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Early Fibrostenosis in Crohn's Disease is Associated with Multiple Susceptibility Loci on Immunochip Analysis
AGA Abstracts
of specific residues in the C-terminal domain of FOXP3 protein including human diseasecausing mutations implicated in IBD (C232G) and IPEX syndrome (L242P, K250D, E251∆) attenuated EZH2 interaction. Conclusively, we showed that deletion of the C-terminal domain of FOXP3 abrogated EZH2 recruitment; however, as expected, both the N- and C-terminal domains were required for FOXP3's ability to repress its known target IL-2 as evident from a DLR assay. Furthermore, these disease-causing FOXP3 mutants lacked their abilities to effectively repress IL-2 expression compared to FOXP3 wild-type as evident from DLR assay and flow cytometric analysis. Finally, in human Tregs, FOXP3-EZH2 interaction was inhibited by approximately 50% upon IL-6 treatment in a JAK/STAT3-dependent manner. Conclusion: Thus, using a private mutation associated with heritable IBD we discovered a more generalizable mechanism by which pro-inflammatory signaling pathways disrupt a critical epigenetic regulatory complex in FOXP3+Tregs.
Overview of identified SNPs associated with early fibrostenosis
Tu1828 DICHOTOMOUS EFFECTS OF ATG16L1 AND LRRK2 IN MODULATING PANETH CELL DEFECT IN JAPANESE AND NORTH AMERICAN CROHN'S DISEASE PATIENTS Ta-Chiang Liu, Takeo Naito, Zhenqiu Liu, Kelli VanDussen, Talin Haritunians, Dalin Li, Katsuya Endo, Yosuke Kawai, Masao Nagasaki, Yoshitaka Kinouchi, Dermot McGovern, Tooru Shimosegawa, Yoichi Kakuta, Thaddeus S. Stappenbeck
Tu1830
Background: Morphological patterns of Paneth cells are a prognostic biomarker in North American Crohn's disease (CD) patients, and are associated with autophagy-associated ATG16L1 and NOD2 variants. Given the different genetic landscape in inflammatory bowel disease (IBD) susceptibility genes across different ethnic groups, we hypothesized that genetic determinants of Paneth cell phenotype in CD cohorts of different ethnic backgrounds are distinct. We also hypothesized that novel genetic determinants of Paneth cell defect are also associated with autophagy as we previously reported. Methods: We performed a hypothesisdriven association on 56 single nucleotide polymorphisms (SNPs) associated with CD susceptibility or known to affect Paneth cell function on 110 Japanese CD patients who underwent ileal resection. We subsequently performed a genome-wide association analysis. Paneth cell phenotype was determined by Defensin-5 immunofluorescence. Selected genotype-Paneth cell defect correlations were compared to a North American CD cohort (n=164). GenotypePaneth cell phenotype correlation was performed by linear regression. Network analysis was performed by Ingenuity Pathway Analysis (QIAGEN). Results: The average percentage of abnormal Paneth cells in Japanese CD was similar to North American CD (P=0.87), and abnormal Paneth cell phenotype was also associated with early recurrence (P=0.013). In contrast to North American CD, ATG16L1 T300A was not associated with Paneth cell defect in Japanese CD (P=0.20). Among the 56 selected SNPs, only LRRK2 M2397T showed significant association with Paneth cell defect (P=3.62x10-4), whereas in North American CD cohort it was not associated (P=0.76). Pathway analysis of LRRK2 and other candidate genes with P<5x10-4 (n=17 total) showed that these genes were connected into 2 distinct networks: the majority of the genes were connected to a PI3K-mTOR-autophagy network, whereas a subset of genes were connected to TNF-α signaling pathway. Finally, we found that the 17 genes potentially act in concert with other known IBD susceptibility genes in promoting Paneth cell defect. Conclusions: We found dichotomous effects of ATG16L1 and LRRK2 on Paneth cell defect between Japanese and North American CD cohorts. Genes affecting Paneth cell phenotype in Japanese CD were also associated with autophagy.
