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.

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 Crohn's 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 Crohn's 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.

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 Crohn's disease (CD) is unclear, although the key metabolite leucotriene B4 (LTB(4)) 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 LTB(4) may be associated with CD. These findings implicate PUFA metabolism as a important pathway in the CD pathogenesis.

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 Crohn's 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 x 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 x 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 x 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.

Associations between ABCB1/MDR1 gene polymorphisms and Crohn's disease: a gene-wide study in a pediatric population.

BACKGROUND: Functional studies support the involvement of the MDR1 gene in the pathways leading to Crohn's disease (CD). Two common single nucleotide polymorphisms (SNPs), C3435T and G2677T/A, thought to alter the function of the corresponding P-glycoprotein, have shown inconsistent associations with CD. We investigated whether DNA variants in the MDR1 gene were associated with susceptibility for CD and specific phenotypes in children. METHODS: A case-control study was conducted at 3 gastroenterology clinics across Canada. Children with CD and population- or hospital-based controls were included. CD cases were classified using the Montreal Classification. Thirteen tag-SNPs and the C3435T variant in the MDR1 gene were genotyped. Single-SNP allelic, genotype as well as gene-wide haplotype associations with CD and its phenotypes at diagnosis were assessed. RESULTS: A total of 270 CD cases and 336 controls were studied. Most cases were male (56.3%), had disease location L3+/-L4 (58.1%), and an inflammatory phenotype B1+/-p (88.5%). Allelic association analysis revealed that SNP rs17327442 was significantly associated with overall susceptibility to CD (odds ratio [OR] = 0.72, 95% confidence interval [CI] = 0.50-0.99, P = 0.04) but this association did not withstand corrections for multiple testing (q-value = 0.56). Genotype-phenotype analysis indicated that 2 SNPs (rs10248420, P = 0.007, q-value = 0.07; rs2032583, P = 0.01, q-value = 0.07) were significantly associated with colonic disease. Five SNPs, rs1128503 (P = 0.02), rs1202184 (P = 0.008), rs1202186 (P = 0.02), rs2091766 (P = 0.03), and rs2235046 (P = 0.03) were nominally associated with noninflammatory CD. Specific haplotypes comprising of the tag-SNPs were significantly associated with either colonic or noninflammatory CD. CONCLUSIONS: Our comprehensive gene-wide analysis suggests that the MDR1 gene may be associated with clinical phenotypes of CD in children.

Investigation of associations between the pregnane-X receptor gene (NR1I2) and Crohn's disease in Canadian children using a gene-wide haplotype-based approach.

BACKGROUND: The pregnane-X-receptor (PXR) is involved in the metabolism and detoxification of numerous xenobiotics and is critical for maintaining intestinal integrity. The NR1I2 gene encoding PXR may confer susceptibility for Crohn's disease (CD) but evidence for associations is conflicting. We investigated whether the NR1I2 gene was associated with susceptibility for pediatric CD. METHODS: A case-control and family-based (case-parent) study was carried out at 3 inflammatory bowel disease (IBD) clinics across Canada. Confirmed cases of CD <20 years were diagnosed using standard criteria. For determination of gene associations parents of the cases and unrelated controls were evaluated. Clinical phenotypes were established based on the Montreal Classification scheme. Eight tag-SNPs (tag-single nucleotide polymorphisms) across the gene were genotyped for allelic or genotypic associations. RESULTS: A total of 270 CD cases, 336 controls, and 395 parents were studied. The mean age (+/-SD) of the cases was 12.1 (+/-3.5) years of age. Most cases were male (56.3%), had disease location L3+/-L4 (58.1%), and an inflammatory phenotype B1+/-p (88.4%) at diagnosis. For 7 SNPs single SNP analysis using case-control or case-parent data did not reveal associations with development of CD and none of the SNPs were significantly associated with disease location or disease behavior at diagnosis. One SNP rs2461823 (P = 0.05) was nominally associated with CD. No overall haplotype association (omnibus P-value = 0.61) or associations with individual haplotypes was evident. CONCLUSIONS: Our gene-wide analysis in a pediatric cohort using both the case-control and case-parent designs suggests that the NR1I2 gene is not associated with CD in Canadian children.

Dietary patterns and risk for Crohn's disease in children.

BACKGROUND: Some dietary foods are considered protective (vegetables and fruits), whereas others (fatty foods) are thought to enhance the risk for Crohn's disease (CD). The evidence, however, is inconsistent. METHODS: We postulated that specific dietary patterns may influence the risk for CD. A case-control study was carried out. Newly diagnosed CD cases with population and/or hospital-based controls < or =20 years were selected from 3 tertiary hospitals across Canada. Pre-disease diet was assessed using a validated food frequency questionnaire (FFQ) administered within 1 month of diagnosis. Factor analyses and unconditional logistic regression (adjusted) was used to determine gender-specific dietary patterns and assess associated risks for CD. Odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs) were estimated. RESULTS: A total of 149 cases and 251 controls were included. The mean age (range) of the cases was 13.3 (2.6-20 years). There were more boys (61.1%). Four dietary patterns each were observed among both boys and girls. Pattern 1 in girls, characterized by meats, fatty foods, and desserts, was positively associated with CD (OR 4.7, 95% CI 1.6-14.2). Pattern 2, common to both boys and girls, was characterized by vegetables, fruits, olive oil, fish, grains, and nuts and was inversely associated with CD in both genders (girls: OR 0.3, 95% CI 0.1-0.9; boys: OR 0.2, 95% CI 0.1-0.5). CONCLUSIONS: Our results suggest that specific dietary patterns could be associated with higher or lower risks for CD in children. Larger prospective studies are required to confirm these findings.

