Jinsong Dong

Autophagy Gene ATG16L1 But Not IRGM Is Associated with Crohn's Disease in Canadian Children

Background: Recent genome‐wide studies have implicated the autophagy genes ATG16L1 and IRGM in the pathogenesis of Crohns disease (CD). We investigated whether these genes were associated with CD in Canadian children. Methods: A case‐control study was carried out at 2 pediatric gastroenterology clinics in Canada. Confirmed cases of CD <20 years diagnosed using standard criteria were classified according to the Montreal Classification scheme. Single nucleotide polymorphisms (SNPs) rs2241880 (ATG16L1) and rs10065172 (IRGM) along with CARD15 SNPs, SNP8, SNP12, and SNP13 were genotyped. Results: A total of 289 CD cases and 290 controls were studied. The mean age (±SD) of the cases was 12.1 (±3.5) years of age. Most cases were male (55.4%), had disease location L3 ± L4 (56.7%), and an inflammatory phenotype B1 ± p (87.2%) at diagnosis. rs2241880 (ATG16L1) was strongly associated with CD (allelic P = 1.24 × 10−6). Children with GG genotype had a more than 3‐fold elevated risk for disease as compared to the wildtype AA homozygotes (odds ratio [OR], 3.1; 95% confidence interval [CI], 1.93–4.94; P = 1.8 × 10−6). Association with SNP rs2241880 was specific for ileal disease (with or without colonic involvement) (case‐based allelic P = 0.02; P‐value versus controls = 9.5 × 10−8). The frequency of IRGM SNP rs10065172 was higher in cases but differences with controls were not statistically significant. No interactions between CARD15 and either ATG16L1 or IRGM were evident. Conclusions: We have confirmed associations between CD and ATG16L1 in a pediatric cohort of Canadian children. Associations with IRGM need to be further evaluated in larger studies. (Inflamm Bowel Dis 2008)

Investigation of Reported Associations Between the 20q13 and 21q22 Loci and Pediatric-Onset Crohn's Disease in Canadian Children

OBJECTIVES:A recent pediatric-focused genome-wide association study has reported novel associations of the 20q13 and 21q22 loci with inflammatory bowel disease (IBD). We aimed to investigate these associations with Crohns disease (CD) in Canadian children.METHODS:A combined case–control and case–parent design was implemented at three pediatric gastroenterology clinics in Canada. Children less than 20 years of age with a confirmed diagnosis of CD were recruited along with controls. For a subset of the patients, biological parents were also recruited. Three single-nucleotide polymorphisms (SNPs) at the 20q13 locus and 1 SNP at the 21q22 locus were genotyped. Associations between individual SNPs and haplotypes were examined.RESULTS:A total of 410 cases, 415 controls, and 302 parents were studied. The mean (±s.d.) age for the cases was 12.3 (±3.2) years. Most cases were men (56.1%) who had ileocolonic disease (L3±L4, 52.2%) and inflammatory behavior (B1±B4, 87.0%) at diagnosis. Single SNP analysis showed that all 3 SNPs at the 20q13 locus were significantly associated with CD (rs2297441, P=2.24×10−4; rs2315008, P=4.77×10−4; rs4809330, P=6.08×10−3). Haplotype analysis suggested that the association signal at 20q13 resided on a common haplotype comprising the minor allele of rs2297441 (P=2.8×10−5). SNP rs2836878 at the 21q22 locus showed a trend for association with CD that was statistically not significant (P=0.06).CONCLUSIONS:Our results support an association between the 20q13 locus and CD in Canadian children. Positional cloning studies are required to further dissect the potential causative genes in the region.

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.

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.