Background: Genome-wide association studies (GWAS) have identified 200 European inflammatory bowel disease (IBD) associated genetic loci. However, the clinical application of GWAS hits in IBD remains unclear. Aim: The aim of this study is to investigate how the inherited genetic risk burden influences IBD disease behavior, complications required intestinal resection, and response to TNF antagonists. Materials and Methods: The Mayo Clinic 3-site IBD GWAS, an ongoing cohort project, was used to perform this study. Clinical factors included in the analysis were gender, age at diagnosis, duration of disease, family history of IBD, smoking at diagnosis, disease location and behavior, perianal disease, bowel resection, and the response to TNF antagonists ("Yes" as being treated with a TNF antagonist and continued use at study enrollment; "No" as being treated with a TNF antagonist but discontinued due to ineffectiveness). Study subjects were genotyped using Illumina Immunochip custom genotyping array. We used the most recently published European 200 IBD susceptibility genetic loci (Nat Genet 2015;47:979) and their effect size to calculate the genetic risk score. The genetic risk score is the summation of the number of risk allele(s), weighted by their effect size (in log odds scale), at each one of the 206 SNPs. The analyses were conducted using the Golden Helix SVS 8.6 and JMP Genetics Pro 11.0. Results: Among a total 653 IBD patients (445 Crohn's disease (CD), 171 ulcerative colitis (UC), 37 indeterminate colitis (IC)), of European ancestry, who had been successfully genotyped, the mean genetic risk score was 0.66 (± s.d. 0.86, range from -2.01 to 2.98)(Figure 1). The mean duration of disease is 13.7 years (± s.d. 11.1). Univariate analysis revealed that younger age at diagnosis (≤25 y/o:0.79 vs. >25 y/o:0.56, P=0.0006), smoking at diagnosis (Yes:0.90 vs. No:0.62, P=0.004), IBD subtype CD (CD:0.72 vs. IC:0.63, UC:0.52, P=0.03), small bowel involvement (Yes:0.75 vs. No:0.53, P=0.002), perianal disease (Yes:0.97 vs. No:0.61, P<0.0001), bowel resection (Yes:0.77 vs. No:0.61, P=0.03), and a non-response to TNF antagonist therapy (nonresponders:0.88 vs. responders:0.63, P=0.01) was associated with a higher genetic risk score (Table 1). Interestingly, the genetic risk score was not different between Crohn's colitis and UC. In multivariate analysis, only perianal disease (adjusted P= 0.001) and response to TNF antagonist (adjusted P=0.04) remained associated with higher genetic risk score. Conclusions: GWAS IBD genetic risk burden is higher in patient who had perianal disease and non-response to TNF antagonist. The genetic risk burden may help predict who is at risk for a complicated disease course and therefore guide therapeutic strategy (e.g. TNF antagonist in lower genetic risk burden patients and different approach in higher genetic risk burden patients).
GENETIC RISK BURDEN AFFECTS INFLAMMATORY BOWEL DISEASE PERIANAL BEHAVIOR AND RESPONSE TO TNF ANTAGONISTS Ming-Hsi Wang, Parakkal Deepak, Jessica Friton, Laura H. Raffals, Jonathan A. Leighton, Shabana F. Pasha, Michael F. Picco, William Faubion
Tu1829 EARLY FIBROSTENOSIS IN CROHN'S DISEASE IS ASSOCIATED WITH MULTIPLE SUSCEPTIBILITY LOCI ON IMMUNOCHIP ANALYSIS Tom Holvoet, Peter Bossuyt, Isabelle Cleynen, Isabelle De Kock, Pieter Hindryckx, Severine Vermeire, Debby Laukens, Martine De Vos Introduction Fibrostenosis is a common complication of Crohn's disease (CD) occurring in about one third of patients. Although the pathophysiology of intestinal fibrosis is incompletely understood, evidence suggests a genetic contribution. Previous genetic association studies and candidate gene studies with fibrostenotic CD were based on clinical definitions which