Circulating sTIM-3 levels were elevated in AIH patients and are associated with AIH disease activity and AIH-related liver damage. These findings indicate that serum sTIM-3 correlated with disease status of AIH and could be useful biomarkers to detect autoimmune-mediated liver injury. Our data suggest a possible link between the TIM-3/GAL-9 pathway and AIH severity or phenotype, and further investigations of the TIM-3 pathway and AIH pathophysiology is warranted.

Serum kynurenine levels are a novel biomarker to predict the prognosis of patients with hepatocellular carcinoma.

BACKGROUND: We examined serum kynurenine levels in patients with chronic hepatitis C virus infection, and the relationship between serum kynurenine and prognosis in patients with hepatocellular carcinoma (HCC) and chronic hepatitis C. METHODS: We retrospectively analyzed 604 patients with HCC diagnosed between January 1999 and December 2015, and 288 patients without HCC who were seen at the National Hospital Organization Nagasaki Medical Center between October 2014 and November 2017. The association between serum kynurenine and prognosis was evaluated using the Cox's proportional hazards regression analysis. RESULTS: Patients with HCC had significantly higher values of serum kynurenine than patients without HCC (median: 557.1 vs. 464.2 ng/mL, p<0.001). Five-year survival rates of HCC patients with serum kynurenine ≥900 (n = 65), 600-899 (n = 194), and <600 ng/mL (n = 345) were 30.6%, 47.4%, and 61.4%, respectively (p = 0.001, log-rank test). Multivariate analysis identified serum kynurenine as an independent predictor for prognosis of HCC patients. The hazard ratio of serum kynurenine ≥900, and 600-899 compared with serum kynurenine <600 ng/mL were 1.91 (p<0.001) and 1.37 (p = 0.015), respectively. CONCLUSIONS: A high level of serum kynurenine correlated with poor prognosis of HCC. Serum kynurenine levels may be a novel biomarker to predict the prognosis of patients with HCC. The development of drugs that inhibit kynurenine production is expected to help improve the prognosis of patients with HCC.

Ethanol Gas Sensing by a Zn-Terminated ZnO(0001) Bulk Single-Crystalline Substrate.

Metal oxide semiconductor gas sensors have been widely studied for the selective detection of various gases with trace concentrations. The identification of the reaction scheme governing the gas sensing response is crucial for further development; however, the mechanism of ethanol (EtOH) gas sensing by ZnO is still controversial despite being one of the most intensively studied target gas and sensing material combinations. In this work, for the first time, the detailed mechanism of EtOH sensing by ZnO is studied by using a bulk single-crystalline substrate, which has a well-defined stoichiometry and atomic arrangement, as the sensing material. The sensing response is substantial on the ZnO substrate even with a millimeter-size thickness, and it becomes larger with resistance of the substrate. The large sensing response is described in terms of the adsorption/desorption of the oxygen species on the substrate surface, namely, oxygen ionosorption. The valence state of the ionosorbed oxygen involved in EtOH sensing is identified to be O2- regardless of the temperature. The increase in the sensing response with the temperature is attributed to the enhanced oxidation rate of the EtOH molecule on the surface as analyzed by pulsed-jet temperature-programmed desorption mass spectrometry, which has been newly developed for analyzing surface reactions in simulated working conditions.

An Integrated Genomic and Transcriptomic Analysis Reveals Candidates of Susceptibility Genes for Crohn's Disease in Japanese Populations.

