CARD15 mutations

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in UC patients in this cohort was low. Conclusion: Whereas CARD15 mutations were so far only reported in CD, we now also demonstrate an increased prevalence in UC patients belonging to mixed families. Also ASCA was more frequent in these UC patients. Inversely, ASCA was less frequent in the CD patients from these mixed families. We therefore show further evidence that mixed IBD families behave differently and that they might represent a distinct entity. A yet unidentified ( genetic or environmental? ) factor must play a role in the different clinical presentations observed in these families.

Crohn's Disease Behavior Changes over the Course of the Disease are Influenced by NOD2/CARD15 Mutations Alicia M. Sambuelli, Laura Murillo, Anilbal Gil, Silvia Negreira, Sergio Huernos, Silvina Goncalves, Julio C. Bai, Amado S. Pena BACKGROUND/AIMS: Current clinical classifications of CD include, as important issue, a disease behavior categorization based on the presence or absence of strictures and/or fistulas. However, behavior variations over the course of the disease, limit the clinical applicability of the classification. We hypothesized that the predisposition to remain with a stable pattern, or to present a rapid, or alternatively, a slowly progression toward stricturing or flstulizing behaviors, may reflect genetically different backgrounds. We aimed to investigate whether the NOD2/CARD15 mutations carriage may influence the predisposition and speed to develop stricturing or fistulizing complications, in order to assess their clinical usefulness to identify CD subgroups with prognosis differences. MATERIAl/METHODS: 143 CD, median duration from onset 10.9 yrs (range 1-57) were analyzed by PCR for the presence of 3020insC frameshift (SNP13), Gly908Arg (SNP12), Arg702trp (SNP8) mutations. CD behavior was categorized in inflammatory, structuring, fistulizing; the evolution time from the CD onset until pattern changes, obtained from an 1BDdata base and clinical records review. Cumulative probabilities of having a stricturing pattern or a fistuflzing pattern in carriers and noncarriers of the mutations were calculated using Kaplan*Meier method and further analyzed by Log-Rank test. RESULTS: Cumulative probabilities of developing stricturing pattern at 1, 5 and 10 yrs from CD onset were higher in carriers of at least an allele of SNP13 vs. non-carriers (59%, 80% and 80% vs. 33%, 33% and 35% respectively, p=O.O01, Log Rank test). Interestingly, SNP13 carriage showed relevant statistical significance to present a stable stricturing pattern (p<0.00001 ), but no an initially stricturing behavior that shift to fistulizing (NS). SNP12 carriers presented higher cumulative probabilities to have abdominal fistulas vs. non-carriers (11% 33% and 66% at 1, 5, 10 yrs vs. 2%, 8% and 19%, p =0.0021), but no perianal fistulas. SNP8 carriers vs. non-carriers showed a trend for intestinal fistulas presentation (7%, 13%, 29% vs. 3%, 10% and 21%, respectively at 1, 5, 10 yrs, p=0.07). CONCLUSION: our study suggests that CARD15 mutations are a useful tool to categorize CD in order to predict the outcome. SNP13 carriers showed predisposition for a rapid development of a stable stricturing behavior, while SNP12 carriers, irrespective of strictures occurrence, showed predisposition to an earlier intestinal fistulas presentation.