lack both sensitivity, specificity and have a high inter-observer disagreement. Additionally, the recent genotype-phenotype analysis by the IIBDGC did not consider the time to development of fibrostenotic disease. As the genetic risk may be more important in patients with early fibrostenosis, in this study we aimed to identify novel genetic markers by focussing on early fibrostenotic disease. Methods In this multicenter, retrospective nested case-control study, performed at the University Hospitals of Ghent and Leuven, computed tomography (CT) and magnetic resonance imaging (MRI) from CD patients obtained between 2002 and 2016 were examined for the presence of fibrostenotic disease. Patients with early ileal fibrostenosis, defined as a the presence of bowel tickening with luminal narrowing and prestenotic dilatation on CT/MRI occurring within 5 years following diagnosis of ileal or ileocolic disease (Montreal L1 or L3), and with available Illumina Immunochip data were included. The control cohort consisted of inflammatory CD patients, also Montreal L1 or L3, without arguments for fibrostenotic disease after min 10 years follow up. Allelic association was assessed using the PLINK v1.07 software. Results In total, 3.024 CT or MRI scans of 2.042 CD patients were screened. 112 patients were selected because of positive arguments for fibrostenosis occurring within 5 years of diagnosis. Of these, Immunochip data were available in 60 cases, and 49 (82%) had confirmed stenosis by histopathology. 343 inflammatory CD controls with genotype data were included in the analysis. Of the 156.500 SNPs analysed, only rs35223850 in the MIS18BP1 genepassed genome-wide significance level for association with early fibrostenosis (P-value<3.3x10^-7, OR 3.9, 17.5% vs 5% in cases vs controls respectively). The protein encoding MIS18BP1 is known to bind to the SP1 transcription factor, and this SNP has been associated with cardiac, liver and kidney fibrosis. Five additional SNPs reached a statistically suggestive significance level of P<5x10^-6, including rs116630177 in the IL23R gene, which is of particular interest as this gene has previously been associated with systemic sclerosis. Conclusions This carefully phenotyped study reveals an important role for genetic contribution to early development of fibrostenotic complications in CD. Our data suggest a role for MIS18BP1 and the SP1 transcription factor as well as the IL23 pathway in the pathogenesis of early intestinal fibrosis.
AGA Abstracts
Table1. Genetic risk scores in different clinical and demographic factors, IBD disease location, disease behavior, bowel resection, perianal disease, and response to TNF antagonists
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of specific residues in the C-terminal domain of FOXP3 protein including human diseasecausing mutations implicated in IBD (C232G) and IPEX syndrome (L242P, K250D, E251∆) attenuated EZH2 interaction. Conclusively, we showed that deletion of the C-terminal domain of FOXP3 abrogated EZH2 recruitment; however, as expected, both the N- and C-terminal domains were required for FOXP3's ability to repress its known target IL-2 as evident from a DLR assay. Furthermore, these disease-causing FOXP3 mutants lacked their abilities to effectively repress IL-2 expression compared to FOXP3 wild-type as evident from DLR assay and flow cytometric analysis. Finally, in human Tregs, FOXP3-EZH2 interaction was inhibited by approximately 50% upon IL-6 treatment in a JAK/STAT3-dependent manner. Conclusion: Thus, using a private mutation associated with heritable IBD we discovered a more generalizable mechanism by which pro-inflammatory signaling pathways disrupt a critical epigenetic regulatory complex in FOXP3+Tregs.
