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OP008 Genotype-phenotype analysis across 130 422 genetic variants identifies RSPO3 as the first genome-wide significant modifier gene in primary sclerosing cholangitis

R. Alberts*1, E. M. de Vries2, G. Alexander3, D. Alvaro4, A. Bergquist5, U. Beuers2, E. Björnsson6, K. M. Boberg7, C. L. Bowlus8, R. W. Chapman9, O. Chazouillères10, A. Cheung11, G. Dalekos12, B. Eksteen13, D. Ellinghaus14, M. Färkkilä15, E. A. Festen1, A. Floreani16, T. Folseraas7, E. Goode17, D. N. Gotthardt18, G. M. Hirschfield19, B. van Hoek20, K. Holm7, S. Hohenester21, J. R. Hov7, F. Imhann1, P. Invernizzi22, X. Jiang7, J. Eaton23, B. D. Juran24, K. N. Lazaridis24, V. Leppa25, J. Z. Liu26, J. Löfberg27, M. P. Manns28, H.-U. Marschall29, M. Marzioni30, A. L. Mason31, E. Melum7, P. Milkiewicz32, T. Müller33, A. Pares34, C. Rupp35, S. M. Rushbrook36, C. Rust37, F. Sampaziotis38, R. N. Sandford39, C. Schramm40, S. Schreiber14, E. Schrumpf7, M. Silverberg41, B. Srivastava39, M. Sterneck42, A. Teufel43, L. Tittmann44, L. Vallier38, A. V. Vila1, B. de Vries1, T. J. Weismüller28, C. Wijmenga45, K. Zachou46, A. Franke14, C. A. Anderson47, T. H. Karlsen7, C. Y. Ponsioen2, R. K. Weersma1

1University of Groningen and University Medical Centre Groningen, Department of Gastroenterology and Hepatology, Groningen, Netherlands, 2Academic Medical Centre, Department of Gastroenterology and Hepatology, Amsterdam, Netherlands, 3University of Cambridge, Department of Medicine, Division of Hepatology, Cambridge, United Kingdom, 4Sapienza University of Rome, Department of Clinical Medicine, Division of Gastroenterology, Rome, Italy, 5Karolinska University Hospital Huddinge, Karolinska Institutet, Department of Gastroenterology and Hepatology, Stockholm, Sweden, 6Landspitali University Hospital, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Reykjavik, Iceland, 7Oslo University Hospital, Rikshospitalet, Norwegian PSC Research Centre, Division of Cancer Medicine, Surgery and Transplantation, Oslo, Norway, 8University of California Davis, Division of Gastroenterology and Hepatology, Davis, California, United States, 9John Radcliffe University Hospitals NHS Trust, Department of Hepatology, Oxford, United Kingdom, 10Hôpital Saint Antoine and U.P.M.C. Univ Paris 06, AP-HP and Department of Hepatology, Paris, France, 11University Health Network - Toronto General Hospital, Gen. Internal Medicine, Toronto, Canada, 12Medical School, University of Thessaly, Department of Medicine and Research Laboratory of Internal Medicine, Larissa, Greece, 13University of Calgary, Snyder Institute of Chronic Diseases, Department of Medicine, Calgary, Canada, 14Christian-Albrechts-University of Kiel, Institute of Clinical Molecular Biology, Kiel, Germany, 15Helsinki University Hospital, Division of Gastroenterology, Department of Medicine, Helsinki, Finland, 16University of Padova, Dept. of Surgical, Oncological and Gastroenterological Sciences, Padova, Italy, 17Norfolk and Norwich University Hospital, Department of Gastroenterology, Norwich, United Kingdom, 18University Hospital of Heidelberg, Department of Medicine, Heidelberg, Germany, 19University of Toronto, Division of Gastroenterology, Department of Medicine, Toronto, Canada, 20Leiden University Medical Centre, Department of Gastroenterology and Hepatology, Leiden, Netherlands, 21University of Munich, Department of Medicine II, Munich, Germany, 22Humanitas Clinical and Research Centre, Centre for Autoimmune Liver Diseases, Rozzano, Italy, 23Mayo Clinic, Division of Gastroenterology and Hepatology, Rochester, Minnesota, United States, 24Mayo Clinic, College of Medicine, Centre for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Rochester, Minnesota, United States, 25Institute for Molecular Medicine Finland F.I.M.M., University of Helsinki and National Institute for Health and Welfare, Public Health Genomics Unit, Helsinki, Finland, 26Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom, 27Karolinska Institute, Department of, Stockholm, Sweden, 28Hannover Medical School, Department of Gastroenterology, Hepatology and Endocrinology, Hannover, Germany, 29Institute of Medicine, Sahlgrenska Academy and University Hospital, Department of Internal Medicine, Gothenburg, Sweden, 30Università Politecnica delle Marche, Ospedali Riuniti University Hospital, Department of Gastroenterology, Ancona, Italy, 31University of Alberta, Division of Gastroenterology and Hepatology, Edmonton, Alberta, Canada, 32Medical University of Warsaw, Liver and Internal Medicine Unit, Warsaw, Poland, 33Charité Universitätsmedizin Berlin, Department of Internal Medicine, Hepatology and Gastroenterology, Berlin, Germany, 34University of Barcelona, Liver Unit, Hospital Clinic, I.D.I.B.A.P.S., C.I.B.E.R.ehd, Barcelona, Spain, 35University Hospital of Heidelberg, Department of Internal Medicine IV, Heidelberg, Germany, 36University Hospitals NHS Trust, Department of Gastroenterology and Hepatology, Norfolk and Norwich, Norwich, United Kingdom, 37University of Munich, Department of Medicine 2, Grosshadern, Munich, Germany, 38Wellcome Trust-Medical Research Council Stem Cell Institute, Anne McLaren Laboratory, Cambridge, United Kingdom, 39University of Cambridge, Academic Department of Medical Genetics, Cambridge, United Kingdom, 40University Medical Centre Hamburg-Eppendorf, 1st Department of Medicine, Hamburg, Germany, 41Mount Sinai Hospital Toronto, Inflammatory Bowel Disease Group, Zane Cohen Centre for Digestive Diseases, Toronto, Ontario, Canada, 42University Medical Centre Hamburg-Eppendorf, Department of Hepatobiliary Surgery and Transplantation, Hamburg, Germany, 43University of Mainz, First Department of Medicine, Mainz, Germany, 44Christian-Albrechts-University of Kiel, Institute for Epidemiology, Kiel, Germany, 45University of Groningen and University Medical Centre Groningen, Department of Genetics, Groningen, Netherlands, 46University of Thessaly, Department of Internal Medicine, Larissa, Greece, 47Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, Massachusetts, United States