Association between genetic variants in the IL-23R gene and early-onset Crohn's disease: results from a case-control and family-based study among Canadian children.

BACKGROUND AND OBJECTIVES: Interleukin (IL)-23 is a key regulator of inflammation and influences the activities of T-helper 17 (Th-17) lymphocytes. Recent reports indicate that variants in the gene coding for its receptor (IL-23R) are strongly associated with Crohn's disease (CD). We investigated whether DNA variants in the IL-23R gene determine susceptibility for CD in Canadian children. DESIGN AND METHODS: A case-control and case-parent trio design was implemented at three pediatric centers across Canada. Cases of CD (

Imbalances in dietary consumption of fatty acids, vegetables, and fruits are associated with risk for Crohn's disease in children.

BACKGROUND AND OBJECTIVES: The role of dietary factors in the etiology of Crohn's disease (CD) is inconsistent largely due to difficulties in acquiring valid information on consumption habits. We examined the impact of diet on new onset CD in children using a validated food-frequency questionnaire (FFQ). METHODOLOGY: A case-control study was carried out. Children < or =20 yr, newly diagnosed with CD, were recruited from 3 pediatric gastroenterology clinics across Canada. Population or hospital controls were selected matched to cases for time of diagnosis (+/-6 months) and area of residence. Dietary consumption 1 yr prior to disease diagnosis was evaluated using a validated FFQ, administered within 1 month of diagnosis. Conditional logistic regression analysis adjusting for potential confounding variables (energy intake, age, gender, body mass index) was carried out. RESULTS: A total of 130 CD patients and 202 controls were studied. Mean age at diagnosis (+/-SD) was 14.2 (2.7). There were more male patients (59%). Comparing the highest to the lowest levels of consumption, higher amounts of vegetables (OR 0.69, 95% CI 0.33-1.44, P= 0.03), fruits (OR 0.49, 95% CI 0.25-0.96, P= 0.02), fish (OR 0.46, 95% CI 0.20-1.06, P= 0.02), and dietary fiber (OR 0.12, 95% CI 0.04-0.37, P < 0.001) protected from CD. Consumption of long-chain omega-3 fatty acids (LCN-omega-3) was negatively associated with CD (OR 0.44, 95% CI 0.19-1.00, P < 0.001). A higher ratio of LCN-omega-3/omega-6 fatty acids was significantly associated with lower risks for CD (OR 0.32, 95% CI 0.14-0.71, P= 0.02). CONCLUSIONS: Our findings indicate that an imbalance in consumption of fatty acids, vegetables, and fruits is associated with increased risks for CD among Canadian children.

Investigating the hygiene hypothesis as a risk factor in pediatric onset Crohn's disease: a case-control study.

BACKGROUND AND OBJECTIVES: Evidence for the hygiene hypothesis in the etiology of Crohn's disease (CD) is unclear. We investigated the relationship between infection-related exposures and risk for CD in children. METHODS: A hospital-based case-control was carried out. Newly-diagnosed cases of CD (n = 194), less than 20 yr of age were recruited from the gastroenterology clinic of a large-pediatric inflammatory bowel disease (IBD) center in Montreal, Canada. Orthopedic patients pair-matched (n = 194) for timing of diagnosis and area of residence were recruited as controls. Information on infection-related exposures between birth and disease diagnosis was ascertained by administering a structured questionnaire to the mother and the index subject. The relationship between the frequency and timing of infection-related exposures with CD was studied. RESULTS: The mean age (SD) at diagnosis was 12.3 (5.1). CD was more common after 10 yr of age. Gender distribution was similar between comparison groups. In multivariate conditional logistic regression, family history of IBD (odds ratio (OR) = 4.6; 95% confidence interval (CI) = 1.6-13.3), age (OR = 1.2; 95% CI = 1.1-1.3), and owning a pet (OR = 2.0; 95% CI = 0.9-4.5) were associated with risk for CD, whereas regular use of a personal towel (OR = 0.5; 95% CI = 0.2-0.9) and lesser crowding in homes (OR = 0.3; 95% CI = 0.1-0.8) were protective. Day-care attendance during the first 6 months of life and "physician-diagnosed infections" between 5 and 10 yr of age were associated with increased risks for CD. CONCLUSIONS: Infection-related exposures seem to enhance risk for CD in children. The timing of these exposures during early childhood may be relevant to the etiology of pediatric CD.