IBD5 Locus Research Articles

Inactivation of Interferon Regulatory Factor 1 Causes Susceptibility to Colitis-Associated Colorectal Cancer

The mechanisms linking chronic inflammation of the gut (IBD) and increased colorectal cancer susceptibility are poorly understood. IBD risk is influenced by genetic factors, including the IBD5 locus (human 5q31), that harbors the IRF1 gene. A cause-to-effect relationship between chronic inflammation and colorectal cancer, and a possible role of IRF1 were studied in Irf1-/- mice in a model of colitis-associated colorectal cancer (CA-CRC) induced by azoxymethane and dextran sulfate. Loss of Irf1 causes hyper-susceptibility to CA-CRC, with early onset and increased number of tumors leading to rapid lethality. Transcript profiling (RNA-seq) and immunostaining of colons shows heightened inflammation and enhanced enterocyte proliferation in Irf1−/− mutants, prior to appearance of tumors. Considerable infiltration of leukocytes is seen in Irf1−/− colons at this early stage, and is composed primarily of proinflammatory Gr1+ Cd11b+ myeloid cells and other granulocytes, as well as CD4+ lymphoid cells. Differential susceptibility to CA-CRC of Irf1−/− vs. B6 controls is fully transferable through hematopoietic cells as observed in bone marrow chimera studies. Transcript signatures seen in Irf1−/− mice in response to AOM/DSS are enriched in clinical specimens from patients with IBD and with colorectal cancer. In addition, IRF1 expression in the colon is significantly decreased in late stage colorectal cancer (stages 3, 4) and is associated with poorer prognosis. This suggests that partial or complete loss of IRF1 expression alters the type, number, and function of immune cells in situ during chronic inflammation, possibly via the creation of a tumor-promoting environment.

P145 INACTIVATION OF IRF1 CAUSES SUSCEPTIBILITY TO COLITIS-ASSOCIATED COLORECTAL CANCER

Colorectal cancer is a highly prevalent malignancy worldwide, with an increased incidence in patients diagnosed with inflammatory bowel disorders (IBD). Risk of IBD is strongly influenced by genetic factors, including the IBD5 locus (5q31), harboring the IRF1 gene. Our studies have shown that in the azoxymethane (AOM)/dextran sodium sulfate (DSS) murine model of colitis-associated colorectal cancer (CA-CRC), loss of interferon regulatory factor 1 (Irf1) results in enhanced tumorigenesis. Transcriptome profiling of AOM/DSS treated colons conducted early in the pathogenesis of CA-CRC showed heightened colonic inflammation and leukocyte infiltration in Irf1-/- mice compared to WT controls, well before the appearance of tumors. Immunoprofiling by flow cytometry revealed this to be a predominantly myeloid influx with an accumulation of proinflammatory Gr1+ Cd11b+ cells in the colons of mutant mice, as well as increases in CD3+ lymphoid cells and mast cell activation. Furthermore, CA-CRC experimentation in bone marrow chimeras proved that it was loss of Irf1 in the hematopoietic compartments that drove tumorigenesis. Finally, human correlative studies showed extensive overlap between genes upregulated in active ulcerative colitis lesions and AOM/DSS treated Irf1-/- mice, as well as decreased expression of IRF1 in later stage colorectal cancer, associated with poorer prognosis in these patients, establishing loss of IRF1 expression as a strong genetic signature to identify susceptible populations. The results from this study establish IRF1 as a major regulator of inflammatory response in situ in the gut, and as a major driver of colitis-associated colorectal cancer in both mice and humans.

Variations in the IBD5 locus confer the risk of inflammatory bowel disease in a Manitoban Caucasian Cohort

Variations in the IBD5 locus confer the risk of inflammatory bowel disease in a Manitoban Caucasian Cohort

Improved integrative framework combining association data with gene expression features to prioritize Crohn's disease genes.

