Irina Costea

Interactions Between the Dietary Polyunsaturated Fatty Acid Ratio and Genetic Factors Determine Susceptibility to Pediatric Crohn's Disease

Increased dietary ratios of ω6/ω3 polyunsaturated fatty acids have been implicated in the pathogenesis of Crohns disease (CD), but epidemiologic data are limited. We investigated whether variants of genes that control polyunsaturated fatty acid metabolism (CYP4F3, FADS1, and FADS2), along with the dietary ratio of ω6/ω3, confers susceptibility to CD. Based on data from 182 children newly diagnosed with CD and 250 controls, we found that children who consumed a higher dietary ratio of ω6/ω3 were susceptible for CD if they were also carriers of specific variants of CYP4F3 and FADS2 genes. Our findings implicate diet-gene interactions in the pathogenesis of CD.

Genome-wide association study signal at the 12q12 locus for Crohn's disease may represent associations with the MUC19 gene.

Background:Genome-wide association studies (GWAS) in Crohn’s disease (CD) have identified associations with single-nucleotide polymorphism (SNP) rs11175593 at chromosome 12q12. The MUC19 and LRRK2 genes reside close to the GWAS signal, but it is as yet unclear which of the 2 genes represent the CD susceptibility genes. Methods:We studied associations between nonsynonymous coding variants in the MUC19 (5) and LRRK2 (3) genes in a case–control sample comprising CD cases aged <18 years at diagnosis. The GWAS lead SNP rs11175593 was also investigated. Allelic, genotype, and haplotype associations were examined assuming different models of inheritance. Results:A total of 530 cases and 600 controls were studied. The mean (±SD) age at diagnosis was 12.4 (±3.3). Most cases were male (57.4%). Most patients had ileocolonic disease location (48.8%) and inflammatory behavior at diagnosis (87.0%). Three MUC19 SNPs were nominally significantly associated with CD (rs11564245, Asp→His: P = 0.02; rs4768261, Ser→Phe: P = 0.0008; and rs2933353, Glu→Ala: P = 0.01). Associations with rs4768261 were maintained after corrections for multiple comparisons (permuted, P = 0.007). None of the LRRK2 SNPs were associated with CD. Haplotype analysis supported the single SNP associations noted with the MUC19 gene. Conclusions:GWAS signal at chromosome 12q12 for CD may represent associations with the MUC19 gene.

Genes Involved in the Metabolism of Poly-Unsaturated Fatty-Acids (PUFA) and Risk for Crohn's Disease in Children & Young Adults

Background and Objectives Epidemiological evidence for the role of polyunsaturated fatty-acids (PUFA) in Crohns disease (CD) is unclear, although the key metabolite leucotriene B4 (LTB4) is closely linked to the inflammatory process. We hypothesized that inherited variation in key PUFA metabolic enzymes may modify susceptibility for CD. Methods and Principal Results A case-control design was implemented at three pediatric gastroenterology clinics in Canada. Children ≤20 yrs diagnosed with CD and controls were recruited. 19 single nucleotide polymorphisms (SNPs) across the ALOX5 (4) CYP4F3 (5) and CYP4F2 (10) genes, were genotyped. Associations between SNPs/haplotypes and CD were examined. A total of 431 cases and 507 controls were studied. The mean (±SD) age of the cases was 12.4 (±3.3) years. Most cases were male (56.4%), had ileo-colonic disease (L3±L4, 52.7%) and inflammatory behavior (B1±p, 87%) at diagnosis. One genotyped CYP4F3 SNP (rs2683037) not in Hardy-Weinberg Equilibrium was excluded. No associations with the remaining 4 CYP4F3 SNPs with CD were evident. However haplotype analysis revealed associations with a two-marker haplotype (TG) (rs3794987 & rs1290617) (p = 0.02; permuted p = 0.08). CYP4F2 SNPs, rs3093158 (OR (recessive) = 0.56, 95% CI = 0.35–0.89; p = 0.01), rs2074902 (OR (trend) = 1.26, 95% CI = 1.00–1.60; p = 0.05), and rs2108622 (OR (recessive) = 1.6, 95% CI = 1.00–2.57; p = 0.05) were significantly associated whereas rs1272 (OR (recessive) = 0.58, 95% CI = 0.30–1.13; p = 0.10) showed suggestions for associations with CD. A haplotype comprising these 4 SNPs was significantly associated (p = 0.007, permuted p = 0.02) with CD. Associations with SNP rs3780901 in the ALOX5 gene were borderline non-significant (OR (dominant) = 1.29, 95% CI = 0.99–1.67; p = 0.056). A haplotype comprising the 4 ALOX5 SNPs (TCAA, p = 0.036) was associated with CD, but did not withstand corrections for multiple comparisons (permuted p = 0.14). Conclusions Inherited variation in enzymes involved in the synthesis/metabolism of LTB4 may be associated with CD. These findings implicate PUFA metabolism as a important pathway in the CD pathogenesis.

