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The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohn's disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohn's disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohn's disease. Here we report that, in human epithelial cells, the Crohn's disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohn's disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy.

Genetic susceptibility is an important contributor to the pathogenesis of Crohn's disease (CD). We investigated multiple CD susceptibility genes in an Australian paediatric onset CD cohort. Newly diagnosed paediatric onset CD patients (n = 72) and controls (n = 98) were genotyped for 34 single nucleotide polymorphisms (SNPs) in 18 genetic loci. Gene-gene interaction analysis, gene-disease phenotype analysis and genetic risk profiling were performed for all SNPs and all genes. Of the 34 SNPs analysed, four polymorphisms on three genes (NOD2, IL23R, and region 3p21) were significantly associated with CD status (p<0.05). All three CD specific paediatric polymorphisms on PSMG1 and TNFRSF6B showed a trend of association with p<0.1. An additive gene-gene interaction involving TLR4, PSMG1, TNFRSF6B and IRGM was identified with CD. Genes involved in microbial processing (TLR4, PSMG1, NOD2) were significantly associated either at the individual level or in gene-gene interactive roles. Colonic disease was significantly associated with disease SNP rs7517847 (IL23R) (p<0.05) and colonic and ileal/colonic disease was significantly associated with disease SNP rs125221868 (IBD5) and SLC22A4 & SLC22A4/5 variants (p<0.05). We were able to demonstrate genetic association of several genes to CD in a paediatric onset cohort. Several of the observed associations have not been reported previously in association with paediatric CD patients. Our findings demonstrate that CD genetic susceptibility in paediatric patients presents as a complex interaction between numerous genes.

Background & Aims

The identification of numerous genes that confer susceptibility to Crohn’s disease (CD) indicates that this complex disease might arise from alterations in several genes with related functions. We examined the functional interaction between the CD risk genes autophagy-related 16-like protein 1 (ATG16L1) and nucleotide-binding oligomerization domain 2 (NOD2) to identify an autophagy-dependent pathway that is altered by disease-associated variants.

Methods

We assessed Nod2 signaling and autophagy activation in response to muramyl dipeptide (MDP) by immunoblot, confocal microscopy, flow cytometry, reporter gene, and gentamycin protection assays in human epithelial cell lines and primary human macrophages and dendritic cells from healthy individuals. The requirement of Nod2 and ATG16L1 expression and the effects of CD-associated variants in MDP-stimulated autophagy and Nod2-dependent signaling were assessed in cell lines manipulated by RNA interference, inhibitors, or ATG16L1 or NOD2 variants and in primary macrophages and dendritic cells from healthy, genotyped donors.

Results

MDP stimulation of epithelial cells, macrophages, and dendritic cells activated autophagy and NF-κB and MAPK signaling; it also increased killing of Salmonella. These responses depended on ATG16L1 and Nod2 expression and were impaired by CD-associated NOD2 variants. Nod2-dependent signaling was not impaired in cells with the ATG16L1 T300A genotype, which is associated with CD. However, the ATG16L1 T300A variant blocked the increase in MDP-mediated killing of Salmonella only in epithelial cell lines and not primary macrophages or dendritic cells.

Conclusions

ATG16L1 and NOD2 are components of an autophagy-mediated, anti-bacterial pathway that is altered in a cell- and function-specific manner by CD-associated mutations.

AIM: To evaluate the association of the autophagy-related 16-like 1 (ATG16L1) T300A polymorphism (rs2241880) with predisposition to inflammatory bowel diseases (IBD) by means of meta-analysis.

METHODS: Publications addressing the relationship between rs2241880/T300A polymorphism of ATG16L1 and Crohn’s disease (CD) and ulcerative colitis (UC) were selected from the MEDLINE and EMBASE databases. To make direct comparisons between the data collected in these studies, the individual authors were contacted when necessary to generate a standardized set of data from these studies. From these data, odds ratio (OR) with 95% confidence interval (CI) were calculated.