Genome-wide association studies in Crohn's disease (CD) have identified 140 genome-wide significant loci. However, identification of genes driving association signals remains challenging. Furthermore, genome-wide significant thresholds limit false positives at the expense of decreased sensitivity. In this study, we explored gene features contributing to CD pathogenicity, including gene-based association data from CD and autoimmune (AI) diseases, as well as gene expression features (eQTLs, epigenetic markers of expression and intestinal gene expression data). We developed an integrative model based on a CD reference gene set. This integrative approach outperformed gene-based association signals alone in identifying CD-related genes based on statistical validation, gene ontology enrichment, differential expression between M1 and M2 macrophages and a validation using genes causing monogenic forms of inflammatory bowel disease as a reference. Besides gene-level CD association P-values, association with AI diseases was the strongest predictor, highlighting generalized mechanisms of inflammation, and the interferon-γ pathway particularly. Within the 140 high-confidence CD regions, 598 of 1328 genes had low prioritization scores, highlighting genes unlikely to contribute to CD pathogenesis. For select regions, comparably high integrative model scores were observed for multiple genes. This is particularly evident for regions having extensive linkage disequilibrium such as the IBD5 locus. Our analyses provide a standardized reference for prioritizing potential CD-related genes, in regions with both highly significant and nominally significant gene-level association P-values. Our integrative model may be particularly valuable in prioritizing rare, potentially private, missense variants for which genome-wide evidence for association may be unattainable.

Interaction between IL23R gene and IBD5 locus in Hungarian ulcerative colitis patients

In previous single-locus association studies, the IBD5 loci were found not to confer susceptibility to UC, while the rs2201841 and rs1004819 IL23R were not investigated in Hungarian UC patients. The aim of our current work was to study two IL23R SNPs (rs1004819 and rs2201841) in Hungarian UC population and to test for possible statistical interaction, stratifying the IL23R genotypes by IBD5 genotypes. 320 unrelated subjects with ulcerative colitis and 316 healthy controls were genotyped. Interactions and specific genotype combinations of a total of seven variants were tested. The variants of IBD5 locus (IGR2198a_1 rs11739135, IGR2096a_1 rs12521868, IGR2230a_1 rs17622208 and SLC22A5 rs2631367) and IL23R (rs1004819, rs2201841) gene were genotyped by PCR-RFLP, the SLC22A4 rs1050152 was determined by direct sequencing. For the IL23R rs1004819 A allele we found significantly higher allele frequency (P = 0.032) in UC patients compared to control subjects. The SNP rs1004819 showed significant association with UC risk for carriers (P = 0.004, OR = 1.606; 95% CI:1.160 – 2.223) and the SNP rs2201842 for homozygotes (P = 0.030, OR = 1.983; 95% CI:1.069 – 3.678). There was no evidence for statistical interaction between IBD5 loci and IL23R genes using logistic regression analysis. After genotype stratification, we could detect a positive association on the background of rs1004819 A allele for SLC22A4 T allele, SLC22A5 C, IGR2198a_1 C or IGR2096_a T allele, the highest OR was calculated in the presence of SLC22A4 T allele (P = 0.005, OR = 2.015; 95% CI:1.230 – 3.300). There was no association with UC for any combinations of rs1004819 and IGR2230a_1. The IL23R rs2201841 homozygous genotype and IBD5 carrier status together did not confer susceptibility for ulcerative colitis. The present study has shown that UC susceptibility genes are likely to act in a complex interactive manner similar to CD.

Susceptibility to ulcerative colitis in Hungarian patients determined by gene-gene interactions

