The Protease Genes Cyld and USP40 Are Associated With Crohn's Disease: Results From a European Consortium

Abstract

Background: Proteases and protease inhibitors affect several components that contribute to mucosal barrier integrity, and therefore may be important players in the pathophysiology of Inflammatory Bowel Disease (IBD). Aim: We aimed to elucidate if and which proteases/ protease inhibitors are involved in IBD pathogenesis. Methods: In a first step, a systematic review was performed of all relevant published genetic studies in Crohn's disease (CD) and ulcerative colitis (UC), to prioritize all known proteases/protease inhibitors based on the amount of available genetic evidence for association with IBD. In a second step, the topranked genes for CD and UC were followed up in a genetic association study. A total of 185 haplotype tagging SNPs in 23 genes were genotyped in an exploratory cohort of 650 CD (CD1), 721 UC (UC1), and 542 healthy controls (HC1). Replication of SNPs with puncorrected<0.1 was performed in 4 independent cohorts: CD2 (n=634), UC2 (n=528), HC2 (n=900); CD3 (n=377), UC3 (n=290), HC3(n=354); CD4 (n=432), UC4 (n=432); and CD5 (n=227), UC5 (n=141). Cases and controls were compared according to the additive genetic model. False Discovery Rate (FDR) correction was applied to correct for multiple testing (R 2.9.1). Results: All SNPs showing significant associations (pFDR<0.05) in the combined cohort [CD1-5 (n=2320) vs HC1-3 (n=1796), and UC1-5 (n=2112) vs HC1-3] are shown in Table 1. Strongest evidence was found in CD for CYLD, located 9kb downstream of CARD15, with 2 risk increasing and 2 protective SNPs. To test whether the signal seen in CYLD is driven by thewell-established CARD15 association, logistic regressionwas performed. CARD15 data were available in 1135 CD and 674 HC. The final model included both CARD15 and CYLD, pointing to independent signals from both genes (pCYLDrs12324931<0.001, OR=2.1[1.6-2.8]; pCARD15rs2066845<0.001, OR=2.9[1.8-4.8]; pCARD15rs2066847<0.001, OR= 3.7[2.5-5.5]). Patients without CARD15 variants showed significant association with rs12324931 (p=0.005, OR=2.9[1.3-6.6]). Another interesting outcome is the strong signal seen in CD for USP40, located near ATG16L1. Logistic regression analysis in CD1 (versus HC1), showed that rs10929178 and rs12472244 (USP40), and rs2241880 (ATG16L1) are independently associated with risk for CD (p=0.009, OR[0.6-0.9]; p=0.05, OR[0.8-1.0]; and p=0.009, OR[0.6-0.9] respectively). Conclusions: We provide strong evidence for association of CYLD on 16q12.1 in CD patients, which is independent from CARD15. CYLD, a cytoplasmic deubiquitinating enzyme, is a key negative regulator of NF-κB, and has been shown to be significantly down-regulated in the intestine of IBD patients. Moreover, we identified several variants in other protease/protease inhibitor genes which are implicated in either CD (USP40, a ubiquitin-specific peptidase), UC (DAG1, MST1, PSMB8), or both (APEH, USP3). Table 1