RESULTS: Twenty-five studies of CD were analyzed, 14 of which involved cases of UC. The variant G allele of ATG16L1 was positively associated with CD (OR = 1.32, 95% CI: 1.26-1.39, P < 0.00001) and UC (OR = 1.06, 95% CI: 1.01-1.10, P = 0.02). For child-onset IBD, a higher G allele frequency was found for cases of CD (OR = 1.35, 95% CI: 1.16-1.57, P = 0.0001) than for cases of UC (OR = 0.98, 95% CI: 0.81-1.19, P = 0.84) relative to controls.

CONCLUSION: The ATG16L1 T300A polymorphism contributes to susceptibility to CD and UC in adults, but different in children, which implicates a role for autophagy in the pathogenesis of IBD.

AIM: To investigate the frequency of NOD2, IL23R and ATG16L1 genetic variants in a case-control panel for inflammatory bowel disease (IBD) from Lithuania.

METHODS: One hundred and eighty unrelated IBD patients [57 Crohn’s disease (CD) and 123 ulcerative colitis (UC)] and 186 healthy controls were genotyped for the following known genetic susceptibility variants: NOD2 - Arg702Trp (rs2066844), Gly908Arg (rs2066845) and Leu1007insC (rs2066847), as well as IL23R - Arg381Gln (rs11209026) and ATG16L1 - Thr300Ala (rs2241880).

RESULTS: The effect that carriership of at least one NOD2 risk allele predisposes to CD was replicated in the Lithuanian population (41.1% CD vs 16.9% controls, P = 2 × 10-4, OR = 3.48, 95% CI: 1.81-6.72). In the allelic single marker analysis, Leu1007insC was strongly associated with CD (21.4% CD vs 4.7% controls, P = 3.687 × 10-8, OR = 5.54, 95% CI: 2.85-10.75). Neither the other two NOD2 variants, nor the known variants in IL23R and ATG16L1 were found to be risk factors for CD, UC or IBD. However, our relatively small study population was underpowered to demonstrate such weak to moderate disease associations.

CONCLUSION: The results support a strong association between CD susceptibility and the Leu1007insC variant in NOD2 in the Lithuanian study population.

Susceptibility to Crohn's disease (CD), a complex inflammatory disease involving the small intestine, is controlled by up to 32 loci1. One CD risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG162. It is not known how Atg16L1 or autophagy contributes to intestinal biology or CD pathogenesis. To address these questions we generated and characterized mice that are hypomorphic for Atg16L1 protein expression, and validated conclusions based on studies in these mice by analyzing intestinal tissues that we collected from CD patients carrying the CD risk allele of ATG16L1. We show that Atg16L1 is a bona fide autophagy protein. Within the ileal epithelium, both Atg16L1 and a second essential autophagy protein Atg5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell which functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment3. Atg16L1 and Atg5-deficient Paneth cells exhibited striking abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to Atg16L1-deficient Paneth cells including increased expression of genes involved in PPAR signaling and lipid metabolism, acute phase reactants, as well as two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, CD patients homozygous for the ATG16L1 CD risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy protein-deficient mice and expressed increased levels of leptin protein. Thus, Atg16L1, and likely the process of autophagy, play their role within the intestinal epithelium of mice and CD patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.

Background

Crohn's disease (CD) has the highest prevalence among individuals of Ashkenazi Jewish (AJ) descent compared to non-Jewish Caucasian populations (NJ). We evaluated a set of well-established CD-susceptibility variants to determine if they can explain the increased CD risk in the AJ population.

Methods

We recruited 369 AJ CD patients and 503 AJ controls, genotyped 22 single nucleotide polymorphisms (SNPs) at or near 10 CD-associated genes, NOD2, IL23R, IRGM, ATG16L1, PTGER4, NKX2-3, IL12B, PTPN2, TNFSF15 and STAT3, and assessed their association with CD status. We generated genetic scores based on the risk allele count alone and the risk allele count weighed by the effect size, and evaluated their predictive value.

Results

Three NOD2 SNPs, two IL23R SNPs, and one SNP each at IRGM and PTGER4 were independently associated with CD risk. Carriage of 7 or more copies of these risk alleles or the weighted genetic risk score of 7 or greater correctly classified 92% (allelic count score) and 83% (weighted score) of the controls; however, only 29% and 47% of the cases were identified as having the disease, respectively. This cutoff was associated with a >4-fold increased disease risk (p < 10e-16).