To study the inflammatory bowel disease-5 locus (IBD5) and interleukin-23 receptor (IL23R) gene variants in UC patients and test for gene-gene interaction. The study population (n = 625) was comprised of 320 unrelated ulcerative colitis (UC) patients with Caucasian origin and 316 age- and gender-matched, healthy controls. Five variants in the IBD5 locus (IGR2198a_1 rs11739135, IGR2096a_1 rs12521868, IGR2230a_1 rs17622208, SLC22A4 rs1050152 and SLC22A5 rs2631367) and two of the IL23R gene (rs1004819, rs2201841) were analysed. PCR and restriction fragment length polymorphism methods were used for genotyping, the SLC22A4 rs1050152 genotypes were determined by direct sequencing. Interactions and specific genotype combinations of the seven variants were tested by binary logistic regression analysis. The IL23R genotypes were stratified by IBD5 genotypes for further interaction analyses. For the IL23R rs1004819 A allele we found significantly higher allele frequency (P = 0.032) in UC patients compared to control subjects. The SNP rs1004819 showed significant association with UC risk for carriers (P = 0.004, OR = 1.606; 95%CI: 1.160-2.223) and the SNP rs2201841 for homozygotes (P = 0.030, OR = 1.983; 95%CI: 1.069-3.678). Individually none of the IBD5 markers conferred risk to UC development. There was no evidence for statistical interaction either between IBD5 loci and IL23R genes using logistic regression analysis. After genotype stratification, we could detect a positive association on the background of rs1004819 A allele for SLC22A4 T, SLC22A5 C, IGR2198a_1 C or IGR2096a_1 T allele, the highest OR was calculated in the presence of SLC22A4 T allele (P = 0.005, OR = 2.015; 95%CI: 1.230-3.300). There was no association with UC for any combinations of rs1004819 and IGR2230a_1. The IL23R rs2201841 homozygous genotype and IBD5 carrier status together did not confer susceptibility for UC. The present study has shown that UC susceptibility genes are likely to act in a complex interactive manner similar to CD.

Functional Variations of Organic Cation Transporters associated to Inflammatory Bowel Disease (IBD) in a Manitoba Cohort

Polymorphisms in SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of IBD. This study sought to investigate the association of common and rare single nucleotide polymorphisms (SNPs) within those genes in Manitoba's IBD population. DNA samples from 160 patients with Crohn's Disease (CD), 149 patients with Ulcerative Colitis (UC) and 142 healthy controls were genotyped by PCR‐RFLP analysis. The IBD5 rs11739136‐C and rs17622208‐A alleles are associated with increased susceptibility to CD (OR=1.8, 95%CI 1.28–2.5, p=0.0006; OR=1.4, 95%CI 1.01–1.92, p=0.04, respectively). Moreover, rs11739136‐CC homozygosity increased the susceptibility of UC (OR=4.74, 95% CI 1.74–12.9, p=0.0009). Both CD (62%) and UC (64%) showed higher frequencies of SLC22A23 rs17309827 T allele than controls (61%).SLC22A4 rs1050152 showed strong Linkage Disequilibrium (LD) (D′ =0.98) with rs11739135. The risk alleles conform a haplotype (TACT) which is carried by 38.8% in CD patients and 29.8% in controls (p=0.02). We report that rs11739135 and rs17622208 best tag IBD5 haplotypes.Two selected rare polymorphisms in SLC22A4 (rs11568500 and rs11568510) were not detected in our population. Rare variations might be relevant in populations with different ethnical background. Further studies on the role of solute transporters in IBD are needed to develop therapeutic interventions.

Contribution of higher risk genes and European admixture to Crohnʼs disease in African Americans

African Americans (AAs) are an admixed population of West African (WA) and European ancestry (EA). Crohn's disease (CD) susceptibility genes have not been established. We therefore evaluated the contribution of European admixture and major established risk genes to AA CD. Ninety-seven admixture informative markers were genotyped for ancestry estimates using STRUCTURE. Overall, 354 AA CD cases and 354 ethnicity-matched controls were genotyped for total 21 single nucleotide polymorphisms (SNPs) in ATG16L1, NOD2, IBD5, IL23R and IRGM by TaqMan or direct sequencing. Association was evaluated by logistic regression, adjusted for ancestry. Mean EA was similar among the CD cases and controls (20.9% and 20.4%, respectively, P = 0.58). No significant admixture differences were observed among 211 to 227 cases stratified by phenotypic subclassifications including onset, surgery, site, and behavior. CD was associated with NOD2 carrier (6.93% CD, 2.15% Controls, P = 0.007), ATG16L1 Thr300Ala (36.1% CD, 29.3% Controls, P = 0.003), SLC22A4 and SLC22A5 (IBD5 locus) functional SNPs (Leu503Phe [10.5% CD, 7.6% Controls, P = 0.05] and g-207c [41.3% CD, 35.7% Controls, P = 0.03], respectively), and IL23R rs2201841 (18.2% CD, 13.8% Controls, P = 0.03), but not IRGM variants, nor three African ancestral NOD2 nonsynonymous variants. IBD5 risk was recessive. An all-minor allele IBD5 haplotype from EA was associated (P = 0.05), whereas a more common haplotype isolating g-207c was not. Specific functional gene variations contribute significantly to AA CD risk. Established NOD2, SLC22A4-A5, and ATG16L1 variants show increased CD risk, with IBD5 recessive. Although CD is more common in whites, European admixture is similar among AA cases and controls.

