M2043 Microarray Study of Mucosal Antimicrobial Peptides in Patients with Inflammatory Bowel Disease Before and After First Infliximab Treatment

plays a role in tissue response to injury and repair, and regulation of T cells and macrophages. MDR-1 codes for the P-glycoprotein drug efflux pump. Genetic variations in both genes have been associated with extensive disease, severe colitis and need for colectomy (1-2). We evaluated the predictive value of GLI-1 and MDR-1 polymorphisms in patients with UC. METHODS: The cohort consisted of 664 patients with UC [46% female, median age at diagnosis 30 (IQR 22-41) years] followed up in our IBD referral centre. All subjects were tested for 4 previously reported GLI-1 haplotype-tagging SNPs [rs3817474 (tSNP1), rs2228225 (tSNP2), rs2228224 (tSNP3), rs2228226 (tSNP4)] and the MDR-1 C3435T SNP using PCR-RFLP. Linkage disequilibrium (LD) was tested using Haploview. GLI-1 haplotypes were determined on the basis of a Bayesian algorithm using the Phase 2.1 software. KaplanMeier analyses were performed to identify predictors of acute severe colitis (modified Truelove and Witts criteria) and need for colectomy. RESULTS: Genotyping was successful in 99% of subjects. All SNPs were in Hardy-Weinberg equilibrium. GLI-1 SNPs were in strong LD (D' 0.97-1.00). During a median follow-up of 7.6 (3.3-13.8) years, 53% of patients developed an extensive colitis, 55% developed an acute severe colitis and 30% needed colectomy for intractable disease. None of the analysed GLI-1 genotypes and haplotypes were predictive of acute severe colitis or need for colectomy (Table). The association between the MDR-1 3435TT genotype and acute severe colitis did not remain significant after correction for multiple testing. CONCLUSION: Despite a large sample size, we could not confirm the reported association between MDR-1 and need for colectomy. Furthermore, we could not confirm the reported association between GLI-1 and extensive disease, acute severe colitis or need for colectomy. The difference with the original GLI-1 study by Lees et al seems to be driven by the lower frequency of the uncommon haplotypes 1211 and 2122 in our cohort (3). (1) Lees et al, JCC Suppl 2007 (2) McGovern et al, Gastroenterology 2004 (3) Ferrante et al, Gut 2007

The Role of Pregnane X Receptor (PXR/NR1i2) Gene Variants in Inflammatory Bowel Disease Julia Seiderer, Jürgen Glas, Julia Diegelmann, Daniel Fischer, Barbara Seitz, Cornelia Tillack, Simone Pfennig, Astrid Konrad, Martin Wetzke, Thomas Griga, Wolfram Klein, Jörg T. Epplen, Uwe Schiemann, Thomas Mussack, Peter Lohse, Burkhard Göke, Thomas Ochsenkuehn, Matthias Folwaczny, Bertram Müller-Myhsok, Stephan Brand Background & Aims: The pregnane X receptor gene (PXR/NR1I2) has recently been reported to be associated with susceptibility for inflammatory bowel disease (IBD). However, since a subsequent case-control study failed to replicate this association in an independent population, further replication studies in different ethnic cohorts are required. We therefore investigated this potential association in a large European IBD cohort. Methods: Genomic DNA from 2823 Caucasian individuals including 859 patients with CD, 464 patients with UC, and 1500 healthy unrelated controls was analyzed for eight single nucleotide polymorphisms (SNPs) in the pregnane X receptor (PXR/NR1I2) gene (rs12721602, rs3814055, rs1523128, rs1523127, rs12721607, rs6785049, rs2276707, rs3814057). Results: With the exception of a weak association of rs2276707 with UC (p=1.02 x 10 -2; OR 1.27 [1.06-1.52]), none of the analyzed variants in the pregnane X receptor (PXR/NR1I2) was associated with CD or UC in our study population. Conclusion: Our data could not confirm the association of PXR gene variants with CD or UC. Further studies investigating the phenotypic effect and potential epistatic interactions of PXR with other IBD susceptibility genes are required to determine the exact role of PXR in IBD. M2043 Microarray Study of Mucosal Antimicrobial Peptides in Patients with Inflammatory Bowel Disease Before and After First Infliximab Treatment Ingrid Arijs, Leentje Van Lommel, Kristel Van Steen, Gert De Hertogh, Roel Quintens, Katleen Lemaire, Marie Joossens, Frans C. Schuit, Karel Geboes, Gert A. Van Assche, Severine Vermeire, Paul J. Rutgeerts Introduction and aim: Antimicrobial peptides (AMPs), host defense peptides, are important components of the innate immune system. Recently, an abnormal expression of AMPs was shown in inflammatory bowel disease (IBD). The aim of the study was to investigate the impact of anti-inflammatory therapy with infliximab (IFX) on the mucosal gene expression of AMPs in IBD patients using oligonucleotide arrays. Methods: The expression of alpha and beta defensins (DEF), lysozyme (LYZ) and phospholipase A2 group IIA (PLA2G2A) were investigated in colonic mucosal biopsies obtained at endoscopy from 41 IBD patients with active colitis (24 ulcerative colitis (UC) and 17 Crohn's disease (CD)) before and 4-6 weeks after first IFX infusion. The patients were classified for response to IFX based on endoscopic and histologic findings: 18 responders (10 CD and 8 UC) and 15 non-responders (5 CD and 10 UC). 6 control patients with normal colonoscopy were included. Total RNA was isolated, labelled and hybridized to Affymetrix HGU133plus2.0 array. Data was analyzed using R software. Results from the array data were validated using real time RT-PCR. Results: Pairwise comparisons (FDR<5%) were performed between controls, patients before and after IFX treatment in UC, CD and IBD (Table). The mRNA levels of DEFA5, DEFA6, DEFB4, LYZ and PLA2G2A were increased before treatment in both active CD and UC compared to controls. The mRNA levels of DEFB4 and LYZ were decreased after treatment in both UC and CD responders compared to baseline. The mRNA levels of DEFA5, LYZ, PLA2G2A remained increased in the IBD responders after treatment compared to controls. In contrast, DEFB1 showed a reduced expression in inflamed colon from both UC and CD patients compared to controls. The expression of DEFB1 was increased in the IBD responders after treatment but remained lower than the expression observed in controls. Conclusion: Our data demonstrate that besides the effect of inflammation on AMPs expression, there is an intrinsic dysregulation of AMPs in colonic mucosa from IBD patients, after controlling for inflammation. These findings support the hypothesis that AMPs play a role in the pathogenesis of IBD. Fold change of the pairwise comparisons