Association between the PTPN2 gene and Crohn's Disease: Dissection of potential causal variants

Background:Although genome-wide association studies (GWAS) and subsequent meta-analyses have confirmed associations between the PTPN2 (protein tyrosine phosphatase, nonreceptor type 2) gene and Crohns disease (CD), the potential causal variants remain unidentified. We aimed to dissect potential causal CD-associated PTPN2 variants, assess their functional significance, and relate PTPN2 protein expression with inflammation in CD. Methods:A 3-stage study was carried out. In stage 1, we genotyped tagging single nucleotide polymorphisms (tag-SNPs) in the PTPN2 gene in a sample of patients with CD (<20 years, n = 556) and controls (n = 602). In stage 2, we resequenced the putative promoter, target exons and introns in the PTPN2 gene, and examined associations with high-frequency variants with CD in the stage 1 cohort. In stage 3 we studied the relationship between PTPN2 protein expression and mucosal inflammation and carried out in silico analyses to study the functional characteristics of the PTPN2 CD-associated SNPs. Results:In stage 1, we observed associations between 5 intronic SNPs and CD including rs1893217 (P = 2 × 10−4), the SNP that is in perfect linkage disequilibrium with the lead genome-wide association studies SNP rs2542151. Resequencing revealed 2 known promoter polymorphisms. No associations between these promoter SNPs and CD were evident. In silico analyses revealed that the 5 associated intronic SNPs influenced PTPN2 expression and binding to important transcription factors. PTPN2 protein was overexpressed in inflamed intestinal tissues of patients with CD. Conclusions:Our findings suggest that noncoding variation in the PTPN2 gene may represent the causal variations influencing susceptibility for CD.

NELL1, NCF4, and FAM92B genes are not major susceptibility genes for Crohn's disease in canadian children and young adults

Background: Genome‐wide association studies (GWAS) and replication studies have shown conflicting associations between the NELL1, NCF4, and FAM92B genes and susceptibility for Crohns disease (CD). We sought to examine whether these genes were associated with CD in Canadian children and young adults. Methods: A case‐control study was carried out at three pediatric gastroenterology clinics across Canada. Patients, ≤20 years at diagnosis, along with controls representative of the general population were selected. Study subjects were genotyped for 22 single nucleotide polymorphisms (SNPs) across the target genes. Allelic and haplotype associations were examined. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were estimated. Results: In all, 566 CD cases and 602 controls were investigated. The mean (±SD) age of the patients was 12.3 (±3.3) years. Most patients were male (57.8%), of Caucasian ancestry (98.2%), and had ileocolonic disease location (48.8%). Barring nominal associations with one FAM92B SNP, none of the other 21 SNPs analyzed were associated with CD either at the allelic or haplotype level. Separate analysis for ileal CD (L1 plus L3) also did not reveal significant associations with any of the SNPs. Similarly, a pooled analysis using data from two recent studies did not demonstrate associations between the NCF4 (OR = 1.10, 95% CI = 0.91–1.32, P = 0.32) and FAM92B (OR = 1.05, 95% CI = 0.95–1.17, P = 0.36) GWAS lead SNPs and ileal CD. Conclusions: GWAS‐reported associations in the NELL1, NCF4, and FAM92B genes could not be replicated in Canadian children and young adults. Further investigation in other populations will be required to confirm the presence/absence of associations, if any. (Inflamm Bowel Dis 2012;)

S1752 The Interleukin (IL)-10 Gene Is Associated with Crohn's Disease in Canadian Children. Findings Based On a Gene-Wide Case-Control Study