Expression and functional analysis of intestinal organic cation/l-carnitine transporter (OCTN) in Crohn's Disease

BackgroundThe IBD5 locus is a genetic risk factor for IBD, particularly Crohn's Disease, coding for the organic cation/carnitine transporters (OCTN1 and 2). Two variants of OCTN are associated with susceptibility to Crohn's Disease. Modified transport of carnitine in vitro has been reported for a polymorphism of OCTN1. The aim was to investigate the function of intestinal OCTNs in IBD in relation to genetic polymorphisms. MethodsIntestinal tissue was obtained from endoscopic biopsies and surgical resections from IBD patients (n=33 and 14, resp.) and controls (n=22 and 14, resp.). OCTN protein levels were measured in intestinal biopsies and carnitine transport was quantified in intestinal resections. ResultsOCTN1 protein levels were significantly higher in ileal versus colonic tissue (2.95%±0.4 vs 0.66%±0.2, resp.; p<0.0002). OCTN1 expression was higher in Crohn's disease patients with mutant homozygous or heterozygous genotypes (0.6%±0.1 vs 3%±0.8, resp., p<0.02). Carnitine transport was very rapid and Na+ dependent (10s). It was not different comparing Crohn's Disease and control groups (0.45±0.12 vs 0.51±0.12nM carnitine/mg prot/min, resp.). Carnitine transport tended to be higher in subjects with mutant homozygous and heterozygous OCTN1 and OCTN2 genotypes (0.19 vs 0.59 and 0.25 vs 0.6, respectively). ConclusionsThe present data reveal that OCTN protein levels appear to be similar in intestinal tissue from Crohn's Disease patients and controls. Overall, ileal carnitine transport appears to as well equal in Crohn's Disease and control groups. However, there was a trend towards higher carnitine transport in subjects with OCTN1 and OCTN2 mutations.

A review of major Crohn’s disease susceptibility genes and their role in disease pathogenesis

Crohn’s disease (CD) is a chronic inflammatory bowel disease whose relevance is increasing in industrialized society. Recent genome wide association studies revealed over seventy one loci associated with disease penetrance. Several variants that increase disease risk encode for altered proteins that diminish bacterial host defense. NOD2 alters intracellular bacterial sensing while ATG16L1 is thought to diminish bacterial clearance by impairing autophagy. Additionally, changes in the IBD5 locus are thought to diminish barrier function. Alternatively, recent data indicate a gain of function genetic variant of IL23R is protective amongst European CD patients. These recent genetic discoveries contradict historical theories that Crohn’s disease results from overactive auto-aggressive responses. Rather, new genetic data suggest disease-associated variants encode for dysfunctional proteins that diminish essential innate immune responses against commensal organisms. This review provides an overview of these critical discoveries and places them in their biological context.

Haplotype in the IBD5 region is associated with refractory Crohn’s disease in Slovenian patients and modulates expression of the SLC22A5 gene