M2045 Reduced Metallothionein Expression in Colonic Crohn's Disease: Evidence for a New Disease-Modifying Gene Debby Laukens, Harald Peeters, Anouk Waeytens, Sara Bogaert, Bert Vander Cruyssen, Dirk Elewaut, Filip De Keyser, Herman Mielants, Claude A. Cuvelier, Paul Van Hummelen, Jurgen Del-Favero, Erik Remaut, Martine De Vos, Pieter Rottiers Background: The identification of genetic determinants of Crohn's disease (CD) remains a challenge. This study aimed at identifying new candidate susceptibility genes for CD by integrating disease loci with gene expression in unaffected colon biopsies of CD patients. We focused on characterizing one of the candidate genes, metallothionein (MT), which belong to a family of highly conserved stress proteins comprising immunomodulating properties. Methods: Sixteen CD patients and 11 controls were subjected to microarray analysis using a focus microarray (VIB Crohn 7K2) containing 6,779 expressed sequence tags. The expression of MT was analyzed by quantitative PCR and immunohistochemistry. To model lowered MT expression In Vitro, a colonic epithelial cell line expressing small interfering RNA against MT was generated. The gene for MRE-binding transcription factor 1 (MTF1) was screened for mutations in 96 patients and the influence of a polymorphism on transcriptional activity was assessed in a luciferase reporter assay. Results: Eighteen differentially expressed genes were identified (Table 1). MT expression was reduced in biopsies (P=0.008) and in PBMCs (P=0.026) of patients with colonic involvement. MT-knockdown HT29 cells showed a reduced IL8 secretion in response to bacterial challenge. Sequence analysis of the main transcriptional regulator of MTs, MTF1, revealed two coding mutations in 7 patients and a frequent polymorphism at the splice site junction of exon 8-9. An intronic polymorphism (IVS1-128A>T) was significantly associated with colonic disease (Chi-square: 8.297, P= 0.004). Moreover, this polymorphism was shown to influence the transcription of a reporter gene, suggesting allele specific transcription of MTF1 which could be responsible for the reduced MT expression observed in colonic CD. Conclusions: We showed that deficient basal MT expression in CD patients with colonic involvement is, at least in part, genetically determined. We identified MTF1 (located within the IBD7 locus) as a new disease-modifying gene. Table 1 Differentially expressed genes in biopsies of CD patients, located near a CD locus

*FDR<5% M2044 Genetic Variations in GLI-1 and MDR-1 Do Not Predict Disease Progression in Patients with Ulcerative Colitis Marc Ferrante, Tamara Coopmans, Isabelle Cleynen, Nathalie Vermeulen, Marie Joossens, Ingrid Arijs, Gert A. Van Assche, Paul Rutgeerts, Séverine Vermeire BACKGROUND: The glioma associated oncogene homologe-1 (GLI-1, 12q13) and the multi drug resistance-1 gene (MDR-1, 7q21) have both been associated with ulcerative colitis (UC). GLI-1 is the major transcriptional target of the Hedgehog signalling pathway which

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AGA Abstracts

M2042