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M2054 A Detailed Haplotype Tagging Investigation of the IL23R Gene Confirms Gene-Wide Association with Childhood Onset IBD and CD
activation. When age of onset and disease susceptibility was tested, none of the SNPs associated with CD in this study were specifically associated with early onset disease. We were unable to replicate 5 other SNPs (rs4958847-IRGM, rs2542151-PTPN2, rs8050910FAM92B, rs16853571-PHOX2B, rs1793004-NELL1). Among these non-replicated SNPs, the allele frequencies in our study mirror the allele frequencies in previous reports. However, we do not have statistical power to refute these findings. Conclusions: Autophagy genes ATG16L1, IRGM, and another gene IL23R are independently associated with early onset CD, and act in an additive fashion. The strong association between early CD and IL23R, ATG16L1 and IRGM further implicate the IL-23/IL-17 axis, and defective autophagy in the pathogenesis of early onset CD. However, these genes were not specifically associated with an earlier susceptibility since the CD risk is similar in our early onset patients compared with previously reported adult CD. M2054 A Detailed Haplotype Tagging Investigation of the IL23R Gene Confirms Gene-Wide Association with Childhood Onset IBD and CD Johan Van Limbergen, Richard K. Russell, Elaine R. Nimmo, Hazel E. Drummond, Linda Smith, Niall H. Anderson, Gail Davies, Peter M. Gillett, Paraic McGrogan, Lawrence Weaver, Michael W. Bisset, Gamal Mahdi, David C. Wilson, Jack Satsangi Introduction: The association of CD with the IL23R (interleukin 23-receptor) Arg381Gln variant has been widely replicated, both in children and in adults. Additional association signals throughout IL23R have been identified. We aimed to assess the gene-wide contribution of germline variation of IL23R to childhood IBD susceptibility and phenotype and to investigate interaction with NOD2/CARD15, by means of a detailed haplotype tagging investigation. Methods: 709 subjects (357 IBD patients <17y at diagnosis (233 CD, 86 UC, 38 IBDU) and 352 population-matched controls) were genotyped for 8 IL23R haplotype tagging single nucleotide polymorphisms (SNPs) (rs3762318, rs4655679, rs12041056, rs6656929, rs10889668, rs10489630, rs1004819, rs790631) based on HapMap data (minor allelic freq >10%, haplotype freq >5%, solid spine of LD). Genotype/haplotype case-control, loglikelihood and genotype-phenotype analyses (Montreal classification) were performed. The IL23R Arg381Gln variant and the three common NOD2/CARD15 variants were previously genotyped. Results: We observed significant associations of four of the tagging SNPs (rs3762318, rs6656929, rs10889668, rs1004819) with IBD/CD on analysis of allelic/genotype frequency. These associations were confirmed on log-likelihood analysis in IBD and CD (recessive model (1000 permutations) p=0.01 and p=0.002, respectively). Haplotype analysis demonstrated a protective effect of the 11221211 haplotype on IBD (2% vs 4% in healthy controls, p=0.02 OR 0.49 (0.26-0.92)) and CD (2%, p=0.02 OR 0.46 (0.22-0.97)). By extending the haplotype analysis to include the previously genotyped Arg381Gln variant, we were able to demonstrate that the protective effect of the 11221211 haplotype was independent of the Arg381Gln variant (r2 with tagging SNPs ≤0.05; IBD: p=0.02 OR 0.50 (0.27-0.93); CD: p=0.02 OR 0.46 (0.22-0.96)). When assessing the association signal for each haplotype block (based on solid spine of LD), we observed associations with both 5'and 3'-end haplotype blocks. After correction for multiple comparisons, no significant genotype-phenotype associations were seen. In wildtype NOD2/CARD15 patients and controls, we observed association with a new risk haplotype (21121212, 5% vs 1% p=0.002 OR 5.17 (1.49-17.90)). Conclusion: We have demonstrated using a gene-wide haplotype tagging strategy that the multiple association signals of the IL23R locus are independent of the Arg381Gln variant in childhood onset IBD and CD. In our high-incidence population characterised by low NOD2/CARD15 variant carriage, we have observed interaction of the IL23R locus with NOD2/CARD15 through the identification of a novel IL23R risk haplotype.