The IBD5 locus (OMIM ID606348) on chromosome 5 was suggested to be one of the most important genetic factors involved in the pathogenesis of inflammatory bowel diseases (IBDs). However the main contributor from this region is still unknown. We investigated the possible association of the IBD5 locus with IBD in Slovenian patients and correlation between disease-associated single nucleotide polymorphisms (SNPs) and quantitative gene expression (eQTL) of candidate genes from the IBD5 locus in peripheral blood lymphocytes and colon tissue biopsies from IBD patients. We genotyped SNPs from the IBD5 locus in 312 healthy controls and 632 IBD patients. We found statistically significant association of polymorphisms rs1050152 in gene SLC22A4 (p=0.005, OR=2.177, 95% CI=1.270-3.526) and rs2631372 in gene SLC22A5 (p=0.001, OR=0.473, 95% CI=0.307-0.731) and TC haplotype of both polymorphisms (p=0.006, OR=1,541, 95% CI=1.130-2.100) with refractory Crohn's disease (CD) in Slovenian patients who do not respond to standard therapy, including patients who develop fistulas. We found decreased expression of SLC22A4 and SLC22A5 genes in peripheral blood lymphocytes from IBD patients compared to control group and decreased expression of SLC22A5 gene in inflamed tissue biopsies compared to noninflamed colon (p=0.009). We found lower expression of SLC22A5 gene in IBD patients with disease-susceptible genotypes for both disease-associated SNPs. Our data suggest that SNPs and haplotype in the IBD5 SLC22A4/SLC22A5 region contribute to the development of particularly refractory Crohn's disease in the Slovenian population, and expression studies in blood lymphocytes and colon tissue biopsies and eQTL analysis suggest that SLC22A5 is the main gene in the IBD5 region contributing to the IBD pathogenesis.

Interaction between CTLA4 gene and IBD5 locus in Hungarian Crohn’s disease patients

The IGR2198a_1 and IGR2096a_1 variants of the IBD5 region were found to be associated with Crohn's disease (CD) in the Hungarian population, while IGR2230a_1 does not seem to confer risk for the disease. In the present study, our aim was to investigate the statistical interaction of these three IBD5 polymorphisms with the +49 A/G substitution within the cytotoxic T lymphocyte antigen-4 (CTLA4) gene, detected previously as neutral gene variant in Hungarian IBD patients. A total of 305 unrelated subjects with CD and 310 healthy controls were genotyped with PCR-RFLP methods. In contrast with single gene effects, after genotype stratification, the IGR2198a_1 C and IGR2096a_1 T variants were found to confer susceptibility only in subjects with CTLA4 +49 AA genotype (P = 0.008; OR = 1.86 and P = 0.016; OR = 1.74, respectively), for IGR2230a_1 no such effect on disease risk could be demonstrated. Analysis of specific genotype combinations unfolded a possible association between the CTLA4 +49 A/G substitution and two of the observed IBD5 variants with respect to disease risk.

Contribution of the IBD5 locus to inflammatory bowel disease: a meta-analysis

To evaluate the association of the IBD5 locus to the predisposition of inflammatory bowel diseases (IBDs), a series of meta-analyses between five IBD5 variants (OCTN1 C1672T, OCTN2 G-207C, OCTN1/2 TC haplotype, IGR2096a_1, IGR2198a_1 and IGR2230a_1) and Crohn's disease (CD) and ulcerative colitis (UC) were performed, which included a total of 26 studies. Overall, five IBD5 variants in a per-allele model of inheritance were significantly associated with elevated CD risk (for OCTN1: OR=1.23, 95% CI=1.16-1.30, P<0.001; for OCTN2: OR=1.20, 95% CI=1.11-1.30, P<0.001; for IGR2096a_1: OR=1.36, 95% CI=1.24-1.46, P<0.001; for IGR2198a_1: OR=1.34, 95% CI=1.24-1.46, P<0.001; for IGR2230a_1: OR=1.35, 95% CI=1.23-1.48, P<0.001) and OCTN1/2 TC haplotype (OR=1.32, 95% CI=1.22-1.43, P<0.001). In the subgroup analysis, the statistically significant associations were also observed in adult- and pediatric-onset CD and in Caucasians for five IBD5 variants and the OCTN1/2 TC haplotype. A statistically significant increase in the risk of UC was detected in a recessive model of inheritances for OCTN1 (OR=1.23, 95% CI=1.08-1.40, P<0.001), OCTN2 (OR=1.18, 95% CI=1.05-1.33, P=0.006), IGR2096a_1 (OR=1.37, 95% CI=1.15-1.62, P<0.001) and IGR2198a_1 (OR=1.35, 95% CI=1.10-1.66, P=0.004); the increased risks of UC were maintained in the adult and Caucasian subgroups, but not the pediatric subgroup. In summary, our results suggested that the IBD5 locus contributes to the susceptibility of CD in a per-allele manner in adults, children and Caucasians, and the locus contributes to the susceptibility of UC in a recessive manner in adult and Caucasian populations.

