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

Abstract

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.

Keywords: Primary sclerosing cholangitis; genetics; liver transplantation.

Figure 1

Association of genetic variants on chromosome 6 with transplant-free survival of patients with PSC. (A) Kaplan-Meier curves of transplant-free survival. Patients are stratified according to their genotype for SNP rs853974. The p value for genotype effect in the Cox proportional hazards model is p=6.07.10⁻⁰⁹. (B) Regional association plot for transplant-free survival. The Y-axis shows the −log₁₀(p value) for genotype effect in the Cox proportional hazards model. PSC, primary sclerosing cholangitis; SNP, single nucleotide polymorphism.

Figure 2

RSPO3 expression in mouse cholangiocytes and in human CLCs, PBD and HSCs. (A) RNA sequencing analysis of RSPO3 expression in DDC-induced cholestatic cholangiocytes, healthy cholangiocytes and multiple organs of normal C57BL/6 mice. (B) Microarray RSPO3 expression in hiPSCs, CLCs and PBD. RSPO3 expression is significantly increased in CLCs and in PBD compared with hiPSCs. n=3; error bars, SD. Asterisks represent statistical significance (****adjusted p<0.0001, ***adjusted p<0.001, Benjamini and Hochberg corrected p values). (C) Quantitative real-time PCR analysis demonstrating the expression of RSPO3 in hiPSC-derived CLCs and PBD samples compared with expression in hiPSCs. Expression levels are fold changes compared with housekeeping gene HMDS calculated using the 2^−ΔCt method. (D) Quantitative real-time PCR analysis showing expression of RSPO3 and cytoglobin B in three patients without PSC. Cytoglobin B messenger RNA expression was evaluated as specific HSC marker. Target genes were normalised using GAPDH as endogenous control and their relative expression was calculated with the 2^−ΔCt method. CLCs, cholangiocyte-like-cells; DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; FPKM, fragments per kilobase of exon per million mapped reads; hIPSC, human-induced pluripotent stem cells; HSC, hepatic stellate cell; PBD, primary bile duct.