course; and the determination of the most appropriate therapeutic options. Therefore thereis a need for new markers that could efficiently diagnose, predict relapses, monitor diseaseactivity or evaluate the therapeutic response to drugs. New techniques in Molecular Biology,such as microarray technology, have become powerful tools for biomarker research as theyallow 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 sulphatesodium (DSS)-induced model, trinitrobencenesulphonic acid (TNBS)-induced model, bothin rats, and IL10-/- knockout mice model). The mechanism of colitis-induction and theimmune response are different in these models. We hypothesized that the comparativeanalysis could identify a set of common biomarker genes, the expression of which is associatedwith 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) andspontaneously in IL10-/- mice (n=3, controls=3). Total RNA was extracted and gene expres-sion changes in each sample were independently analyzed with Affymetrix Rat Genome 2302.0 and Mouse Genome 430 2.0 GeneChips® at Progenika Biopharma S.A. Data werenormalized 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 sequencesin DSS model, 941 in TNBS model and 1454 in IL10-/- knockout model. Finally, weperformed a biomarker analysis using Ingenuity Pathways Analysis software consideringgenes with a FC>1.5. The analysis identified a group of 22 genes, most of them overexpressed, as potential biomarkers of colitis. This gene signature includes classical colitismarkers, as IL1b, S100a8, S100a9 or Mmp13, and other novel genes not previously relatedto this disease. We are currently validating the expression changes by real time PCR andwestern blot in animals and human samples. We will correlate gene expression changeswith disease progression and clinical data on IBD patients. This gene signature might beuseful for classifying IBD patients and might provide an objective measure of disease statusas well as specific therapeutic response.

M2052

IRAK-1 Single Nucleotide Polymorphism in Patients with Inflammatory BowelDiseaseStephan 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 apivotal role in modulating the expression of proinflammatory cytokines involved in inflam-matory bowel disease (IBD). Recently, it was shown that the functional IRAK-1 singlenucleotide polymorphism (rs1059703) - located on the X-chromosome - is associated withincreased NF-κB transcriptional activity and expression of NF-κB-dependent proinflammat-ory cytokines (Liu et al., J Immunol 2007). The same IRAK-1 variant was found to beassociated 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 nonsynonymoussingle nucleotide polymorphism (SNP) rs1059703 is associated with IBD susceptibility.Patients and methods: In total, 3016 IBD patients comprising 1889 patients with Crohn'sdisease (CD) and 1127 patients with ulcerative colitis (UC) were enrolled in the study. Thecontrol group was represented by 1474 healthy subjects. For segregation analysis, 490 CDand UC trios (father-mother-child) were included. After DNA isolation genotyping wasperformed using a TaqMan MGB biallelic discrimination system. Differences between patientsand controls and Hardy-Weinberg equilibrium were assessed by a chi-square test. Forsegregation analysis a transmission disequilibrium test (TDT) was applied. Results: Compar-ing IBD patients with controls no significant difference was found (p=0.77). The comparisonof IRAK-1 genotype frequencies of CD patients with controls and UC patients with controlsrevealed no significant difference (p=0.57 and p=0.83, respectively). Interestingly, TDTanalysis of 490 CD family trios demonstrated segregation of the rs1059703 SNP withCD (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 susceptibilityfactor for IBD. Considering the significant segregation in the cohort of CD patients acontributory role in this condition cannot be ruled out. Therefore, studies in CD patientsfrom different geographical areas seem to be worthwhile for further clarification.

M2053

Autophagy Like Genes (ATG16L1 & IRGM), IL23R and Three Other VariantsAre Associated with Pediatric Onset Crohn's Disease (CD) with Similar Risks(Odds Ratio) to Adult OnsetNicholas Peterson, Stephen L. Guthery, Lee Denson, Jessica J. Lee, Shehzad Saeed,Vincent F. Biank, Rebecca Ehlert, Gitit Tomer, Richard J. Grand, Colin D. Rudolph, SubraKugathasan

Background & Aims: A genetic predisposition to CD is strongly supported by the recentidentification of several novel susceptibility loci by genome wide association studies. Thesestudies were performed in subjects with heterogeneous ages of onset. We tested the hypothesisthat very early onset CD is more strongly associated with genetic variants than previouslyreported in adult cohorts by performing replication studies in a large exclusively pediatriconset CD cohort. Methods: 555 pediatric onset cases (mean age of onset 11.7 years, 309parent-child trios) and 486 controls were enrolled. Eleven newly described single nucleotidepolymorphisms (SNPs) were genotyped. Both case-control and TDT analyses were used.The global pattern of gene expression in diseased colonic mucosa was determined andrelated 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 NKX2-3), and SNP rs9858542 located in a region of extended linkage disequilibrium on chromo-some 3q21 were associated with pediatric CD. The odds ratio for CD risk for each loci wassimilar 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 anadditive 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 homogenousglobal pattern of gene expression, which was enriched for pathways regulating lymphocyte