Two independent genetic factors responsible for the associations of the IBD5 locus with Crohnʼs disease in the Czech population

The role of the IBD5 locus in development of Crohn's disease (CD) has not been clarified. In the Czech population we examined its genetic association using variants of the SLC22A4 (rs1050152), SLC22A5 (rs2631367), two single nucleotide polymorphisms (SNPs) shown to be associated with CD in genome-wide studies (rs6596075 and rs2188962), and four SNPs previously shown to tag the haplotype blocks 4, 7, 9, 10 of the IBD5 locus (IGR2063b_1, IGR2230a_1, IGR100Xa_1, IGR3236a_1). The genotype, phenotype, and allelic frequencies were compared between 469 unrelated patients with CD (177 pediatric-onset, 292 adult-onset) and 470 unrelated healthy controls, all Caucasians of Czech ancestry. The most significant difference between patients and controls was found for the SNP rs6596075 (odds ratio [OR] = 0.70 for the G allele; 95% CI 0.52-0.94) in the dominant model and SNP IGR2063b_1 (OR = 1.38 for the G allele; 95% CI 1.14-1.67) in the log-additive model. We found a strong linkage disequilibrium across the IBD5 locus except rs6596075. The haplotype consisting of minor alleles of all tested SNPs except rs6596075 was carried by 31% patients and 23% control subjects (OR = 1.35, 95% CI 1.06-1.72). The association of variants in SLC22A4 and SLC22A5 was dependent on this risk haplotype, while the strong association of the rs6596075 was seemingly independent. In the analysis of subphenotypes we found only an association of the penetrating disease with rs6596075 (OR = 2.13; 95% CI 1.31-3.47). Our study confirms the importance of IBD5 in determining CD susceptibility, and demonstrates that two independent genetic factors may be responsible for the association observed within this locus.

PWE-004 AK155: a candidate gene for inflammatory bowel disease

<h3>Introduction</h3> AK155 (interleukin-26) is a member of the “IL-10 superfamily”. It maps to the IBD2 locus (chromosome 12q15). AK155 is expressed by Th1 and Th17 lymphocytes. The receptor is expressed on epithelial cells, including colon carcinoma and keratinocyte-derived cell lines.1 Activation of the receptor complex results in secretion of IL-10 and IL-18 with surface expression of CD54.1 AK155 mRNA expression is elevated in Crohn9s colitis. This expression correlates with IL-8 and IL-22.2 <h3>Methods</h3> We sequenced AK155 from genomic DNA (gDNA) of patients with IBD to identify novel disease-associated single nucleotide polymorphisms (SNPs). These were then used for allele association studies. gDNA was obtained from peripheral blood mononuclear cells of 30 IBD patients (15 UC and 15 CD). Combinations of primers were used which amplified the open reading frame, introns 1, 2 and 4, 35 base pairs of the 59UTR immediately proximal to the start codon, 115 bp of intron 3 and 124 bp of the 39UTR beyond the stop codon. PCR products were sequenced using the ABI 377 automated sequencer. <h3>Results</h3> Four novel SNPs were identified. Each SNP was found in a single individual from the 30 patients studied. The first coding SNP (C87T) in exon one was silent. The second coding SNP (C316T) in exon three lead to a substitution in the amino acid sequence of AK155 of an arginine residue to a cysteine. The other two SNPs were identified within introns two and three. The C316T SNP polymorphism was used for an allelic association study in IBD. gDNA samples were obtained from 130 IBD patients and 50 controls. The C316T SNP was identified in one of the 130 patients and no control. This patient was a 38-year-old woman with Crohn9s colitis. C316T is in proximity to the intron two SNP but again the intronic SNP was identified in a single individual from the 180 samples. Each SNP was checked on five separate occasions using different oligonucleotide primer combinations and DNA polymerase enzymes. It was identified with each of these sequencing primers and polymerase combinations. <h3>Conclusion</h3> AK155 is both a positional and functional candidate gene for IBD. We were unable to identify a disease-associated polymorphism. There are limitations to the techniques used to perform our association studies. We are limited by the numbers of patients and genetically heterogeneous nature of our IBD population. We believe these SNPs should be studied in large numbers of multiply affected families.