M2052 IRAK-1 Single Nucleotide Polymorphism in Patients with Inflammatory Bowel Disease Stephan L. Haas, Andreas Ruether, Manfred V. Singer, Stefan Schreiber, Ulrich Böcker Background: IL-1R-associated kinase (IRAK)-1 is a critical mediator of toll like receptor/ interleukin-1 receptor-induced activation of the transcription factor NF-κB which plays a pivotal role in modulating the expression of proinflammatory cytokines involved in inflammatory bowel disease (IBD). Recently, it was shown that the functional IRAK-1 single nucleotide polymorphism (rs1059703) - located on the X-chromosome - is associated with increased NF-κB transcriptional activity and expression of NF-κB-dependent proinflammatory cytokines (Liu et al., J Immunol 2007). The same IRAK-1 variant was found to be associated with a significantly higher mortality rate in patients with sepsis (Arcaroli et al., Am J Respir Crit Care Med 2006). Aim of the study: To evaluate whether the nonsynonymous single nucleotide polymorphism (SNP) rs1059703 is associated with IBD susceptibility. Patients and methods: In total, 3016 IBD patients comprising 1889 patients with Crohn's disease (CD) and 1127 patients with ulcerative colitis (UC) were enrolled in the study. The control group was represented by 1474 healthy subjects. For segregation analysis, 490 CD and UC trios (father-mother-child) were included. After DNA isolation genotyping was performed using a TaqMan MGB biallelic discrimination system. Differences between patients and controls and Hardy-Weinberg equilibrium were assessed by a chi-square test. For segregation analysis a transmission disequilibrium test (TDT) was applied. Results: Comparing IBD patients with controls no significant difference was found (p=0.77). The comparison of IRAK-1 genotype frequencies of CD patients with controls and UC patients with controls revealed no significant difference (p=0.57 and p=0.83, respectively). Interestingly, TDT analysis of 490 CD family trios demonstrated segregation of the rs1059703 SNP with CD (p=0.0262), whereas no significant segregation was found in UC patients (p=0.147). Conclusion: The tested IRAK-1 genetic variant does not appear to be a strong susceptibility factor for IBD. Considering the significant segregation in the cohort of CD patients a contributory role in this condition cannot be ruled out. Therefore, studies in CD patients from different geographical areas seem to be worthwhile for further clarification.
M2055 Sex-Related Inheritance and Transmission Pattern in Inflammatory Bowel Disease Zuzana Zelinkova, Pieter C. Stokkers, Klaas van der Linde, E. J. Kuipers, Christien J. van der Woude Background: Extensive epidemiological data indicate a female predominance in inflammatory bowel disease (IBD). However, the exact character of sex-related inheritance in IBD is unknown. Aim: To characterize the sex-related inheritance and transmission pattern in inflammatory bowel disease. Methods: IBD patients with family history of IBD were identified in the IBD outpatient clinic database in two academic medical centers. Family relationship, sex and the type of the disease (Crohn`s disease - CD, ulcerative colitis - UC, unclassified - U) were retrieved from the database and the sex and disease type distribution were compared to the baseline IBD population. The binomial distribution test was used to analyze the imprinting pattern in the families in which both, a parent and a child had IBD. Results: In total, 608 IBD (CD/UC/U; 363/233/12) patients from 289 families were included. The baseline IBD patients' database comprised 2700 patients (CD/UC/U; 1609/1009/82). In the familial IBD patients' population, a higher female predominance (F/M; 369/239; ratio 1.5) was observed compared with the baseline population (F/M; 1444/1174; ratio 1.2). This familial female predominance applied for both CD (F/M; 232/131; ratio 1.8) and UC (F/M; 128/105; ratio 1.2). Subsequently, the imprinting pattern was analyzed. In total, 87 families in which both, a parent and a child were affected were identified. A significantly higher number of mother to child transmissions (55 vs. 32 of father to child transmissions) was observed (p=0.004). The female imprinting was specifically related to CD (mother/father to child transmissions; 31/14; p=0.001), in UC no significant differences between mother and father to child transmission numbers were observed. The analysis of offspring sex distribution pattern revealed significantly higher female to female transmission compared with female to male transmission rate (female to female/female to male transmission; 36/18; p=0.005). No specific offspring sex-related transmission pattern was observed in the paternal transmission families. Conclusion: In inflammatory bowel disease, the female predominance is rather associated with familial than sporadic occurence of the disease. In Crohn`s disease, this predominance may be related to the imprinting of the disease predisposition with a specific female to female transmission pattern.