T : 11501$$CH204-02-08 16:47:10 Page 459Layout: 11501B : o

A-459 AGA Abstracts

activation. When age of onset and disease susceptibility was tested, none of the SNPsassociated with CD in this study were specifically associated with early onset disease. Wewere unable to replicate 5 other SNPs (rs4958847-IRGM, rs2542151-PTPN2, rs8050910-FAM92B, 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 genesATG16L1, IRGM, and another gene IL23R are independently associated with early onsetCD, 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 thepathogenesis of early onset CD. However, these genes were not specifically associated withan earlier susceptibility since the CD risk is similar in our early onset patients comparedwith previously reported adult CD.

M2054

A Detailed Haplotype Tagging Investigation of the IL23R Gene ConfirmsGene-Wide Association with Childhood Onset IBD and CDJohan Van Limbergen, Richard K. Russell, Elaine R. Nimmo, Hazel E. Drummond, LindaSmith, Niall H. Anderson, Gail Davies, Peter M. Gillett, Paraic McGrogan, LawrenceWeaver, Michael W. Bisset, Gamal Mahdi, David C. Wilson, Jack Satsangi

Introduction: The association of CD with the IL23R (interleukin 23-receptor) Arg381Glnvariant has been widely replicated, both in children and in adults. Additional associationsignals throughout IL23R have been identified. We aimed to assess the gene-wide contributionof germline variation of IL23R to childhood IBD susceptibility and phenotype and to investig-ate 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) and352 population-matched controls) were genotyped for 8 IL23R haplotype tagging singlenucleotide polymorphisms (SNPs) (rs3762318, rs4655679, rs12041056, rs6656929,rs10889668, rs10489630, rs1004819, rs790631) based on HapMap data (minor allelicfreq >10%, haplotype freq >5%, solid spine of LD). Genotype/haplotype case-control, log-likelihood and genotype-phenotype analyses (Montreal classification) were performed. TheIL23R Arg381Gln variant and the three common NOD2/CARD15 variants were previouslygenotyped. Results: We observed significant associations of four of the tagging SNPs(rs3762318, rs6656929, rs10889668, rs1004819) with IBD/CD on analysis of allelic/geno-type frequency. These associations were confirmed on log-likelihood analysis in IBD andCD (recessive model (1000 permutations) p=0.01 and p=0.002, respectively). Haplotypeanalysis demonstrated a protective effect of the 11221211 haplotype on IBD (2% vs 4% inhealthy 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 wasindependent 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 foreach 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 significantgenotype-phenotype associations were seen. In wildtype NOD2/CARD15 patients and con-trols, we observed association with a new risk haplotype (21121212, 5% vs 1% p=0.002OR 5.17 (1.49-17.90)). Conclusion: We have demonstrated using a gene-wide haplotypetagging strategy that the multiple association signals of the IL23R locus are independent ofthe Arg381Gln variant in childhood onset IBD and CD. In our high-incidence populationcharacterised by low NOD2/CARD15 variant carriage, we have observed interaction of theIL23R locus with NOD2/CARD15 through the identification of a novel IL23R risk haplotype.

M2055

Sex-Related Inheritance and Transmission Pattern in Inflammatory BowelDiseaseZuzana Zelinkova, Pieter C. Stokkers, Klaas van der Linde, E. J. Kuipers, Christien J. vander Woude

Background: Extensive epidemiological data indicate a female predominance in inflammatorybowel disease (IBD). However, the exact character of sex-related inheritance in IBD isunknown. Aim: To characterize the sex-related inheritance and transmission pattern ininflammatory bowel disease. Methods: IBD patients with family history of IBD were identifiedin 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 werecompared to the baseline IBD population. The binomial distribution test was used to analyzethe 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. Thebaseline IBD patients' database comprised 2700 patients (CD/UC/U; 1609/1009/82). In thefamilial 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). Thisfamilial 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 familiesin which both, a parent and a child were affected were identified. A significantly highernumber of mother to child transmissions (55 vs. 32 of father to child transmissions) wasobserved (p=0.004). The female imprinting was specifically related to CD (mother/father tochild transmissions; 31/14; p=0.001), in UC no significant differences between mother andfather to child transmission numbers were observed. The analysis of offspring sex distributionpattern revealed significantly higher female to female transmission compared with femaleto male transmission rate (female to female/female to male transmission; 36/18; p=0.005). Nospecific offspring sex-related transmission pattern was observed in the paternal transmissionfamilies. Conclusion: In inflammatory bowel disease, the female predominance is ratherassociated with familial than sporadic occurence of the disease. In Crohn`s disease, thispredominance may be related to the imprinting of the disease predisposition with a specificfemale to female transmission pattern.

AG

AA

bst

ract

s