Interaction of the major inflammatory bowel disease susceptibility alleles in Crohn’s disease patients

To investigate the interaction of interleukin-23 receptor (IL23R) (rs1004819 and rs2201841), autophagy-related 16-like 1 (ATG16L1) (rs2241880), caspase recruitment domain-containing protein 15 (CARD15) genes, and IBD5 locus in Crohn's disease (CD) patients. A total of 315 unrelated subjects with CD and 314 healthy controls were genotyped. Interactions and specific genotype combinations of a total of eight variants were tested. The variants of IBD5 locus (IGR2198a_1 rs11739135 and IGR2096a_1 rs12521868), CARD15 (R702W rs2066845 and L1007fs rs2066847), ATG16L1 (rs2241880) and IL23R (rs1004819, rs2201841) genes were genotyped by PCR-RFLP, the G908R (rs2066844) in CARD15 was determined by direct sequencing. The association of ATG16L1 T300A with CD was confirmed [P = 0.004, odds ratio (OR) = 1.69, 95% CI: 1.19-2.41], and both IL23R variants were found to represent significant risk for the disease (P = 0.008, OR = 2.05, 95% CI: 1.20-3.50 for rs1004819 AA; P < 0.001, OR = 2.97, 95% CI: 1.65-5.33 for rs2201841 CC). Logistic regression analysis of pairwise interaction of the inflammatory bowel disease (IBD) loci indicated that IL23R, ATG16L1, CARD15 and IBD5 (IGR2198a_1) contribute independently to disease risk. We also analysed the specific combinations by pair of individual ATG16L1, IL23R rs1004819, rs2201841, IGR2198a_1, IGR2096a_1 and CARD15 genotypes for disease risk influence. In almost all cases, the combined risk of susceptibility pairs was higher in patients carrying two different risk-associated gene variants together than individuals with just one polymorphism. The highest OR was found for IL23R rs2201841 homozygous genotype with combination of positive CARD15 status (P < 0.001, OR = 9.15, 95% CI: 2.05-40.74). The present study suggests a cumulative effect of individual IBD susceptibility loci.

Plasma carnitine ester profiles in Crohn's disease and ulcerative colitis patients with different IGR2230a_1 genotypes

An association has been repeatedly demonstrated between inflammatory bowel disease (IBD) and the IBD5 locus in the 5q31 chromosomal region. The aim of the present study was to examine the prevalence of the IGR2230a_1 intronic nucleotide polymorphism of the slc22a5 gene (coding for the OCTN2 carnitine transporter protein) lying within this region, and its possible relationship with the carnitine metabolism in Hungarian IBD patients and controls. We genotyped by restriction fragment length polymorphism 200 Crohn's disease (CD) and 246 ulcerative colitis (UC) patients, as well as 187 healthy controls. From plasma samples we determined detailed carnitine ester profiles of 76 CD, 43 UC patients and 45 control persons using electrospray ionization triple quadruple tandem mass spectrometry. The distribution of the genotypes was not significantly different in the CD or the UC group compared with the controls. We found no significant alterations of the carnitine profile in the carrier/non-carrier or the homozygote/non-homozygote comparisons in both the CD and the UC groups, stratified by IGR2230a_1 genotype. Our data suggest that this polymorphism alone is not associated with CD and UC in the Hungarian population, and has no effect on the carnitine metabolism.

Interleukin‐27 polymorphisms are associated with inflammatory bowel diseases in a Korean population

The cytokine interleukin (IL)-27 is composed of two subunits, Epstein-Barr virus-induced gene 3 (EBI3) and p28, and IL-27 is a novel IL-12 family member that mediates between the innate and adaptive immune systems. We previously identified four polymorphisms in the human IL-27 gene and we suggested that the polymorphism of IL-27 is associated with the susceptibility to asthma. IL-27 transcripts are significantly elevated in active Crohn's disease (CD) but not in ulcerative colitis (UC). To determine whether these IL-27 single nucleotide polymorphisms are associated with the susceptibility to inflammatory bowel disease (IBD), the genotype and allelic frequencies of the IL-27 polymorphisms were analyzed between the IBD patients and the healthy controls. Genotype analysis of the IL-27 gene was performed by the single-base extension (SBE) method. The haplotype frequencies of IL-27 for multiple loci were estimated using the expectation maximization (EM) algorithm. The genotype frequencies of the g.-964A > G polymorphism in the IBD patients were significantly different from those of the healthy control group (P = 0.001). In both the UC and CD patients, the genotype frequencies of the g.-964A > G polymorphism were also significantly different from the frequencies of the healthy control group (P = 0.009). The frequencies of the AGT and GGT haplotypes were significantly different between the healthy control group and the IBD patient group (P = 0.00004 and 0.021, respectively). Our results suggest that the g.-964A > G polymorphism of the IL-27 gene located on the IBD1 locus might be associated with the susceptibility to IBD.