Conclusions

CD-associated genetic risks were similar to those reported in NJ population and are unlikely to explain the excess prevalence of the disease in AJ individuals. These results support the existence of novel, yet unidentified, genetic variants unique to this population. Understanding of ethnic and racial differences in disease susceptibility may help unravel the pathogenesis of CD leading to new personalized diagnostic and therapeutic approaches.

BACKGROUND/OBJECTIVE:

Variants in the interleukin-23 receptor (IL23R) and the autophagy-related 16-like 1 (ATG16L1) genes have been associated with an increased risk of Crohn’s disease (CD). Both genes were identified through genome-wide association scans and subsequent studies have validated these associations. To assess the effect size of these variants, an independent case-control association study and meta-analysis were performed.

METHODS:

British Caucasian subjects with inflammatory bowel disease (n=500) and 877 ethnically matched controls were genotyped for the disease-associated variants in IL23R and ATG16L1. In addition, meta-analyses of 12,991 patients and 14,598 controls, and 11,909 patients and 15,798 controls, were conducted on independently published data for the associations between IL23R and ATG16L1 variants and CD, respectively.

RESULTS:

In the present cohort, both susceptibility variants showed highly significant associations, including IL23R (rs11209026, P=0.0006; OR 0.37; 95% CI 0.21 to 0.67) and ATG16L1 (rs2241880, P=0.0017; OR 1.36; 95% CI 1.12 to 1.66). The meta-analysis based on the random effects model showed similar combined effects for rs11209026 (n=26, OR 0.41; 95% CI 0.37 to 0.46) and rs2241880 (n=25, OR 1.33; 95% CI 1.28 to 1.39). There was no statistically significant gene-gene interaction between caspase recruitment domain (CARD15) variants and the IL23R or ATG16L1 polymorphisms (P=0.44 and P=0.24, respectively).

CONCLUSION:

The present cohort and meta-analysis provides strong evidence that, in addition to CARD15, polymorphisms in both IL23R and ATG16L1 alter susceptibility to CD and that these effects are consistent across all populations of European ancestry; however, only ATG16L1 is relevant to inflammatory bowel disease in the Asian population.

Recently identified genetic determinants for enhanced susceptibility to Crohn’s disease (CD) included polymorphisms in the ATG16L1 and IRGM1 loci suggesting that the autophagy pathway plays a role in the pathogenesis of this disease. We have generated and analyzed three mouse models with diminished expression of autophagy proteins and show how the loss of function of various autophagy components contributes to CD pathogenesis. In the mouse small intestine, the common cellular target of Atg16L1, Atg5, and Atg7 is the Paneth cell, a specialized epithelial cell whose main function is the delivery of antimicrobial factors into the intestinal lumen by production and secretion of its characteristic cytoplasmic granules. Autophagy-deficient Paneth cells exhibited a striking loss of function in this granule exocytosis pathway. Transcriptional analysis revealed a gain of function whereby the gene expression associated with inflammatory responses was increased in autophagy-deficient Paneth cells. Importantly, we validated these findings by analyzing intestinal tissues from CD patients. Similar Paneth cell abnormalities were observed in CD patients homozygous for the ATG16L1 risk allele. Thus, one role for the autophagy pathway in CD pathogenesis is through selective effects on the biology and specialized properties of Paneth cells.

Background

IBD5, IL23R, and ATG16L1 genetic variations are established Crohn’s disease (CD) risks alleles. We evaluated these in a population-based case-control study within a cohort to determine their penetrance, population attributable risk, independence, and relationship to other established CD risk factors, including NOD2.

Methods

DNA from 213 CD, 118 ulcerative colitis, and 315 healthy control subjects from the population-based University of Manitoba IBD Research Registry were genotyped for IBD5 and IL23R single-nucleotide polymorphisms (SNPs), and for the Thr300Ala ATG16L1 SNP. Univariate and multivariate analyses were performed for these and nongenetic risk factors. We introduce multidimensionality reduction (MDR) to explore gene– gene interactions.

