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schanges vary by outcome. Methods: A cohort of 245 patients who had undergone IPAAand closure of ileostomy at least one year prior to recruitment was prospectively accruedat Mount Sinai Hospital in Toronto, Ontario. Biopsy specimens from the pouch and afferentlimb of the ileum were collected at the time of recruitment and preserved in RNAlater.Based on clinical and endoscopic data at time of enrolment, 40 individuals from this cohortwere allocated into 4 outcome groups - no pouchitis (NP), acute pouchitis (AP), Crohn'sdisease - like phenotype (CDL) and familial adenomatous polyposis controls (FAP). ThemiRNeasy Mini Kit was used to extract total RNA from all samples. The eluted transcriptomeswere analyzed on Affymetrix Human Gene 1.0 ST arrays and the data were backgroundcorrected and normalized in Affymetrix Gene Expression Console. A repeated measuresANOVA model in the statistical package R was used to assess the significance of each gene.Raw p-values were corrected for multiple comparisons by the false discovery rate (FDR)method. Results: The expression levels of 144 gene transcripts were significantly associatedwith outcome at a corrected p-value cut-off of <0.05. There was also evidence that 117 ofthese were differentially regulated in the pouch compared to the afferent limb. Approximately20% of the genes associated with outcome have known xenobiotic activity related to metabol-izing and removing various drugs, toxins and other compounds from the cytoplasm of cells.All of these genes exhibited virtually the same expression pattern of up to 4 fold reductionin relative pouch mRNA abundance in the AP and CDL groups compared to the NP andFAP groups. (Figure 1.) A prominent example is the gene ABCB1 also known as multi-drugresistance 1 (MDR1). Previous reports have shown that single nucleotide polymorphismswhich result in attenuated ABCB1 expression are associated with UC. Conclusion: Tran-scriptome analysis of UC patients following IPAA showed that acute pouchitis and theCrohn's disease-like phenotype are associated with significantly repressed drug and toxinefflux activity in the affected tissue. These findings corroborate the hypothesis that impairedbarrier function could lead to development of intestinal inflammation.

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Autophagy Gene Polymorphisms Influence the Interaction of E.Coli andMacrophages in Crohn's DiseaseTim Elliott, Natalie J. Prescott, Barry N. Hudspith, Neil B. Rayment, John Hermon-Taylor,Jonathan Brostoff, Christopher G. Mathew, Jeremy D. Sanderson

Introduction: A well established expression of the proposed defect in innate immunity inCrohn's disease (CD) is the prolonged survival of strains of E.coli in peripheral bloodmonocytes/macrophages (PBM) from patients with CD compared to those from healthycontrols (HC). Altered handling of E.coli in CD has been demonstrated in PBM and in-situin CD by laser dissection of E.coli infected macrophages. Several of the gene polymorphismsconferring an increased risk of CD occur in genes involved in bacteria-immune cell interac-tions. The aim of this study was to determine the influence of these polymorphisms on theinteraction of CD-derived E.coli with monocyte / macrophages from CD. Methods: PBM'sand lamina propria macrophages were obtained from 43 and 17 CD patients respectively(mean age 39yrs / range 20-70yrs). PBM's were challenged with E.coli and then the responseof these PBM's measured by levels of bacterial killing, using the gentamicin protectionassay. Phagocytosis and reactive oxygen radicals (ROR's) production were measured byflow cytometry using the phagotest and phagoburst assays. Cytokine (TNFα, IL10, IL23)production was measured by ELISA. In parallel experiments, TNFα, IL10 and CD163expression was measured by qRT-PCR in laser dissected E.coli laden and unladen laminaproprial macrophages. All patients were genotyped for the 3 common NOD-2 SNP's (G908R,L1007fs & R702W), and the autophagy related SNP's (ATG16L1 and IRGM (rs13361189).Genotypes and monocyte/macrophage markers were compared. Results: Presence of theATG16L1 risk variant was strongly associated with a reduced E.coli induced TNFα expressionin CD PBM's (p=0.0017) and increased levels of ROR's on uptake of CD derived E.coli (p=0.0027). Presence of IRGM risk variant was associated with increased IL10 expression fromPBM's (p=0.027). NOD-2 mutations were not associated with a difference in any variablestudied. No genotype altered survival of E.coli in PBM at 2 or 4 hours after infection or in-situ lamina propria macrophage cytokine expression. Conclusion: These results show thatdefective autophagy modifies the handling of intracellular bacteria in CD although theprimary defect leading to prolonged bacterial survival remains unknown. Further studiesare needed to elucidate the nature of this innate defect and it's role in CD pathogenesis.

