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 Crohn's 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.

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.

Association between genome-wide association studies reported SNPs and pediatric-onset Crohn's disease in Canadian children.

A recent pediatric-focused genome-wide association study has implicated three novel susceptibility loci for Crohn' disease (CD).We aimed to investigate whether the three recently reported and other previously reported genes/loci were also associated with CD in Canadian children. A case-control design was implemented at three pediatric gastroenterology clinics in Canada. Children <19 years of age with a confirmed diagnosis of CD were recruited along with controls. Single nucleotide polymorphisms (SNPs) in 19 reported genes/loci were genotyped. Associations between individual SNPs and CD were examined. A total of 563 cases and 553 controls were studied. The mean (+/-SD) age of the cases was 12.3 (+/-3.2) years. Most cases were male (56.0%), had ileo-colonic disease (L3 +/- L4, 48.8%) and inflammatory behavior (B1 +/- p, 87.9%) at diagnosis. Allelic association analysis (two-tailed) showed that 8 of the 19 targeted SNPs were significantly associated with overall susceptibility for CD. Associations with one additional SNP was borderline non-significant. Significantly associated SNPs included SNPs rs1250550 (p = 0.026) and rs8049439 (p = 0.04), recently reported to be specifically associated with pediatric-onset CD.Based on the results, we confirmed associations between two of the three novel pediatric-CD loci and other regions reported for associations with either pediatric and/or adult-onset CD.

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 Crohn's 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=2.24x10(-4); rs2315008, P=4.77x10(-4); rs4809330, P=6.08x10(-3)). Haplotype analysis suggested that the association signal at 20q13 resided on a common haplotype comprising the minor allele of rs2297441 (P=2.8x10(-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.

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.

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 (

Folate cycle gene variants and chemotherapy toxicity in pediatric patients with acute lymphoblastic leukemia.

The gene polymorphisms of the methotrexate (MTX) action pathway influence event-free survival (EFS) in children with acute lymphoblastic leukemia (ALL). Here we assessed whether the gene variants associated with lower EFS also correlate with lower rates of episodes of toxicity. Homozygous individuals for cyclin D1 (CCND1) A870 allele and carriers of at least one methylenetetrahydrofolate reductase (MTHFR) T677 variant had a significantly lower frequency of weeks with high-grade hematologic and liver toxicity during consolidation and maintenance treatment, as based on the analysis of 186 pediatric ALL patients. This finding may have importance for MTX dose adjustment.

Polymorphisms in genes involved in the corticosteroid response and the outcome of childhood acute lymphoblastic leukemia.

BACKGROUND: Considerable variability in sensitivity to corticosteroids (CS) has been observed among individuals with regard to both the natural and synthetic compounds. The role of genetic polymorphisms in modulating CS function, and hence in disease susceptibility, has been extensively analyzed. Their impact on therapeutic response still remains to be explored. The role of cytochrome P450 (CYP) 3A4 in corticosteroid metabolism, and that of the glucocorticoid receptor (NR3C1) in regulation of responsive genes, renders CYP3A4 and NR3C1 polymorphisms as potential candidates for pharmacogenetic analysis. AIM: The aim of the study was to analyze the role of these polymorphisms in the outcome of a disease treated with CS drugs. METHODS: Towards this aim we analyzed the CYP3A4-290A/G substitution and three NR3C1 polymorphisms (200G/A, 1220A/G and BclI RFLP) in 222 children with acute lymphoblastic leukemia (ALL) whose treatment protocols, among other components, contained corticosteroid drugs. RESULTS: The analysis of survival probabilities in relation to the indicated genotypes showed only an association between homozygosity for allele G of the NR3C1 BclI RFLP polymorphism and overall survival (univariate and multivariate hazard ratio [HR] 2.7, 95% confidence interval [CI] 1.0, 7.6 and 5.2, 95% CI 1.4, 18.9, respectively). The association reflects a correlation with disease progression and prognosis, and may vary depending on risk of relapse. CONCLUSION: A reduction in survival probability in children with ALL was associated with homozygosity for G allele of the NR3C1BclI RFLP polymorphism, particularly in certain patient subgroups. Further analysis is required to replicate this finding and to understand the mechanism underlying the observed association.

The influence of cyclin D1 (CCND1) 870A>G polymorphism and CCND1-thymidylate synthase (TS) gene-gene interaction on the outcome of childhood acute lymphoblastic leukaemia.

The 870A>G polymorphism in the cyclin D1 (CCND1) gene modulates mRNA splicing, leading to altered protein that may affect the regulation of the G1/S cell-cycle checkpoint. This polymorphism has been reported to influence susceptibility to and progression of several malignancies. Furthermore, the change of retinoblastoma protein regulation mediated by CCND1 may play a role in the development of methotrexate (MTX) resistance via an associated higher activity of enzymes that are inhibited by MTX. This study shows that children with acute lymphoblastic leukaemia (ALL) who are homozygous for the CCND1 A variant have a lower probability of event-free survival (P = 0.006) compared to carriers of the G variant. A significant result is retained in the presence of other prognostic factors. This impact is even more apparent in individuals who are also homozygous for thymidylate synthase (TS) triple repeat (P < 0.00005), which has previously been shown to influence the outcome of childhood ALL.

Polymorphism G80A in the reduced folate carrier gene and its relationship to methotrexate plasma levels and outcome of childhood acute lymphoblastic leukemia.

Methotrexate (MTX) is a key compound of chemotherapeutic regimens used in the treatment of childhood acute lymphoblastic leukemia (ALL). Resistance to this drug may arise by, among other factors, altered cellular uptake that may hamper the efficacy of the treatment. Recently, a G(80)A polymorphism has been described in the reduced folate carrier gene (RFC1), which encodes the major MTX transporter. Here, we assessed the association between the genetic polymorphisms G(80)A and both MTX plasma levels and childhood ALL outcome. Children with the A(80) variant had worse prognoses than patients with the GG genotype (P =.04), as shown by event-free survival estimates. Patients homozygous for A(80) had higher levels of MTX (P =.004) than the other genotype groups. Possible explanations for observed associations are discussed; however, additional experiments are required to achieve understanding of the underlying mechanism.