Table I CD Mixed CD UC Mixed UC

CARDI~ % 48.4 45.1 42.3 19.3

ASCA+ % 35.5 54.1 26.9 6,1

Fa.NCA+% 6.5 7.8 42.3 34.2

M1548 Crohn disease is associated with the Toll-like receptor (TLR)-4 polymorphism Asp299Gly : further evidence for a deficient bacterial recognition in the triggering of the disease Denis P. Franchimont, Severine Vermeire, Hakim E1 Honsui, Marieke Pierik, Thierry Gustot, Olivier Lemoine, Gilbert Vassart, Jacques Deviere, Andre Van Gossum, Paul Rutgeerts Background and Aim. Elicitation of an innate immune response to bacterial products is mediated through families of pattern-recognition receptors (PRRa) including cell surface receptors, the toll-like receptors, and cytosolic receptors, the Nods. Mutations in the cytosolic receptor Nod2 (CARD15) have been associated with Crohn disease (CD). The recently characterized TLR4 Asp299Gly polymorphism impairs LPS signaling and is associated with increased susceptibility to gram-negative bacteria in humans. We sought to determine if this TLR4 polymorphism was associated with CD and impact, alone or in interaction with Nod2, on a particular CD phenotype. Patients and methods. Two independent cohorts of CD patients (cohort 1 n = 334 and cohort 2 n = 88), originating from 2 university 1BD hospitals and 136 healthy controls were genotyped after informed consent for the TLR4 variant Asp299Gly using Taqman PCR and for the three disease-associated NOd2/CARD15 variants (Arg702Trp, Gly908Arg and Leul007fsinsC) using PCR-RFLPs. Results. In cohort 1, 68/ 334 (20.4%) CD patients carried TLR4-Asp299Gly compared to only 13/136 (9.5%) controls (p = 0.0049). Allele frequencies for the wild type and mutated allele respectively were 10.8% vs. 5.2% (p<0 001). Of this cohort, 150/334 (44.9%) CD patients carried at least one NOd2 variant compared to 30/136 (22.0%) controls (p<0.0001). These results were confirmed in cohort 2 with 18/88 (20.4%) CD patients carrying the TLR4-Asp299Gly variant (p = 0.021 compared to controls). The allele frequency for the mutated allele in this cohort was 10.8%. NOD2 prevalence was 42% (p = 0.0014). Uinvariate analysis performed on the total cohort of 422 patients failed to show any significant association between TLR4- Asp299Gly and a particular CD phenotype. In the combined cohort, 56.6% of patients carried CARD15 or NOD2 variants with 12.1% of patients NOd2-/TLR4 + , 36.5% Nod2 +/TLR4-, 43.1% NOd2-/ TLR4- and 8.3% Nod2+/TLR4+ without phenotype association. Conclusion. We report on a novel association between the TLR4 Asp299Gfy polymorphism and CD. The currently studied clinical characteristics could not identify a particular phenotype associated with this polymorphism. This strong association further underscores the important genetic influence of PRRs in the host-bacteria interactions in Crohn disease.

M1551 Association between Polymorphisms in the IKBL Gene And Ulcerative Colitis in Japan Shinya Ohmori, Yoshitaka Kinouchi, Seiichi Takahashi, Keisuke Matsumoto, Yasuhiro Kojima, Mika Hiroki, Mikako Sugimura, Tatsuya Kikuchi, Eiki Nomura, Hiroynki Aihara, Sho Takagi, Kenichi Negoro, Tooru Shimosegawa Background & Aims: HLA.DRB1"1502 is strongly associated with ulcerative colitis (UC) in Japan, therefore, the gene variant conferring susceptibility to UC is thought to be in the DRB1 gene itself or in a linkage disequilibrium with DRB1"1502. Our association mapping using microsatelfite markers narrowed the susceptibility locus ranging from the DRB1 gene to the HLA.B gene (unpublished data). The IKBLgene is located between the TNF gene and the HLA.B gene and is thought to play an important role in immune regulation on the basis of its homology to I-kappa-B family. This study aimed to perform a candidate gene analysis of the IKBL gane including linkage disequilibrium analysis between HLA-DRBI"1502 and polymorphisms of the IKBLgene. Methods: The sequences of the 5'-flanking region (-1 to 731) and its entire exons of the IKBL gene were examined in 2 patients with UC and 14 healthy controls using direct sequencing method. The genotypings of 3 single nucleotide polymorphisms (SNPs, -325 C to G, -263 A to G, -63 A to T) detected in the 5'-flanking region were performed on 141 healthy controls and 143 patients with UC using PCRsequence specific primer method. The DRBI genotype of this cohort had already detenmned in our previous study. Disequilibrium parameter D' was calculated using Estimated Haplotype program. Results: A total of 4 SNPs were identified. One SNP was a synonymous variant in exon 3, the others were located in the position -325 (C to G), -263 (A to G), -63 (A to T) to the transcription start site, respectively. Allele frequencies of -263G and -63T were significantly higher in UC (-263G 36.0%, -63T 69.5%) than those in healthy controls (263G 17.6%; p=0.000001; odds ratio=2.6, -63T 54.4%; p=0.00024; odds ratio= 1.9), while allele frequency of -325C/G showed no significant difference (p = 0.094). The -263G allele was in a strong linkage disequilibrium (D'=0.65 p=0.0000053) to DRBl*1502. Conclusion: These findings suggested that the IKBL-263G qualified as one of the candidate SNPs conferring susceptibility to UC in Japan.