M2053 Autophagy Like Genes (ATG16L1 & IRGM), IL23R and Three Other Variants Are Associated with Pediatric Onset Crohn's Disease (CD) with Similar Risks (Odds Ratio) to Adult Onset Nicholas Peterson, Stephen L. Guthery, Lee Denson, Jessica J. Lee, Shehzad Saeed, Vincent F. Biank, Rebecca Ehlert, Gitit Tomer, Richard J. Grand, Colin D. Rudolph, Subra Kugathasan Background & Aims: A genetic predisposition to CD is strongly supported by the recent identification of several novel susceptibility loci by genome wide association studies. These studies were performed in subjects with heterogeneous ages of onset. We tested the hypothesis that very early onset CD is more strongly associated with genetic variants than previously reported in adult cohorts by performing replication studies in a large exclusively pediatric onset CD cohort. Methods: 555 pediatric onset cases (mean age of onset 11.7 years, 309 parent-child trios) and 486 controls were enrolled. Eleven newly described single nucleotide polymorphisms (SNPs) were genotyped. Both case-control and TDT analyses were used. The global pattern of gene expression in diseased colonic mucosa was determined and related to risk allele carriage. Results: Six variants including autophagy related gene rs2241880 (ATG16L1) and rs13361189 (IRGM), another SNP rs11209026 (IL23R) and 3 other variants (an intergenic variant in chromosome 10 (rs224136), rs10883365 (located within NKX23), and SNP rs9858542 located in a region of extended linkage disequilibrium on chromosome 3q21 were associated with pediatric CD. The odds ratio for CD risk for each loci was similar in pediatric CD compared with the adult onset (age of onset 24-26 years - NIDDK, WTCCC and a German cohort)CD. In addition, ATG16L1, IRGM, and IL23R acted in an additive effect to increase the CD risk in a multiple logistic regression model (Chi sQ 9.1, p=0.003). Patients carrying 2 or more ATG16L1/IRGM risk alleles exhibited a homogenous global pattern of gene expression, which was enriched for pathways regulating lymphocyte
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AGA Abstracts
AGA Abstracts
course; and the determination of the most appropriate therapeutic options. Therefore there is a need for new markers that could efficiently diagnose, predict relapses, monitor disease activity or evaluate the therapeutic response to drugs. New techniques in Molecular Biology, such as microarray technology, have become powerful tools for biomarker research as they allow a genome-wide screening of differentially expressed genes between several conditions. Here we present a comparative study of three animal models of acute colitis (dextran sulphate sodium (DSS)-induced model, trinitrobencenesulphonic acid (TNBS)-induced model, both in rats, and IL10-/- knockout mice model). The mechanism of colitis-induction and the immune response are different in these models. We hypothesized that the comparative analysis could identify a set of common biomarker genes, the expression of which is associated with colitis, irrespective of the etiology of the diseasE. colitis was induced with 5% DSS (n= 9, controls=4), by a single enema of 10mg of TNBS in 50% ethanol (n=6, controls=4) and spontaneously in IL10-/- mice (n=3, controls=3). Total RNA was extracted and gene expression changes in each sample were independently analyzed with Affymetrix Rat Genome 230 2.0 and Mouse Genome 430 2.0 GeneChips® at Progenika Biopharma S.A. Data were normalized with Bioconductor, and differentially expressed genes (fold change, -1>FC>1, p<0.05) were obtained using SpotFire Decision Site v9.0 (Integromics S.L.): 5102 sequences in DSS model, 941 in TNBS model and 1454 in IL10-/- knockout model. Finally, we performed a biomarker analysis using Ingenuity Pathways Analysis software considering genes with a FC>1.5. The analysis identified a group of 22 genes, most of them over expressed, as potential biomarkers of colitis. This gene signature includes classical colitis markers, as IL1b, S100a8, S100a9 or Mmp13, and other novel genes not previously related to this disease. We are currently validating the expression changes by real time PCR and western blot in animals and human samples. We will correlate gene expression changes with disease progression and clinical data on IBD patients. This gene signature might be useful for classifying IBD patients and might provide an objective measure of disease status as well as specific therapeutic response.