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Whole Genome Sequence of a Crohn Disease Trio - A Paradigm forIndividualized Disease Etiology DiscoveryPhilip C. Rosenstiel, Matthias Barann, Andre Franke, Bjoern Stade, Ingo Thomsen,Markus B. Schilhabel, Vrunda Sheth, Clarence Lee, Ulrich C. Klostermeier, KevinMcKernan, Annette Fritscher-Ravens, Stefan Schreiber

Background: Discovery of the genetic etiology of Crohn disease, a human chronic inflammat-ory bowel disease, represents a paradigm for polygenic traits in humans with over 30replicated disease genes/loci identified mostly by genome-wide association studies (GWAS).However, not more than 20% of the cumulative genetic variance can be explained by thefindings made. Further discovery of low penetrance disease loci, individual genetic variation

S-28AGA Abstracts

patterns and epistatic effects have all been limited due to mostly statistical issues, whichlimits clinical translation of the findings. Gathering knowledge on entire individual diseasegenomes is a logical advancement of GWAS as it includes rare and potentially causativevariants “hidden” on common haplotypes. Methods: For this benchmarking study, a Falk-Rubinstein trio with an extreme phenotype was selected, i.e. early, severe onset in theaffected child. Genome sequencing was carried out on an Applied Biosystems SOLiD 4System. We analyzed approx. 1.5 billion shotgun mapped reads from 50+25 bp paired endlibraries, 0.5 billion 50+50 mate pair and 0.2 billion exome fragment reads for each of theindividuals. Results: With a minimum mapped individual throughput of 90 Gb, over 99%of the reference genome was covered with at least one uniquely placed pair of reads. Thegenome data sets resulted in approx. 3.3 million SNP calls in each individual with highconfidence, approx. 20% of which are not annotated in current databases. An Illumina SNP-Chip (HumanOmni1-Quad v1-0) was applied as a benchmark for the SNP identificationmethods. SNP calling by sequence analysis and SNP-chip data showed a correlation of>99.3% for all three individuals. Categorization of the SNPs in relation to functional elementsin the genome was performed and identified >10,000 novel SNPs in gene-associated regions.After exclusion of relevant de novo mutations in the child, we utilized the pedigree structureto prioritize putatively functional SNPs which follow a recessive mode of inheritance (gainof homozygosity or compound heterozygous SNPs). The individual genomes are related tocorresponding RNAseq data from peripheral blood mononuclear cells and intestinal tissueresulting in a comprehensive map of expression levels, alternative splicing and allelic imbal-ance that was further used to filter the genomic information. By using this approach, wedemonstrate an interesting cluster of missense variants in genes from the IL12/23 and TH17pathways. Conclusion: We present the first study aiming for the extraction of the full geneticvariability in a Crohn disease trio and utilizing this data together with genetical genomicsanalyses by RNAseq as a basis for personalized understanding of the genetic variants thatcause Crohn disease. Ultimately, such approaches may develop into novel diagnostic andtherapeutic algorithms.