Results

ATG16L1, IBD5, and IL23R SNPs were significantly associated with CD. Multivariate analysis showed independent CD association for carriers of ATG16L1 (odds ratio [OR] = 1.8, 95% confidence interval [CI] 1.09–3.24), IBD5-IGR2230 (OR = 2.16, 95% CI 1.30–3.59), and IL23R-rs10889677 (OR = 2.13, 95% CI 1.39–3.28) while retaining association for NOD2 mutation carriers (OR = 4.45, 95% CI 2.68–7.38), IBD family history (OR = 2.75, 95% CI 1.42–5.31), tobacco (OR = 2.06, 95% CI 1.35–3.14), and Jewish ethnicity (OR = 20.1, 95% CI 2.16–186.8). IL23R minor variants for Arg381Gln and Intron 6 rs7517848 showed independent, CD protection and 3′ untranslated variant rs108896778 showed risk. MDR analysis suggested an interaction between IBD5, ATG16L1, and IL23R risk alleles. Penetrance values for ATG16L1 and IBD5 were 0.27% for heterozygotes, and 0.35% and 0.44%, respectively, for homozygotes. IL23R rs108896778 penetrance was 0.37%.

Conclusions

A population-based analysis of CD risk factors is useful for characterizing the epidemiology of multiple CD genetic and nongenetic risk factors. Gene– gene interactions are likely, but require further evaluation in large population-based cohorts.

Background: Variants of the caspase activating recruitment domain 15/nucleotide oligomerisation domain 2 (CARD15/NOD2) gene have been associated with susceptibility to Crohn’s disease (CD).

Aim: Our aim was to evaluate the allele frequencies of the CARD15 variants R702W, G908R, and 1007fs in Finnish inflammatory bowel disease (IBD) patients and to search for possible associations between CARD15 variants and occurrence of familial forms of IBD or complicated forms of CD.

Patients and methods: We investigated 198 sporadic CD patients, 46 probands with familial CD, 27 CD probands from mixed IBD families, 99 unrelated patients with ulcerative colitis (UC), and 300 control individuals for the occurrence of the CARD15 gene variants R702W, G908R, and 1007fs.

Results: In CD patients, the allele frequencies for the rare variants of these polymorphisms were 3.3%, 0.6%, and 4.8% (total 8.7%), and the corresponding frequencies in healthy controls were 1.8%, 0%, and 1.7% (total 3.5%) (8.7% v 3.5%; p<0.01). In UC patients allele frequencies were comparable with those in controls. The frequency of the 1007fs polymorphism variant allele was significantly higher among all CD patients than in controls (4.8% v 1.7%; p<0.01) but there was no significant difference in allele frequencies between the CD and UC groups. The 1007fs allele frequency was higher in familial CD than in non-familial cases with CD (10.9% v 3.5%; p<0.01). There were no significant differences in the allele frequencies of the R702W and G908R polymorphisms between CD patients, UC patients, and controls. We found that 15.5% of CD patients, 9.1% of UC patients, and 6.7% of controls carried at least one of the CARD15 variants. In CD patients carrying at least one of the three NOD2 variants, the ileum was affected more often than in non-carrier CD patients (90% v 73%; p<0.05), they had stricturing or penetrating disease more often than non-carriers (88% v 56%; p<0.01), and they had an increased need for bowel surgery.

Conclusions: The frequency of NOD2 gene variants was lower in genetically homogenous Finns than in other populations. The 1007fs variant was associated with CD. The occurrence of CARD15 variants predicted ileal location as well as stricturing and penetrating forms of CD.

AIM: To study whether high-sensitivity C-reactive protein (hs-CRP) measurement can aid the assessment of disease activity and glucocorticoid treatment in paediatric inflammatory bowel disease (IBD).

METHODS: CRP levels were measured in 39 children with IBD undergoing colonoscopy [median age 12.8 years, Crohn’s disease (CD) n = 20], in 22 other children with IBD followed for acute response to glucocorticoids, and in 33 paediatric non-IBD patients. When standard CRP level was below detection limit (< 5 mg/L), hs-CRP was analyzed.