Expression quantitative trait locus (eQTL) analyses have enabled us to predict the function of disease susceptibility SNPs. However, eQTL for the effector memory T cells (TEM) located in the lamina propria mononuclear cells (LPMCs), which play an important role in Crohn's disease (CD), are not yet available. Thus, we conducted RNA sequencing and eQTL analyses of TEM cells located in the LPMCs from IBD patients (n = 20). Genome-wide association study (GWAS) was performed using genotyping data of 713 Japanese CD patients and 2,063 controls. We compared the results of GWAS and eQTL of TEM, and also performed a transcriptome-wide association study using eQTL from Genotype Tissue Expression project. By eQTL analyses of TEM, correlations of possible candidates were confirmed in 22,632 pairs and 2,463 genes. Among these candidates, 19 SNPs which showed significant correlation with tenascin-XA (TNXA) expression were significantly associated with CD in GWAS. By TWAS, TNFSF15 (FDR = 1.35e-13) in whole blood, ERV3-1 (FDR = 2.18e-2) in lymphocytes, and ZNF713 (FDR = 3.04e-2) in the sigmoid colon was significantly associated with CD. By conducting integration analyses using GWAS and eQTL data, we confirmed multiple gene transcripts are involved in the development of CD.

Integrated GWAS and mRNA Microarray Analysis Identified IFNG and CD40L as the Central Upstream Regulators in Primary Biliary Cholangitis.

Genome-wide association studies (GWASs) in European and East Asian populations have identified more than 40 disease-susceptibility genes in primary biliary cholangitis (PBC). The aim of this study is to computationally identify disease pathways, upstream regulators, and therapeutic targets in PBC through integrated GWAS and messenger RNA (mRNA) microarray analysis. Disease pathways and upstream regulators were analyzed with ingenuity pathway analysis in data set 1 for GWASs (1,920 patients with PBC and 1,770 controls), which included 261 annotated genes derived from 6,760 single-nucleotide polymorphisms (P < 0.00001), and data set 2 for mRNA microarray analysis of liver biopsy specimens (36 patients with PBC and 5 normal controls), which included 1,574 genes with fold change >2 versus controls (P < 0.05). Hierarchical cluster analysis and categorization of cell type-specific genes were performed for data set 2. There were 27 genes, 10 pathways, and 149 upstream regulators that overlapped between data sets 1 and 2. All 10 pathways were immune-related. The most significant common upstream regulators associated with PBC disease susceptibility identified were interferon-gamma (IFNG) and CD40 ligand (CD40L). Hierarchical cluster analysis of data set 2 revealed two distinct groups of patients with PBC by disease activity. The most significant upstream regulators associated with disease activity were IFNG and CD40L. Several molecules expressed in B cells, T cells, Kupffer cells, and natural killer-like cells were identified as potential therapeutic targets in PBC with reference to a recently reported list of cell type-specific gene expression in the liver. Conclusion: Our integrated analysis using GWAS and mRNA microarray data sets predicted that IFNG and CD40L are the central upstream regulators in both disease susceptibility and activity of PBC and identified potential downstream therapeutic targets.

Galectin-9 in autoimmune hepatitis: Correlation between serum levels of galectin-9 and M2BPGi in patients with autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a disorder of unknown etiology in which immune-mediated liver damage progresses to cirrhosis or hepatocellular carcinoma (HCC). The mainstay therapy for AIH is steroids and other immunosuppressive treatments. Currently, there are no validated markers for monitoring immune-mediated hepatic inflammation. Galectin-9 has recently been identified as a potential biomarker in patients with chronic liver disease. The objective of this study was to determine whether Galectin-9 and other serum proteins are associated with active disease in AIH patients.We enrolled 77 Japanese patients with well-documented AIH who were identified from the National Hospital Organization-AIH-liver-network database, as well as 32 patients with chronic hepatitis C (CHC), 27 patients with SLE, and 17 healthy control subjects. Serum levels of galectin-9, and markers of liver injury were measured and compared between groups.Serum levels of galectin-9 were significantly higher in AIH patients than in CHC patients (13.8 ±â€Š4.9 ng/mL vs 8.9 ±â€Š3.0 ng/mL, P < .001) or healthy controls (13.8 ±â€Š4.9 ng/mL vs 5.0 ±â€Š1.3 ng/mL, P < .001). In AIH group, serum galectin-9 levels weakly correlated with alanine aminotransferase levels or total bilirubin (TB) and strongly correlated with C-X-C motif chemokine 10 (CXCL10) and Mac-2 binding protein glycosylation isomer (M2BPGi) levels, but did not correlate with the histological grade of liver fibrosis. Steroid treatment of AIH patients significantly reduced serum galectin-9 levels (14.1 ±â€Š4.9 ng/mL vs 8.3 ±â€Š3.8 ng/mL, P < .001). SLE patients exhibited higher galectin-9 levels, whereas the galectin-9 levels did not correlate with liver function tests such as alanine aminotransferase levels.Serum galectin-9 correlated with disease status in AIH patients and could thus be useful biomarkers to detect hepatic autoimmunity. Because circulating galectin-9 reflects autoimmune-mediated inflammation, it may have additional utility as a biomarker for other autoimmune disorders.