Possible role of selected IGR and SLC22A4/SLC22A5 loci in development of inflammatory bowel diseases

The IBD5 locus (MIM#606348) on chromosome 5q31 has been demonstrated to confer increased risk for inflammatory bowel disease. Controversial reports have been published about the significance of individual loci located in this region. Here we investigated the possible genetic association of inflammatory bowel diseases with C1672T of SLC22A4 and G-207C SLC22A5 alleles, and with IGR2096a_1 (rs12521868) and IGR2198a_1 (rs11739135) susceptibility variants of the IBD5 region located on chromosome 5q31. Total of 440 patients, 206 with Crohn's disease, 234 with ulcerative colitis, and 279 controls were studied by PCR-RFLP methods. Neither the C1672T, and G-207C alleles, nor the TC haplotype were found to confer risk for Crohn's disease or ulcerative colitis. By contrast, both of the minor allele frequencies of IGR2096a_1 T (48.1%) and IGR2198a_1 C (46.1%) were increased in Crohn's disease subjects as compared with the controls (38.5% and 38.4%, respectively; p<0.05). Using regression analysis adjusted to age and gender these alleles were found to confer risk for Crohn's disease (OR=1.694, 95% CI: 1.137-2.522; p=0.010 for T allele, OR=1.644, 95% CI=1.103-2.449; p=0.015 for C allele of IGRs). In UC no such associations were found. Our results revealed the susceptibility nature of the examined IGR minor alleles in Hungarians, which nation differs historically from the surrounding Caucasian populations in origin of the founders of the state.

Epistasis Between Toll-Like Receptor-9 Polymorphisms and Variants in NOD2 and IL23R Modulates Susceptibility to Crohn's Disease

Recent data suggest functional interactions between NOD2 and other receptors of the innate immune system modulating inflammatory responses. Here we analyzed the role of Toll-like receptor 9 (TLR-9) gene variants with respect to susceptibility to inflammatory bowel disease (IBD) and tested for genetic interactions with NOD2 and other susceptibility genes for Crohn's disease (CD). The single-nucleotide polymorphisms (SNPs) -1237T/C (rs5743836) and 2848A/G (rs352140=p.Pro545Pro) in TLR9, the main CD-associated variants within the genes for NOD2, IL23R, ATG16L1, and variants in the IBD5 locus and in the DLG5 gene were assessed in 956 patients with IBD (606 CD and 350 ulcerative colitis) and in 792 healthy controls. The associations with disease susceptibility and phenotype, and epistatic gene-gene interactions, were analyzed. The TLR9 -1237T/C polymorphism showed significant interactions with NOD2 mutations. The frequency of -1237C was significantly higher in CD patients with at least one NOD2 mutation (P=0.004 vs. controls, odds ratio (OR) 1.60, 95% confidence interval (CI) (1.15-2.21)) and further increased in CD patients with two mutated NOD2 alleles (P=0.002 vs. controls, OR 2.37, 95% CI (1.35-4.15)). Significant gene-gene interactions were also observed for the TLR9 polymorphism -1237T/C with IL23R variants (most significantly with rs1004819, P=0.0007), with a particular high frequency of -1237C in CD patients carrying CD-protective IL23R variants. Epistatic interactions of the TLR9 -1237T/C SNP were also noted with the DLG5 113G/A variant (P=0.0007). Our results provide evidence for genetic interactions between polymorphisms in TLR9 and CD-associated variants in NOD2, IL23R, and DLG5, differentially modulating CD susceptibility.