Brijesh Srivastava

Genome-wide association study of primary sclerosing cholangitis identifies new risk loci and quantifies the genetic relationship with inflammatory bowel disease

Primary sclerosing cholangitis (PSC) is a rare progressive disorder leading to bile duct destruction; ∼75% of patients have comorbid inflammatory bowel disease (IBD). We undertook the largest genome-wide association study of PSC (4,796 cases and 19,955 population controls) and identified four new genome-wide significant loci. The most associated SNP at one locus affects splicing and expression of UBASH3A, with the protective allele (C) predicted to cause nonstop-mediated mRNA decay and lower expression of UBASH3A. Further analyses based on common variants suggested that the genome-wide genetic correlation (rG) between PSC and ulcerative colitis (UC) (rG = 0.29) was significantly greater than that between PSC and Crohns disease (CD) (rG = 0.04) (P = 2.55 × 10−15). UC and CD were genetically more similar to each other (rG = 0.56) than either was to PSC (P < 1.0 × 10−15). Our study represents a substantial advance in understanding of the genetics of PSC.

Fine mapping and replication of genetic risk loci in primary sclerosing cholangitis

Abstract Background and aims. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts eventually leading to biliary cirrhosis. Recent genetic studies in PSC have identified associations at 2q13, 2q35, 3p21, 4q27, 13q31 and suggestive association at 10p15. The aim of this study was to further characterize and refine the genetic architecture of PSC. Methods. We analyzed previously reported associated SNPs at four of these non-HLA loci and 59 SNPs tagging the IL-2/IL-21 (4q27) and IL2RA (10p15) loci in 992 UK PSC cases and 5162 healthy UK controls. Results. The most associated SNPs identified were rs3197999 (3p21 (MST1), p = 1.9 × 10-6, ORA vs G = 1.28, 95% CI (1.16–1.42)); rs4147359 (10p15 (IL2RA), p = 2.6 × 10-4, ORA vs G = 1.20, 95% CI (1.09–1.33)) and rs12511287 (4q27 (IL-2/IL-21), p = 3.0 × 10-4, ORA vs T = 1.21, 95% CI (1.09–1.35)). In addition, we performed a meta-analysis for selected SNPs using published summary statistics from recent studies. We observed genome-wide significance for rs3197999 (3p21 (MST1), P combined = 3.8 × 10-12) and rs4147359 (10p15 (IL2RA), P combined = 1.5 × 10-8). Conclusion. We have for the first time confirmed the association of PSC with genetic variants at 10p15 (IL2RA) locus at genome-wide significance and replicated the associations at MST1 and IL-2/IL-21 loci in a large homogeneous UK population. These results strongly implicate the role of IL-2/IL2RA pathway in PSC and provide further confirmation of MST1 association.

Genetic association analysis identifies variants associated with disease progression in primary sclerosing cholangitis

Objective Primary sclerosing cholangitis (PSC) is a genetically complex, inflammatory bile duct disease of largely unknown aetiology often leading to liver transplantation or death. Little is known about the genetic contribution to the severity and progression of PSC. The aim of this study is to identify genetic variants associated with PSC disease progression and development of complications. Design We collected standardised PSC subphenotypes in a large cohort of 3402 patients with PSC. After quality control, we combined 130 422 single nucleotide polymorphisms of all patients—obtained using the Illumina immunochip—with their disease subphenotypes. Using logistic regression and Cox proportional hazards models, we identified genetic variants associated with binary and time-to-event PSC subphenotypes. Results We identified genetic variant rs853974 to be associated with liver transplant-free survival (p=6.07×10–9). Kaplan-Meier survival analysis showed a 50.9% (95% CI 41.5% to 59.5%) transplant-free survival for homozygous AA allele carriers of rs853974 compared with 72.8% (95% CI 69.6% to 75.7%) for GG carriers at 10 years after PSC diagnosis. For the candidate gene in the region, RSPO3, we demonstrated expression in key liver-resident effector cells, such as human and murine cholangiocytes and human hepatic stellate cells. Conclusion We present a large international PSC cohort, and report genetic loci associated with PSC disease progression. For liver transplant-free survival, we identified a genome-wide significant signal and demonstrated expression of the candidate gene RSPO3 in key liver-resident effector cells. This warrants further assessments of the role of this potential key PSC modifier gene.