Role of deleterious single nucleotide variants in the coding regions of TNFAIP3 for Japanese autoimmune hepatitis with cirrhosis.

Autoimmune hepatitis (AIH) is an autoimmune liver disease and cirrhosis is sometimes complicated with AIH at diagnosis, influencing its prognosis. TNFAIP3 gene encodes A20, an inhibitor of nuclear factor-κB pathway, and is a susceptibility gene for autoimmune diseases. We investigated deleterious variants in the coding regions of TNFAIP3 gene of Japanese AIH patients or those with cirrhosis. The deleterious variants in the coding regions of TNFAIP3 gene were analyzed by the cycle sequencing method and the frequencies of deleterious TNFAIP3 alleles of AIH or AIH with cirrhosis were compared with those of Japanese controls. The deleterious alleles in TNFAIP3 were not associated with AIH. A significant association was shown for the deleterious alleles in TNFAIP3 (P = 0.0180, odds ratio (OR) 4.28, 95% confidence interval (CI) 1.53-11.95) with AIH with cirrhosis at presentation. The serum IgM levels in AIH patients with deleterious alleles in TNFAIP3 were tended to be lower than those without (P = 0.0152, Q = 0.1216). The frequency of deleterious alleles in TNFAIP3 was higher in the AIH subset without the DRB1 risk alleles than that with (P = 0.0052, OR 5.10, 95%CI 1.55-16.74). The deleterious alleles in TNFAIP3were associated with AIH with cirrhosis.

NFKB1 and MANBA Confer Disease Susceptibility to Primary Biliary Cholangitis via Independent Putative Primary Functional Variants.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is a chronic and cholestatic liver disease that eventually leads to cirrhosis and hepatic failure. We recently identified several susceptibility genes included NFKB1 and MANBA for PBC in the Japanese population by genome-wide association study. However, the primary functional variants in the NFKB1/MANBA region and the molecular mechanism for conferring disease susceptibility to PBC have not yet been clarified. METHODS: We performed high-density association mapping based on a single-nucleotide polymorphism (SNP) imputation analysis, using data from a whole-genome sequence reference panel of 1070 Japanese individuals and the previous genome-wide association study (1389 PBC patients, 1508 healthy controls). Among SNPs (P < 5.0 × 10-7) in the NFKB1/MANBA region, putative primary functional variants and the molecular mechanism for conferring disease susceptibility to PBC were identified by in silico/in vitro functional analysis. RESULTS: Among the SNPs in the NFKB1/MANBA region, rs17032850 and rs227361, which changed the binding of transcription factors lymphoid enhancer-binding factor 1 (LEF-1) and retinoid X receptor α (RXRα), respectively, were identified as putative primary functional variants that regulate gene expression. In addition, expression-quantitative trait locus data and gene editing using a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system supported the potential role of rs17032850 and rs227361 in regulating NFKB1 and MANBA expression, respectively. CONCLUSIONS: We identified independent putative primary functional variants in NFKB1/MANBA and showed the distinct molecular mechanism by which each putative primary functional variant conferred susceptibility to PBC. Our approach was useful to dissect the pathogenesis not only of PBC, but also other digestive diseases in which NFKB1/MANBA has been reported as a susceptibility locus.

A Genome-wide Association Study Identifying RAP1A as a Novel Susceptibility Gene for Crohn's Disease in Japanese Individuals.