M2052 IRAK-1 Single Nucleotide Polymorphism in Patients with Inflammatory Bowel Disease Stephan L. Haas, Andreas Ruether, Manfred V. Singer, Stefan Schreiber, Ulrich Böcker Background: IL-1R-associated kinase (IRAK)-1 is a critical mediator of toll like receptor/ interleukin-1 receptor-induced activation of the transcription factor NF-κB which plays a pivotal role in modulating the expression of proinflammatory cytokines involved in inflammatory bowel disease (IBD). Recently, it was shown that the functional IRAK-1 single nucleotide polymorphism (rs1059703) - located on the X-chromosome - is associated with increased NF-κB transcriptional activity and expression of NF-κB-dependent proinflammatory cytokines (Liu et al., J Immunol 2007). The same IRAK-1 variant was found to be associated with a significantly higher mortality rate in patients with sepsis (Arcaroli et al., Am J Respir Crit Care Med 2006). Aim of the study: To evaluate whether the nonsynonymous single nucleotide polymorphism (SNP) rs1059703 is associated with IBD susceptibility. Patients and methods: In total, 3016 IBD patients comprising 1889 patients with Crohn's disease (CD) and 1127 patients with ulcerative colitis (UC) were enrolled in the study. The control group was represented by 1474 healthy subjects. For segregation analysis, 490 CD and UC trios (father-mother-child) were included. After DNA isolation genotyping was performed using a TaqMan MGB biallelic discrimination system. Differences between patients and controls and Hardy-Weinberg equilibrium were assessed by a chi-square test. For segregation analysis a transmission disequilibrium test (TDT) was applied. Results: Comparing IBD patients with controls no significant difference was found (p=0.77). The comparison of IRAK-1 genotype frequencies of CD patients with controls and UC patients with controls revealed no significant difference (p=0.57 and p=0.83, respectively). Interestingly, TDT analysis of 490 CD family trios demonstrated segregation of the rs1059703 SNP with CD (p=0.0262), whereas no significant segregation was found in UC patients (p=0.147). Conclusion: The tested IRAK-1 genetic variant does not appear to be a strong susceptibility factor for IBD. Considering the significant segregation in the cohort of CD patients a contributory role in this condition cannot be ruled out. Therefore, studies in CD patients from different geographical areas seem to be worthwhile for further clarification.
M2055 Sex-Related Inheritance and Transmission Pattern in Inflammatory Bowel Disease Zuzana Zelinkova, Pieter C. Stokkers, Klaas van der Linde, E. J. Kuipers, Christien J. van der Woude Background: Extensive epidemiological data indicate a female predominance in inflammatory bowel disease (IBD). However, the exact character of sex-related inheritance in IBD is unknown. Aim: To characterize the sex-related inheritance and transmission pattern in inflammatory bowel disease. Methods: IBD patients with family history of IBD were identified in the IBD outpatient clinic database in two academic medical centers. Family relationship, sex and the type of the disease (Crohn`s disease - CD, ulcerative colitis - UC, unclassified - U) were retrieved from the database and the sex and disease type distribution were compared to the baseline IBD population. The binomial distribution test was used to analyze the imprinting pattern in the families in which both, a parent and a child had IBD. Results: In total, 608 IBD (CD/UC/U; 363/233/12) patients from 289 families were included. The baseline IBD patients' database comprised 2700 patients (CD/UC/U; 1609/1009/82). In the familial IBD patients' population, a higher female predominance (F/M; 369/239; ratio 1.5) was observed compared with the baseline population (F/M; 1444/1174; ratio 1.2). This familial female predominance applied for both CD (F/M; 232/131; ratio 1.8) and UC (F/M; 128/105; ratio 1.2). Subsequently, the imprinting pattern was analyzed. In total, 87 families in which both, a parent and a child were affected were identified. A significantly higher number of mother to child transmissions (55 vs. 32 of father to child transmissions) was observed (p=0.004). The female imprinting was specifically related to CD (mother/father to child transmissions; 31/14; p=0.001), in UC no significant differences between mother and father to child transmission numbers were observed. The analysis of offspring sex distribution pattern revealed significantly higher female to female transmission compared with female to male transmission rate (female to female/female to male transmission; 36/18; p=0.005). No specific offspring sex-related transmission pattern was observed in the paternal transmission families. Conclusion: In inflammatory bowel disease, the female predominance is rather associated with familial than sporadic occurence of the disease. In Crohn`s disease, this predominance may be related to the imprinting of the disease predisposition with a specific female to female transmission pattern.