Background and Aim: RNAi is a powerful biological tool whereby any specific genes of interest can be specifically targeted. However, application of RNAi for silencing genes of the GI tract has proven difficult. We recently described a bacteria-based RNAi technology (transkingdom RNAi, tkRNAi) to simultaneously produce and deliver RNAi to the GI tract (Xiang, Nat Biotech 2006, 697). The aim of this study was to examine whether tkRNAi can be used to silence the expression of IL-10, an anti-inflammatory cytokine, and induce a pro-inflammatory state In Vivo. Methods: After selection of themost powerful siRNA sequence, tkRNAi bacteria were created producing shRNA against mouse IL-10 (TRIP-sh7) and control (TRIP-scrambled). J774.A1 cells were incubated with TRIP-bearing bacteria for 2h at various MOIs, and then stimulated with LPS for 24h. IL-10 gene silencing was analyzed by quantitative RT-PCR and ELISA. To assess In Vivo gene silencing, 12 Balb/c mice were randomized into 3 groups: untreated (n=4); TRIP-sh7 (n=4); TRIP-scrambled (n=4). Following bacteria treatment for 2 weeks, colons were harvested and analyzed by qRT-PCR and IHC staining for IL-10 and γ-IFN. Results: treatment of J774.A1 cells with TRIP-sh7 reduced IL-10 mRNA and protein levels by >80% (p 70% (p < 0.05) compared to animals treated with TRIPscrambled control bacteria. IHC staining of colon tissue demonstrated a marked reduction of IL-10 protein expression in TRIP-sh7 treated mice compared to untreated or mice treated with control bacteria. Moreover, treatment with TRIP-sh7 was associated with increased colonic production of the TH1 cytokine γ-IFN. Treatment with TRIP-sh7 for two weeks was also associated with a significant loss of body weight compared to untreated or mice treated with control bacteria (p < 0.05 for both). Summary and Conclusion: bacteria expressing shRNA against IL-10 can mediate potent gene silencing in LPS-stimulated J774.A1 cells, and in colonic epithelial cells In Vivo. Furthermore colonic levels of γ-IFN were markedly increased following bacterial RNAi against IL-10. tkRNAi approach may be useful for In Vivo functional genomics studies in the GI tract, and for developing RNAi therapies for GI diseases. Since tkRNAi can be adapted to induce silencing in multiple animal species, this approach may be suited for development of animal models in rodents or non-rodent, for diseases such as IBD.

S1754 Interactions Between PTPN2, CARD15 and ATG16L1 Genes Are Associated with Pediatric-Onset Crohn's Disease. Findings Based On a Gene-Wide Case-Control Study in Canadian Children

Background and Aim: RNAi is a powerful biological tool whereby any specific genes of interest can be specifically targeted. However, application of RNAi for silencing genes of the GI tract has proven difficult. We recently described a bacteria-based RNAi technology (transkingdom RNAi, tkRNAi) to simultaneously produce and deliver RNAi to the GI tract (Xiang, Nat Biotech 2006, 697). The aim of this study was to examine whether tkRNAi can be used to silence the expression of IL-10, an anti-inflammatory cytokine, and induce a pro-inflammatory state In Vivo. Methods: After selection of themost powerful siRNA sequence, tkRNAi bacteria were created producing shRNA against mouse IL-10 (TRIP-sh7) and control (TRIP-scrambled). J774.A1 cells were incubated with TRIP-bearing bacteria for 2h at various MOIs, and then stimulated with LPS for 24h. IL-10 gene silencing was analyzed by quantitative RT-PCR and ELISA. To assess In Vivo gene silencing, 12 Balb/c mice were randomized into 3 groups: untreated (n=4); TRIP-sh7 (n=4); TRIP-scrambled (n=4). Following bacteria treatment for 2 weeks, colons were harvested and analyzed by qRT-PCR and IHC staining for IL-10 and γ-IFN. Results: treatment of J774.A1 cells with TRIP-sh7 reduced IL-10 mRNA and protein levels by >80% (p 70% (p < 0.05) compared to animals treated with TRIPscrambled control bacteria. IHC staining of colon tissue demonstrated a marked reduction of IL-10 protein expression in TRIP-sh7 treated mice compared to untreated or mice treated with control bacteria. Moreover, treatment with TRIP-sh7 was associated with increased colonic production of the TH1 cytokine γ-IFN. Treatment with TRIP-sh7 for two weeks was also associated with a significant loss of body weight compared to untreated or mice treated with control bacteria (p < 0.05 for both). Summary and Conclusion: bacteria expressing shRNA against IL-10 can mediate potent gene silencing in LPS-stimulated J774.A1 cells, and in colonic epithelial cells In Vivo. Furthermore colonic levels of γ-IFN were markedly increased following bacterial RNAi against IL-10. tkRNAi approach may be useful for In Vivo functional genomics studies in the GI tract, and for developing RNAi therapies for GI diseases. Since tkRNAi can be adapted to induce silencing in multiple animal species, this approach may be suited for development of animal models in rodents or non-rodent, for diseases such as IBD.