BACKGROUND AND AIMS: Genome-wide association studies [GWASs] of European populations have identified numerous susceptibility loci for Crohn's disease [CD]. Susceptibility genes differ by ethnicity, however, so GWASs specific for Asian populations are required. This study aimed to clarify the Japanese-specific genetic background for CD by a GWAS using the Japonica array [JPA] and subsequent imputation with the 1KJPN reference panel. METHODS: Two independent Japanese case/control sets (Tohoku region [379 CD patients, 1621 controls] and Kyushu region [334 CD patients, 462 controls]) were included. GWASs were performed separately for each population, followed by a meta-analysis. Two additional replication sets [254 + 516 CD patients and 287 + 565 controls] were analysed for top hit single nucleotide polymorphisms [SNPs] from novel genomic regions. RESULTS: Genotype data of 4 335 144 SNPs from 713 Japanese CD patients and 2083 controls were analysed. SNPs located in TNFSF15 (rs78898421, Pmeta = 2.59 × 10-26, odds ratio [OR] = 2.10), HLA-DQB1 [rs184950714, pmeta = 3.56 × 10-19, OR = 2.05], ZNF365, and 4p14 loci were significantly associated with CD in Japanese individuals. Replication analyses were performed for four novel candidate loci [p <1 × 10-6], and rs488200 located upstream of RAP1A was significantly associated with CD [pcombined = 4.36 × 10-8, OR = 1.31]. Transcriptome analysis of CD4+ effector memory T cells from lamina propria mononuclear cells of CD patients revealed a significant association of rs488200 with RAP1A expression. CONCLUSIONS: RAP1A is a novel susceptibility locus for CD in the Japanese population.

Serum cytokine profiles and Mac-2 binding protein glycosylation isomer (M2BPGi) level in patients with autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an autoimmune liver disease that is characterized by a progressive destruction of the liver parenchyma and the development of liver fibrosis. We aimed to examine the relationship between circulating cytokines/chemokines and the Mac-2 binding protein glycosylation isomer (M2BPGi) levels in Japanese patients with autoimmune hepatitis (AIH).We investigated the relationship between circulating cytokines/chemokines and M2BPGi levels in Japanese patients with AIH. Seventy-seven patients with well-documented AIH were enrolled in the National Hospital Organization (NHO)-AIH-liver-network database. We measured the serum levels of 20 cytokines in 31 selected AIH patients before and after steroid treatment using multisuspension cytokine array.Eleven cytokines and soluble adhesion molecules were increased in untreated AIH patients compared with treated AIH patients. Among these cytokines and soluble adhesion molecules, soluble intercellular adhesion molecule-1 (sICAM-1) and interferon-γ-inducible protein 10 (IP-10) were most downregulated by steroid therapy in AIH patients. We measured serum sICAM-1 and IP-10 by ELISA and found the levels were significantly higher in AIH patients (n = 77) compared with chronic viral hepatitis C patients (n = 32). Furthermore, there was a positive correlation between sICAM-1 or IP-10 and alanine aminotransferase, total bilirubin, and circulating M2BPGi levels. M2BPGi levels were increased in AIH patients with high stages of liver fibrosis. Additionally, M2BPGi levels were correlated with the histological grade of inflammation in AIH. Circulating M2BPGi levels were significantly reduced by steroid treatment in AIH patients.sICAM-1 and IP-10 are useful markers to assess immune-mediated hepatitis activity in AIH and they correlate with circulating M2BPGi. Serum M2BPGi levels increased in untreated AIH patients with active hepatitis and were decreased by steroid therapy. M2BPGi reflects autoimmune-mediated hepatic inflammation as well as liver fibrosis.

Identification of the functional variant driving ORMDL3 and GSDMB expression in human chromosome 17q12-21 in primary biliary cholangitis.