M2053 Autophagy Like Genes (ATG16L1 & IRGM), IL23R and Three Other Variants Are Associated with Pediatric Onset Crohn's Disease (CD) with Similar Risks (Odds Ratio) to Adult Onset Nicholas Peterson, Stephen L. Guthery, Lee Denson, Jessica J. Lee, Shehzad Saeed, Vincent F. Biank, Rebecca Ehlert, Gitit Tomer, Richard J. Grand, Colin D. Rudolph, Subra Kugathasan Background & Aims: A genetic predisposition to CD is strongly supported by the recent identification of several novel susceptibility loci by genome wide association studies. These studies were performed in subjects with heterogeneous ages of onset. We tested the hypothesis that very early onset CD is more strongly associated with genetic variants than previously reported in adult cohorts by performing replication studies in a large exclusively pediatric onset CD cohort. Methods: 555 pediatric onset cases (mean age of onset 11.7 years, 309 parent-child trios) and 486 controls were enrolled. Eleven newly described single nucleotide polymorphisms (SNPs) were genotyped. Both case-control and TDT analyses were used. The global pattern of gene expression in diseased colonic mucosa was determined and related to risk allele carriage. Results: Six variants including autophagy related gene rs2241880 (ATG16L1) and rs13361189 (IRGM), another SNP rs11209026 (IL23R) and 3 other variants (an intergenic variant in chromosome 10 (rs224136), rs10883365 (located within NKX23), and SNP rs9858542 located in a region of extended linkage disequilibrium on chromosome 3q21 were associated with pediatric CD. The odds ratio for CD risk for each loci was similar in pediatric CD compared with the adult onset (age of onset 24-26 years - NIDDK, WTCCC and a German cohort)CD. In addition, ATG16L1, IRGM, and IL23R acted in an additive effect to increase the CD risk in a multiple logistic regression model (Chi sQ 9.1, p=0.003). Patients carrying 2 or more ATG16L1/IRGM risk alleles exhibited a homogenous global pattern of gene expression, which was enriched for pathways regulating lymphocyte
A-459
AGA Abstracts
AGA Abstracts
course; and the determination of the most appropriate therapeutic options. Therefore there is a need for new markers that could efficiently diagnose, predict relapses, monitor disease activity or evaluate the therapeutic response to drugs. New techniques in Molecular Biology, such as microarray technology, have become powerful tools for biomarker research as they allow a genome-wide screening of differentially expressed genes between several conditions. Here we present a comparative study of three animal models of acute colitis (dextran sulphate sodium (DSS)-induced model, trinitrobencenesulphonic acid (TNBS)-induced model, both in rats, and IL10-/- knockout mice model). The mechanism of colitis-induction and the immune response are different in these models. We hypothesized that the comparative analysis could identify a set of common biomarker genes, the expression of which is associated with colitis, irrespective of the etiology of the diseasE. colitis was induced with 5% DSS (n= 9, controls=4), by a single enema of 10mg of TNBS in 50% ethanol (n=6, controls=4) and spontaneously in IL10-/- mice (n=3, controls=3). Total RNA was extracted and gene expression changes in each sample were independently analyzed with Affymetrix Rat Genome 230 2.0 and Mouse Genome 430 2.0 GeneChips® at Progenika Biopharma S.A. Data were normalized with Bioconductor, and differentially expressed genes (fold change, -1>FC>1, p<0.05) were obtained using SpotFire Decision Site v9.0 (Integromics S.L.): 5102 sequences in DSS model, 941 in TNBS model and 1454 in IL10-/- knockout model. Finally, we performed a biomarker analysis using Ingenuity Pathways Analysis software considering genes with a FC>1.5. The analysis identified a group of 22 genes, most of them over expressed, as potential biomarkers of colitis. This gene signature includes classical colitis markers, as IL1b, S100a8, S100a9 or Mmp13, and other novel genes not previously related to this disease. We are currently validating the expression changes by real time PCR and western blot in animals and human samples. We will correlate gene expression changes with disease progression and clinical data on IBD patients. This gene signature might be useful for classifying IBD patients and might provide an objective measure of disease status as well as specific therapeutic response.