RESULTS: Sixty-four percent (25/39) of the children with IBD undergoing colonoscopy displayed undetectable (< 5 mg/L) standard CRP levels. Of these, the hs-CRP measurement could not differentiate between active (median, 0.2 mg/L, range, 0.007-1.37, n = 17) or quiescent (0.1 mg/L, 0.01-1.89, n = 8, P = NS) disease. Patients with ileocolonic CD had higher CRP levels (14 mg/L, 0.06-45, n = 13) than patients with no ileal involvement (0.18 mg/L, 0.01-9, n = 7, P < 0.01) or ulcerative colitis (UC) (0.13 mg/L, 0.007-23, P < 0.05). In children with active IBD treated with systemic glucocorticoids, the standard CRP was undetectable in 59% of the patients. The hs-CRP levels did not differ between patients that responded to steroid therapy and in non-responders.

CONCLUSION: The measurement of hs-CRP did not prove useful in the assessment of disease activity or glucocorticoid treatment in paediatric IBD patients that had undetectable standard CRP.

CTLA-4 is a key factor in regulating and maintaining self tolerance, providing a negative signal to the T cell and thus limiting immune responses. Several polymorphisms within the CTLA-4 gene have been associated with an increased risk of developing autoimmune diseases and, very recently, with susceptibility to human cancer. Acute lymphoblastic leukemia is a clonal disorder of lymphoid progenitors representing the most frequent malignancy of childhood. Here, we show the presence at significantly elevated levels of a circulating soluble form of CTLA-4 in 70% of B-ALL pediatric patients with active disease, the positive correlation between the percentage of leukemic B lymphocytes and the amount of serum sCTLA-4, and the expression of sCTLA-4 transcript by B cells in patients. Finally, a correlation between CD1d expression (a negative prognostic marker) and the sCTLA-4 in B-ALL patients was observed. This suggests a possible role of this soluble molecule as a marker of progression or severity of the neoplastic disease.

SUMMARY

It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn’s disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn’s disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFα and IFNγ and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.

The present study was performed to determine what proportion of people in Sardinia with or without Crohn's disease were infected with Mycobacterium avium subspecies paratuberculosis and had a preponderance of allelic variants of Nod2, an intracellular protein involved in Crohn's disease susceptibility. Genetic analysis of the alleles of the NOD2/CARD15 gene (insC3020, G908R, and R702W alleles), linked to susceptibility or genetic predisposition to Crohn's disease in humans, was carried out on specimens from 37 Crohn's disease patients and 34 patients without Crohn's disease. Our results show that more than 70 percent of people in Sardinia with Crohn's disease carry at least one of the susceptibility-associated NOD2/CARD15 alleles and were infected with Mycobacterium avium subspecies paratuberculosis.

Considering the epidemiological, genetic and immunological data, we can conclude that the inflammatory bowel diseases are heterogeneous disorders of multifactorial etiology in which hereditability and environment interact to produce the disease. It is probable that patients have a genetic predisposition for the development of the disease coupled with disturbances in immunoregulation. Several genes have so far been related to the diagnosis of Crohn’s disease. These genes are related to innate pattern recognition receptors, to epithelial barrier homeostasis and maintenance of epithelial barrier integrity, to autophagy and to lymphocyte differentiation. So far, the strongest and most replicated associations with Crohn’s disease have been demonstrated with NOD2, IL23R and ATG16L1 genes. Many genes have so far been implicated in the prognosis of Crohn’s disease and many attempts have been made for classification of genetic profiles in Crohn’s disease. CARD15 seems to be not only a susceptibility gene, but also a disease-modifier gene for Crohn’s disease. Enriching our understanding of Crohn’s disease genetics is of value, but when combining genetic data with functional data the outcome could be of major importance to clinicians.

Considering epidemiological, genetic and immunological data, we can conclude that the inflammatory bowel diseases are heterogeneous disorders of multifactorial etiology in which hereditability and environment interact to produce the disease. It is probable that patients have a genetic predisposition for the development of the disease coupled with disturbances in immunoregulation. Several genes have been so far related to the diagnosis of Crohn’s disease. Those genes are related to innate pattern recognition receptors, to epithelial barrier homeostasis and maintenance of epithelial barrier integrity, to autophagy and to lymphocyte differentiation. So far, the most strong and replicated associations with Crohn’s disease have been done with NOD2, IL23R and ATG16L1 genes. Many genes have so far been implicated in prognosis of Crohn’s disease and many attempts have been made to classify genetic profiles in Crohn’s disease. CARD15 seems not only a susceptibility gene, but also a disease-modifier gene for Crohn’s disease. Enriching our understanding on Crohn’s disease genetics is important but when combining genetic data with functional data the outcome could be of major importance to clinicians.