Overview of identified SNPs associated with early fibrostenosis
Tu1828 DICHOTOMOUS EFFECTS OF ATG16L1 AND LRRK2 IN MODULATING PANETH CELL DEFECT IN JAPANESE AND NORTH AMERICAN CROHN'S DISEASE PATIENTS Ta-Chiang Liu, Takeo Naito, Zhenqiu Liu, Kelli VanDussen, Talin Haritunians, Dalin Li, Katsuya Endo, Yosuke Kawai, Masao Nagasaki, Yoshitaka Kinouchi, Dermot McGovern, Tooru Shimosegawa, Yoichi Kakuta, Thaddeus S. Stappenbeck
Tu1830
Background: Morphological patterns of Paneth cells are a prognostic biomarker in North American Crohn's disease (CD) patients, and are associated with autophagy-associated ATG16L1 and NOD2 variants. Given the different genetic landscape in inflammatory bowel disease (IBD) susceptibility genes across different ethnic groups, we hypothesized that genetic determinants of Paneth cell phenotype in CD cohorts of different ethnic backgrounds are distinct. We also hypothesized that novel genetic determinants of Paneth cell defect are also associated with autophagy as we previously reported. Methods: We performed a hypothesisdriven association on 56 single nucleotide polymorphisms (SNPs) associated with CD susceptibility or known to affect Paneth cell function on 110 Japanese CD patients who underwent ileal resection. We subsequently performed a genome-wide association analysis. Paneth cell phenotype was determined by Defensin-5 immunofluorescence. Selected genotype-Paneth cell defect correlations were compared to a North American CD cohort (n=164). GenotypePaneth cell phenotype correlation was performed by linear regression. Network analysis was performed by Ingenuity Pathway Analysis (QIAGEN). Results: The average percentage of abnormal Paneth cells in Japanese CD was similar to North American CD (P=0.87), and abnormal Paneth cell phenotype was also associated with early recurrence (P=0.013). In contrast to North American CD, ATG16L1 T300A was not associated with Paneth cell defect in Japanese CD (P=0.20). Among the 56 selected SNPs, only LRRK2 M2397T showed significant association with Paneth cell defect (P=3.62x10-4), whereas in North American CD cohort it was not associated (P=0.76). Pathway analysis of LRRK2 and other candidate genes with P<5x10-4 (n=17 total) showed that these genes were connected into 2 distinct networks: the majority of the genes were connected to a PI3K-mTOR-autophagy network, whereas a subset of genes were connected to TNF-α signaling pathway. Finally, we found that the 17 genes potentially act in concert with other known IBD susceptibility genes in promoting Paneth cell defect. Conclusions: We found dichotomous effects of ATG16L1 and LRRK2 on Paneth cell defect between Japanese and North American CD cohorts. Genes affecting Paneth cell phenotype in Japanese CD were also associated with autophagy.
Background: Genome-wide association studies (GWAS) have identified 200 European inflammatory bowel disease (IBD) associated genetic loci. However, the clinical application of GWAS hits in IBD remains unclear. Aim: The aim of this study is to investigate how the inherited genetic risk burden influences IBD disease behavior, complications required intestinal resection, and response to TNF antagonists. Materials and Methods: The Mayo Clinic 3-site IBD GWAS, an ongoing cohort project, was used to perform this study. Clinical factors included in the analysis were gender, age at diagnosis, duration of disease, family history of IBD, smoking at diagnosis, disease location and behavior, perianal disease, bowel resection, and the response to TNF antagonists ("Yes" as being treated with a TNF antagonist and continued use at study enrollment; "No" as being treated with a TNF antagonist but discontinued due to ineffectiveness). Study subjects were genotyped using Illumina Immunochip custom genotyping array. We used the most recently published European 200 IBD susceptibility genetic loci (Nat Genet 2015;47:979) and their effect size to calculate the genetic risk score. The genetic risk score is the summation of the number of risk allele(s), weighted by their effect size (in log odds scale), at each one of the 206 SNPs. The analyses were conducted using the Golden Helix SVS 8.6 and JMP Genetics Pro 11.0. Results: Among a total 653 IBD patients (445 Crohn's disease (CD), 171 ulcerative colitis (UC), 37 indeterminate colitis (IC)), of European ancestry, who had been successfully genotyped, the mean genetic risk score was 0.66 (± s.d. 0.86, range from -2.01 to 2.98)(Figure 1). The mean duration of disease is 13.7 years (± s.d. 11.1). Univariate analysis revealed that younger age at diagnosis (≤25 y/o:0.79 vs. >25 y/o:0.56, P=0.0006), smoking at diagnosis (Yes:0.90 vs. No:0.62, P=0.004), IBD subtype CD (CD:0.72 vs. IC:0.63, UC:0.52, P=0.03), small bowel involvement (Yes:0.75 vs. No:0.53, P=0.002), perianal disease (Yes:0.97 vs. No:0.61, P<0.0001), bowel resection (Yes:0.77 vs. No:0.61, P=0.03), and a non-response to TNF antagonist therapy (nonresponders:0.88 vs. responders:0.63, P=0.01) was associated with a higher genetic risk score (Table 1). Interestingly, the genetic risk score was not different between Crohn's colitis and UC. In multivariate analysis, only perianal disease (adjusted P= 0.001) and response to TNF antagonist (adjusted P=0.04) remained associated with higher genetic risk score. Conclusions: GWAS IBD genetic risk burden is higher in patient who had perianal disease and non-response to TNF antagonist. The genetic risk burden may help predict who is at risk for a complicated disease course and therefore guide therapeutic strategy (e.g. TNF antagonist in lower genetic risk burden patients and different approach in higher genetic risk burden patients).