Background

Primary sclerosing cholangitis (PSC) is a severe disease of the bile ducts without curative medical treatment, often leading to liver transplantation (LTx) or death in young patients. PSC is strongly associated with inflammatory bowel disease (IBD). About 2% to 7.5% of patients with ulcerative colitis have PSC, whereas 70% to 80% of patients with PSC have IBD. Genetic variants that are associated with PSC susceptibility have been identified through genome-wide association analyses. Little is known on the role of genetics in disease behaviour or progression in complex diseases in general or PSC in particular.

Methods

To identify genetic variants driving PSC sub phenotypes, the International PSC Study Group has collected extensive clinical data for 3 402 PSC patients from 13 countries. Phenotypes include dates of birth and diagnosis, cause of death, dates of LTx, co-occurrence with IBD, etc. Patients were genotyped using the Illumina Immunochip microarray. We performed within-cases association analysis for binary outcomes and Cox-proportional hazards modelling for time-to-event phenotypes using 130 422 single nucleotide polymorphisms (SNP) after quality control. We corrected for clinical parameters, such as smoking status, by adding them to the model. We imputed the HLA using SNP2HLA.

Results

We identify a genomic region on chromosome 6, harbouring RSPO3, to be associated significantly with liver transplant-free survival (SNP rs853974, p = 2.87 × 10–8) When including cholangiocarcinoma as an endpoint, the association remained significant (p = 2.04 × 10–9). Kaplan–Meier analysis shows that homozygous AA allele carriers of rs853974 have a 55% chance of liver transplant-free survival of 10 years, as compared with GG carriers, which have a 75% chance. Using RNA-Seq on cholangiocytes (control and DDC–treated mice), as well as multiple tissues in mice, we showed significantly elevated expression of RSPO3 in cholangiocytes. In addition, we were able to demonstrate high expression of RPSO3 in cholangiocyte-like cells as compared with human induced pluripotent stem cells, and confirmed this by qPCR. Binary outcomes revealed several suggestive associations. We confirm published associations of classical HLA alleles for AIH in patients with PSC-AIH–overlap features (DQA1*05:01, p = 1.87 × 10–7 and DQB1*02:01, p = 3.81 × 10–11).

Conclusion

In this large International PSC Study Group cohort, we show that a genetic variant next to RSPO3 is associated with liver transplant-free survival. The protein RSPO3 is an agonist of the canonical Wnt/β-catenin signalling pathway, which plays a central role in stem cell maintenance and pro- and regression of liver fibrosis. Findings of RSPO3 being highly expressed in both mouse and human cholangiocytes warrant further assessments in querying the role of this potential key PSC modifier gene.