Autophagy is a homeostatic pathway that processes and recycles damaged organelles and other cytoplasmic contents. While studies have implicated autophagy in the immune response to infection, the understanding of how the autophagic machinery specifically targets intracellular pathogens has remained elusive. Two recent studies have uncovered an autophagy-mediated immune response to bacteria through their detection by Nod receptors. In particular, Nod1 and Nod2 recruit the autophagic protein ATG16L1 to the plasma membrane at the bacterial entry site to promote an autophagy-dependent elimination of bacteria. In addition, Nod2 and ATG16L1 synergize to initiate an adaptive immune response to bacterial invasion by enhancing major histocompatibility complex (MHC) class II antigen presentation. These findings link two Crohn disease-associated susceptibility genes and reveal that cells expressing the risk-associated variants of ATG16L1 are defective in autophagy-mediated bacterial handling and antigen presentation. This could lead to bacterial persistence and contribute to the pathogenesis of the disease.

Background

Clinical and experimental observations in animal models indicate that intestinal commensal bacteria are involved in the initiation and amplification of inflammatory bowel disease (IBD). No paediatric reports are available on intestinal endogenous microflora in IBD.

Aims

To investigate and characterise the predominant composition of the mucosa‐associated intestinal microflora in colonoscopic biopsy specimens of paediatric patients with newly diagnosed IBD.

Methods

Mucosa‐associated bacteria were quantified and isolated from biopsy specimens of the ileum, caecum and rectum obtained at colonoscopy in 12 patients with Crohn's disease, 7 with ulcerative colitis, 6 with indeterminate colitis, 10 with lymphonodular hyperplasia of the distal ileum and in 7 controls. Isolation and characterisation were carried out by conventional culture techniques for aerobic and facultative‐anaerobic microorganisms, and molecular analysis (16S rRNA‐based amplification and real‐time polymerase chain reaction assays) for the detection of anaerobic bacterial groups or species.

Results

A higher number of mucosa‐associated aerobic and facultative‐anaerobic bacteria were found in biopsy specimens of children with IBD than in controls. An overall decrease in some bacterial species or groups belonging to the normal anaerobic intestinal flora was suggested by molecular approaches; in particular, occurrence of Bacteroides vulgatus was low in Crohn's disease, ulcerative colitis and indeterminate colitis specimens.

Conclusion

This is the first paediatric report investigating the intestinal mucosa‐associated microflora in patients of the IBD spectrum. These results, although limited by the sample size, allow a better understanding of changes in mucosa‐associated bacterial flora in these patients, showing either a predominance of some potentially harmful bacterial groups or a decrease in beneficial bacterial species. These data underline the central role of mucosa‐adherent bacteria in IBD.

AIM: 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.

METHODS: 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.

RESULTS: 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).

CONCLUSION: The present study suggests a cumulative effect of individual IBD susceptibility loci.

The aim of this study was to integrate human clinical, genotype, mRNA microarray and 16 S rRNA sequence data collected on 84 subjects with ileal Crohn’s disease, ulcerative colitis or control patients without inflammatory bowel diseases in order to interrogate how host-microbial interactions are perturbed in inflammatory bowel diseases (IBD). Ex-vivo ileal mucosal biopsies were collected from the disease unaffected proximal margin of the ileum resected from patients who were undergoing initial intestinal surgery. Both RNA and DNA were extracted from the mucosal biopsy samples. Patients were genotyped for the three major NOD2 variants (Leufs1007, R702W, and G908R) and the ATG16L1T300A variant. Whole human genome mRNA expression profiles were generated using Agilent microarrays. Microbial composition profiles were determined by 454 pyrosequencing of the V3–V5 hypervariable region of the bacterial 16 S rRNA gene. The results of permutation based multivariate analysis of variance and covariance (MANCOVA) support the hypothesis that host mucosal Paneth cell and xenobiotic metabolism genes play an important role in host microbial interactions.