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Role of Haptoglobin in Susceptibility of IBD and in Triggering Murine ColitisLucía Marquez, Chong Shen, Kathleen Machiels, Clémentine Perrier, Vera Ballet, SophieOrgane, Marc Ferrante, Liesbet Henckaerts, Paul J. Rutgeerts, Severine Vermeire, Jan L.Ceuppens, Isabelle Cleynen

Background: Haptoglobin (Hp) is a hemoglobin-binding protein with immunomodulatoryproperties. Its gene is located on 16q22 and harbours a common polymorphism with 2structurally different alleles: Hp1 and Hp2. Hp2 has been shown to be overrepresented inimmune diseases like Rheumatoid Arthritis and Systemic Lupus Erythematosus. Results inCrohn's disease (CD) are contradictory. Aims: We studied this polymorphism in CD andUlcerative Colitis (UC), as well as its influence on circulating serum levels of Hp. We alsodetermined if Hp has an effect on the course of experimentaly-induced murine DSS andoxazolone colitis. Methods: 2419 patients (CD exploratory cohort n=1061; UC exploratorycohort n=755; CD confirmatory cohort n=452 trios; UC confirmatory cohort n=151 trios)and 452 healthy controls (HC) were genotyped. Hp serum levels were determined in 20randomly selected individuals of each genotypE. colitis was induced in mice wild-type (WT)and knock-out (KO) for Hp, with DSS and Oxazolone (OXA). mRNA expression in colonictissue of IL17, IFNγ, TNF, IL6 and IL13 was measured by Quantitative Reverse TranscriptasePCR. IL17 production by isolated mesenteric lymph node cells was measured by ELISA.Results were analyzed using Chi2 and FBAT (Family Based Association Tests) for categoricaldata, and Mann-Whitney and Kruskall-Wallis tests for continuous data. Results: Table 1shows allele and genotype frequencies in CD, UC and HC. Prevalence of Hp2 was higherin CD (p=0.0001) and UC (p=0.0009), compared to HC. Genotype 22 was overrepresentedin UC (p=0.003) and CD (p=0.0001), again compared to HC. In the confirmatory cohorts,allele 2 was overtransmitted to the affected offspring (CD, T:UT=247:186, p=0.003; UC,T:UT=87:65, p=0.07). A trend was observed for higher Hp serum levels in patients withgenotypes 11 and 21 (median 1.4g/L), compared to 22 (median 0.8g/L; p=0.07). No signific-ant differences were found among Hp genotypes and phenotypic characteristics of IBDpatients. DSS and OXA induced-colitis was more severe in KO than in WT mice, evidencedby more weight loss, and higher macroscopic and microscopic scores. mRNA levels of IL17,IFNγ, TNF and IL6 were higher in DSS treated KO mice, and IL13 in OXA KO mice,compared to DSS and OXA treated WT mice. IL17 production was significantly higher inthe presence of Hp-deficient serum compared to WT (median 9123 vs 3583 pg/ml, p<0.05).Conclusions: The higher frequency of the functional allele 2 in IBD patients, and its over-transmission in CD and UC trios suggests that the Hp gene plays a role in susceptibility toIBD. The Hp gene was previously implicated in other immune diseases, underscoring thecommon pathways between these diseases. Experimental models of colitis showed that Hphas a protective role on inflammatory colitis, most likely by inhibition of the production ofTh1 and Th17 cytokines.Table 1

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Outcome After Pelvic Pouch Surgery is Associated With Differences in theMicrobiome as Characterized by 16s rRNA PyrosequencingAndrea D. Tyler, Natalie Berard, Boyko Kabakchiev, Raquel Milgrom, Smita L. Halder,Zane Cohen, Robin S. McLeod, Denis O. Krause, Mark S. Silverberg

Introduction: Ileal inflammation affecting the pelvic pouch following ileal pouch-anal ana-stomosis(IPAA) in patients with ulcerative colitis(UC) is a relatively common yet poorlyunderstood phenomenon. Patients with ileostomies rarely develop inflammation, suggesting