Numerous genome-wide association studies (GWAS) have been performed to identify susceptibility genes to various human complex diseases. However, in many cases, neither a functional variant nor a disease susceptibility gene have been clarified. Here, we show an efficient approach for identification of a functional variant in a primary biliary cholangitis (PBC)-susceptible region, chromosome 17q12-21 (ORMDL3-GSDMB-ZPBP2-IKZF3). High-density association mapping was carried out based on SNP imputation analysis by using the whole-genome sequence data from a reference panel of 1,070 Japanese individuals (1KJPN), together with genotype data from our previous GWAS (PBC patients: n = 1,389; healthy controls: n = 1,508). Among 23 single nucleotide polymorphisms (SNPs) with P < 1.0 × 10-8, rs12946510 was identified as the functional variant that influences gene expression via alteration of Forkhead box protein O1 (FOXO1) binding affinity in vitro. Moreover, expression-quantitative trait locus (e-QTL) analyses showed that the PBC susceptibility allele of rs12946510 was significantly associated with lower endogenous expression of ORMDL3 and GSDMB in whole blood and spleen. This study not only identified the functional variant in chr.17q12-21 and its molecular mechanism through which it conferred susceptibility to PBC, but it also illustrated an efficient systematic approach for post-GWAS analysis that is applicable to other complex diseases.

Hydrophobic bile acids suppress expression of AE2 in biliary epithelial cells and induce bile duct inflammation in primary biliary cholangitis.

Understanding the mechanisms of chronic inflammation in primary biliary cholangitis (PBC) is essential for successful treatment. Earlier work has demonstrated that patients with PBC have reduced expression of the anion exchanger 2 (AE2) on biliary epithelial cells (BEC) and deletion of AE2 gene has led to a PBC-like disorder in mice. To directly address the role of AE2 in preventing PBC pathogenesis, we took advantage of our ability to isolate human BEC and autologous splenic mononuclear cells (SMC). We studied the influence of hydrophobic bile acids, in particular, glycochenodeoxycholic acid (GCDC), on AE2 expression in BEC and the subsequent impact on the phenotypes of BEC and local inflammatory responses. We demonstrate herein that GCDC reduces AE2 expression in BEC through induction of reactive oxygen species (ROS), which enhances senescence of BEC. In addition, a reduction of AE2 levels by either GCDC or another AE2 inhibitor upregulates expression of CD40 and HLA-DR as well as production of IL-6, IL-8 and CXCL10 from BEC in response to toll like receptor ligands, an effect suppressed by inhibition of ROS. Importantly, reduced AE2 expression enhances the migration of autologous splenic mononuclear cells (SMC) towards BEC. In conclusion, our data highlight a key functional role of AE2 in the maintenance of the normal physiology of BEC and the pathogenic consequences of reduced AE2 expression, including abnormal intrinsic characteristics of BEC and their production of signal molecules that lead to the chronic inflammatory responses in small bile ducts.

Fine-mapping analysis revealed complex pleiotropic effect and tissue-specific regulatory mechanism of TNFSF15 in primary biliary cholangitis, Crohn's disease and leprosy.

Genetic polymorphism within the 9q32 locus is linked with increased risk of several diseases, including Crohn's disease (CD), primary biliary cholangitis (PBC) and leprosy. The most likely disease-causing gene within 9q32 is TNFSF15, which encodes the pro-inflammatory cytokine TNF super-family member 15, but it was unknown whether these disparate diseases were associated with the same genetic variance in 9q32, and how variance within this locus might contribute to pathology. Using genetic data from published studies on CD, PBC and leprosy we revealed that bearing a T allele at rs6478108/rs6478109 (r(2) = 1) or rs4979462 was significantly associated with increased risk of CD and decreased risk of leprosy, while the T allele at rs4979462 was associated with significantly increased risk of PBC. In vitro analyses showed that the rs6478109 genotype significantly affected TNFSF15 expression in cells from whole blood of controls, while functional annotation using publicly-available data revealed the broad cell type/tissue-specific regulatory potential of variance at rs6478109 or rs4979462. In summary, we provide evidence that variance within TNFSF15 has the potential to affect cytokine expression across a range of tissues and thereby contribute to protection from infectious diseases such as leprosy, while increasing the risk of immune-mediated diseases including CD and PBC.