1314 OUTCOMES OF LIVER TRANSPLANTATION IN PRIMARY SCLEROSING CHOLANGITIS (PSC) – A SINGLE UK CENTRE EXPERIENCE

Eleven of them were common targets of antibodies present in all patient sera and often found in auto-immune disorders. Conclusion: This is the first immunological description of a new type of de novo-AIH occurring after BMT. The eleven common antigenic targets differ from those reported in de novo-AIH occurring after hepatic transplantation. The existence of an hepatic variant of GVHD can be put under consideration.

Genotype-phenotype analysis across 130 422 genetic variants identifies RSPO3 as the first genome-wide significant modifier gene in primary sclerosing cholangitis

Background Ulcerative colitis (UC), a complex polygenic disorder, is one of the main subphenotypes of inflammatory bowel disease. A comprehensive dissection of the genetic etiology of UC needs to assess the contribution of rare genetic variants including copy number variations (CNVs) to disease risk. In this study, we performed a multi-step genome-wide case-control analysis to interrogate the presence of disease-relevant rare copy number variants. Methods One thousand one hundred twenty-one German UC patients and 1770 healthy controls were initially screened for rare deletions and duplications employing SNP-array data. Quantitative PCR and high density custom array-CGH were used for validation of identified CNVs and fine mapping. Two main follow-up panels consisted of an independent cohort of 451 cases and 1274 controls, in which CNVs were assayed through quantitative PCR, and a British cohort of 2396 cases versus 4886 controls with CNV genotypes based on array data. Additional sample sets were assessed for targeted and in silico replication. Results Twenty-four rare copy number variants (14 deletions and 10 duplications), overrepresented in UC patients were identified in the initial screening panel. Follow-up of these CNV regions in four independent case-control series as well as an additional public in silico control group (totaling 4439 UC patients and 15,961 healthy controls) revealed three copy number variants enriched in UC patients; a 15.8 kb deletion upstream of ABCC4 and CLDN10 at13q32.1 (0.43 % cases, 0.11 % controls), a 119 kb duplication at 7p22.1, overlapping RNF216, ZNF815, OCM and CCZ1 (0.13 % cases, 0.01 % controls) and a 134 kb large duplication upstream of the KCNK9 gene at 8q24.3 (0.22 % carriers among cases, 0.03 % carriers among controls). The trend of association with UC was present after the P-values were corrected for combining data from different subpopulations. Break-point mapping of the deleted region suggested non-allelic homologous recombination as the mechanism underlying its formation. Conclusion Our study presents a pragmatic approach for effective rare CNV screening of SNP-array data sets and implicates the potential contribution of rare structural variants in the pathogenesis of UC.

956 RISK FACTOR PREDICTIVE OF TRANSPLANT FREE SURVIVAL IN A LARGE COHORT OF PSC PATIENTS IN THE UK

Conclusion: We here demonstrate that the protection from autoimmune cholangitis which was observed in male mice was mediated by testosterone. Indeed, testosterone supplementation induced resistance to disease in otherwise susceptible female mice. The increased inflammation observed in female mice was associated with increased IL-17 expression by endogenous CD4 positive T cells which seems to be recruited to the liver via an increased expression of chemokines that recruit T cells to the liver. These results may stimulate future investigations into gender differences observed in autoimmune liver diseases.

PTU-070 Fine mapping of the IL-2/IL-21 and IL2RA loci in primary sclerosing cholangitis