GENETIC RISK BURDEN AFFECTS INFLAMMATORY BOWEL DISEASE PERIANAL BEHAVIOR AND RESPONSE TO TNF ANTAGONISTS Ming-Hsi Wang, Parakkal Deepak, Jessica Friton, Laura H. Raffals, Jonathan A. Leighton, Shabana F. Pasha, Michael F. Picco, William Faubion
Tu1829 EARLY FIBROSTENOSIS IN CROHN'S DISEASE IS ASSOCIATED WITH MULTIPLE SUSCEPTIBILITY LOCI ON IMMUNOCHIP ANALYSIS Tom Holvoet, Peter Bossuyt, Isabelle Cleynen, Isabelle De Kock, Pieter Hindryckx, Severine Vermeire, Debby Laukens, Martine De Vos Introduction Fibrostenosis is a common complication of Crohn's disease (CD) occurring in about one third of patients. Although the pathophysiology of intestinal fibrosis is incompletely understood, evidence suggests a genetic contribution. Previous genetic association studies and candidate gene studies with fibrostenotic CD were based on clinical definitions which lack both sensitivity, specificity and have a high inter-observer disagreement. Additionally, the recent genotype-phenotype analysis by the IIBDGC did not consider the time to development of fibrostenotic disease. As the genetic risk may be more important in patients with early fibrostenosis, in this study we aimed to identify novel genetic markers by focussing on early fibrostenotic disease. Methods In this multicenter, retrospective nested case-control study, performed at the University Hospitals of Ghent and Leuven, computed tomography (CT) and magnetic resonance imaging (MRI) from CD patients obtained between 2002 and 2016 were examined for the presence of fibrostenotic disease. Patients with early ileal fibrostenosis, defined as a the presence of bowel tickening with luminal narrowing and prestenotic dilatation on CT/MRI occurring within 5 years following diagnosis of ileal or ileocolic disease (Montreal L1 or L3), and with available Illumina Immunochip data were included. The control cohort consisted of inflammatory CD patients, also Montreal L1 or L3, without arguments for fibrostenotic disease after min 10 years follow up. Allelic association was assessed using the PLINK v1.07 software. Results In total, 3.024 CT or MRI scans of 2.042 CD patients were screened. 112 patients were selected because of positive arguments for fibrostenosis occurring within 5 years of diagnosis. Of these, Immunochip data were available in 60 cases, and 49 (82%) had confirmed stenosis by histopathology. 343 inflammatory CD controls with genotype data were included in the analysis. Of the 156.500 SNPs analysed, only rs35223850 in the MIS18BP1 genepassed genome-wide significance level for association with early fibrostenosis (P-value<3.3x10^-7, OR 3.9, 17.5% vs 5% in cases vs controls respectively). The protein encoding MIS18BP1 is known to bind to the SP1 transcription factor, and this SNP has been associated with cardiac, liver and kidney fibrosis. Five additional SNPs reached a statistically suggestive significance level of P<5x10^-6, including rs116630177 in the IL23R gene, which is of particular interest as this gene has previously been associated with systemic sclerosis. Conclusions This carefully phenotyped study reveals an important role for genetic contribution to early development of fibrostenotic complications in CD. Our data suggest a role for MIS18BP1 and the SP1 transcription factor as well as the IL23 pathway in the pathogenesis of early intestinal fibrosis.
AGA Abstracts
Table1. Genetic risk scores in different clinical and demographic factors, IBD disease location, disease behavior, bowel resection, perianal disease, and response to TNF antagonists
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