M1549 NOd2 Gene Mutations Are Associated with Growth Failure in Children with Crohn's Disease Gitit Tomer, Clare Cehallos, Erlinda Concepcion, Keith J. Benkov Background: The CARD15/NOD2 gene on chromosome 16 encodes an intracellular protein involved in bacterial recognition by monocytes. Mutations in NOD2 have been found in patients with Crohn's disease (CD). Aims: to analyze the NOD2 gene mutations in children with CD, and to perform genotype-phenotype correlations. Methods: We studied 80 children with CD and 136 healthy controls at Mount Sinai Medical Center. CD was diagnosed based on established clinical, endoscopic, radiological, and histological criteria. Clinical records of all patients were systematically reviewed and detailed phenotypic information was obtained. Patients' DNA samples were analyzed for the 3 NOD2 single nucleotide polymorphism (SNP's) R702W (SNP8), Gg08R (SNPI2), 1007fs (SNP13). Genotype-phenotype correlations were analyzed according to demographic and clinical criteria including ethnicity, anthropometric measurements, disease location, and response to treatment. Results: Eighty patients with CD were analyzed. The mean age at diagnosis was 11.5 years (range 6-18). We found 28 NOD2 mutations in 25 (31%) patients, compared with 14 in 136 controls (10%)(p<0.0001). Three patients were compound heterozygote. There were 16 males and 9 females with mutations. The three main variants (RT02W, G908R, and lO07fs) represented 18%, 28% and 54% of the total mutations in patients (n = 28), respectively. There was no statistically significant difference in the frequency of NOD2 mutations between Jewish and non-Jewish patients. NOD2 mutations were more common in patients with ileal disease (42%) as compared to patients with colonic disease (20%; p = 0.09). Fifty three percent of children with NOD2 gene mutation were below 5th percentile for weight at diagnosis while only 26% of children without mutations were below 5th percentile (p = 0.03). Similar trends were observed for height but they did not reach statistical significance. Conclusions: This is the first study of NOD2 mutation screening in children with CD. The study confirms that three sequence variants in the NOD2 gene confer risk to CD in children, and that the NOD2 genotype influences disease phenotype in CD. Our study showed an association between NOD2 mutations and growth laflure. This association may be secondary to the correlation of NOD2 mutations with ileal disease or it may reflect a more severe disease. 2"he underlying mechanisms by which the NOD2 gene confers susceptibility to CD remain incompletely understood and need to be further elucidated.

M1552 Crohn's Disease Patients with CARD15 Variants Have Higher Risk of Developing Axial Joint Involvement Martine De Vos, Harald Peeters, Bert Vander Cruyssen, Debby Laukens, Denis Marichal, Marthe Vandenbergbe, Tessa Van Oostveldt, Dirk Elewaut, Filip De Keyser, Claude Cuvelier, Eric Veys, Erik Remant, Lothar Steidler, Herman Mielants Aim of the study : We investigated whether mutations in the CARD15 gene in Crohn's disease (CD) patients can be linked with the presence of articular involvement. Method : 91 consecutive patients with CD from our gastroenterology unit were evaluated by a rheumatologist for the presence of peripheral arthritis and/or axial involvement. Radiographies of sacroiliac joints were performed in all patients and blindly assessed by the rhenmatologist. RFLP-PCR technique was used to genotype all patients for three single nucleotide polymorphisms in CARD15 gene. Every SNP was verified by direct sequencing. HLA-B27 phenotype was determined. Results : Overall carrier frequencies of R702W, G908R and 1007fs in CD patients was 52%. Prevalences of mutations in CD patients with and without articular involvement were respectively 65% and 44% (p=O,09). Radiologic evidence of sacroiliitis (at least grade 2) with or without ankylosmg spondylitis was found in 23 patients (25%). Seventeen (74%) of these patients were carriers of CARD15 gene mutation (p = 0,02

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AGA Abstracts