Introduction Recent genetic studies in Primary sclerosing cholangitis (PSC), a chronic bile duct disease, have shown suggestive association at IL-2/21 (4q27) and IL2RA (10p15). IL-2 and IL2RA are key regulators of immune tolerance. To further refine association at 4q27 and 10p15, a fine mapping study was undertaken in 1030 British PSC cases and 5162 healthy controls. Methods For SNP selection, 80 Kbp and 564 Kbp regions were selected on 10p15 and 4q27, respectively, and SNP data from HapMap Data Rel 24/phase II was used to identify tag SNPs with Haploview v4.2. 62 tag SNPs were genotyped on a Sequenom platform. Control genotype data were available for 62 SNPs, previously genotyped in the Wellcome Trust Case Control Consortium 2 (WTCCC 2). 59 SNPs (28 at 4q27 and 31 at 10p15) passed quality control and were analysed using logistic regression in PLINK v1.07. For selected SNPs, previously published summary statistics1 were used to perform a meta-analysis. Results Significant association (p<8.5×10−4) corrected for multiple testing (Bonferroni method) was observed for one SNP at 4q27 and three SNPs at 10p15 (Abstract PTU-070 table 1). In addition, nominal significance (p<0.05) was seen for 9/27 SNPs at 4q27 and 10/28 SNPs at 10p15. Genome-wide significance (p<5×10−8) was observed for rs4147359 (10p15) in the combined analysis.Abstract PTU-070 Table 1 Analysis results of associated SNPs Chr SNP Locus Alleles (minor/major) Minor allele frequency (cases/controls) p Value (UK cohort) OR UK cohort (95% CI) OR (combined) (95% CI) p Value (combined) 4 rs12511287 IL2/IL21 A/T 0.30/0.26 2.9×10−4 1.21 (1.09 to 1.34) – – 10 rs4147359* IL2RA A/G 0.38/0.34 2.6×10−4 1.20 (1.08 to 1.32) 1.25 (1.16 to 1.36) 1.5×10−8 10 rs706778* IL2RA T/C 0.44/0.39 4.3×10−4 1.19 (1.08 to 1.31) 1.24 (1.14 to 1.35) 3.4×10−7 10 rs7090530 IL2RA C/A 0.35/0.39 6.9×10−4 0.83 (0.75 to 0.92) – – *Meta-analysis using previously published summary statistics1 was performed in R statistical software package using the Metagen package. Conclusion This study confirms the IL2RA and IL-2/IL-21 locus association in PSC suggesting a role of adaptive immune responses. Genetic variants at these loci are associated with autoimmune diseases. Association of PSC with variants at these loci could imply not only a shared genetic risk with other diseases but also identify the immunological pathways favouring disease development. Functional studies are now required to identify the causative gene or genes to facilitate rapid translation to the discovery of novel therapeutics. Competing interests None declared. Reference 1. Melum E, et al. Nat Genet 2011;43:17–19.

OP07 Phenotypic description of a large cohort of PSC patients in the UK

Introduction Primary Sclerosing cholangitis (PSC), a chronic cholestatic liver disease of unknown aetiology or pathogenesis, remains an area for active research. Aim The demographic and phenotypic characteristics of a UK cohort of 1194 patients with PSC are described. Method All patients were recruited as part of an ongoing national collaborative effort (PSC-UK) between October 2008 and May 2011. The diagnosis of PSC was confirmed on the basis of characteristic ERCP, MRCP or histology. Patients with small-duct PSC were also included. All patients completed a descriptive phenotypic questionnaire sent at recruitment. Demographic, phenotypic data and family history were extracted from the participant questionnaires. Results 1194 patients have returned completed questionnaires. Median age at recruitment was 59 years and 63% were male (male to female ratio =1.7:1). 64% of patients were lifelong non-smokers and only 4.3% were smoking at recruitment. 63.5% reported inflammatory bowel disease, split into 87% with Ulcerative colitis and 13% with Crohns disease. 1.5% had a sibling with PSC and 14% had a sibling with inflammatory bowel disease. Further, 0.5% had one or more children with PSC and 4.6% had children with inflammatory bowel disease. 24% were asymptomatic, but over 50% reported itching and fatigue as the presenting symptom. Jaundice was present in 35%. The most common associated autoimmune disease was thyroid disease, present in 9% followed by Coeliac disease in 2.1% and type 1 diabetes mellitus in 1.8%. 16% of patients reported pan-procto or sub-total colectomy and 12.2% had undergone cholecystectomy. 2.7% of patients reported a history of colon cancer and 1.25% patients had a history of skin cancer. Conclusion This is the largest reported demographic and phenotypic description of a single cohort of PSC patients. PSC is more common in young, non-smoking male patients. The frequency of associated inflammatory bowel disease is similar to that reported in other studies. It is plausible that siblings of patients with PSC have an increased risk of not only PSC but also inflammatory bowel disease. These data are consistent with increasing evidence pointing to a role of genetic factors in the pathogenesis of PSC.