We report results of a nonsynonymous SNP scan for ulcerative colitis and identify a previously unknown susceptibility locus at ECM1. We also show that several risk loci are common to ulcerative colitis and Crohn’s disease (IL23R, IL12B, HLA, NKX2-3 and MST1), whereas autophagy genes ATG16L1 and IRGM, along with NOD2 (also known as CARD15), are specific for Crohn’s disease. These data provide the first detailed illustration of the genetic relationship between these common inflammatory bowel diseases.

Inflammatory bowel disease 5 (IBD5) is a 250 kb haplotype on chromosome 5 that is associated with an increased risk of Crohn’s disease in Europeans. The OCTN1 gene is centrally located on IBD5 and encodes a transporter of the antioxidant ergothioneine (ET). The 503F variant of OCTN1 is strongly associated with IBD5 and is a gain-of-function mutation that increases absorption of ET. Although 503F has been implicated as the variant potentially responsible for Crohn’s disease susceptibility at IBD5, there is little evidence beyond statistical association to support its role in disease causation. We hypothesize that 503F is a recent adaptation in Europeans that swept to relatively high frequency and that disease association at IBD5 results not from 503F itself, but from one or more nearby hitchhiking variants, in the genes IRF1 or IL5. To test for evidence of recent positive selection on the 503F allele, we employed the iHS statistic, which was significant in the European CEU HapMap population (P = 0.0007) and European Human Genome Diversity Panel populations (P ≤ 0.01). To evaluate the hypothesis of disease-variant hitchhiking, we performed haplotype association tests on high-density microarray data in a sample of 1,868 Crohn’s disease cases and 5,550 controls. We found that 503F haplotypes with recombination breakpoints between OCTN1 and IRF1 or IL5 were not associated with disease (odds ratio [OR]: 1.05, P = 0.21). In contrast, we observed strong disease association for 503F haplotypes with no recombination between these three genes (OR: 1.24, P = 2.6 × 10−8), as expected if the sweeping haplotype harbored one or more disease-causing mutations in IRF1 or IL5. To further evaluate these disease-gene candidates, we obtained expression data from lower gastrointestinal biopsies of healthy individuals and Crohn’s disease patients. We observed a 72% increase in gene expression of IRF1 among Crohn’s disease patients (P = 0.0006) and no significant difference in expression of OCTN1. Collectively, these data indicate that the 503F variant has increased in frequency due to recent positive selection and that disease-causing variants in linkage disequilibrium with 503F have hitchhiked to relatively high frequency, thus forming the IBD5 risk haplotype. Finally, our association results and expression data support IRF1 as a strong candidate for Crohn’s disease causation.

AIM: To assess whether the polymorphisms of NOD2/CARD15, autophagy-related 16-like 1 (ATG16L1), and interleukin-23 receptor (IL23R) genes play a more critical role in the susceptibility of childhood-onset than in adult-onset Crohn’s disease (CD).

METHODS: Polymorphisms R702W, G908R, and 3020insC of NOD2/CARD15; rs2241880 A/G of ATG16L1, and rs11209026 (R381Q) of IL23R gene were assessed in 110 childhood-onset CD, 364 adult-onset CD, and 539 healthy individuals. Analysis of polymorphisms R702W, G908R, and 3020insC of NOD2/CARD15 genotyping was performed by allele specific polymerase chain reaction (PCR) or by PCR-restriction fragment length polymorphism analysis. The polymorphisms rs2241880 A/G of the ATG16L1, and rs11209026 (R381Q) of the IL23R gene in the children’s cohort were genotyped by PCR and melting curve analysis whereas adult group genotyping was performed using the Affymetrix Genome-Wide Human SNP Array 5.0 (500K).

RESULTS: The 3020insC allele in NOD2/CARD15 was significantly higher in childhood than in adult-onset CD (P = 0.0067). Association with at least 1 NOD2/CARD15 variant was specific for ileal disease (with or without colonic involvement). Even if the frequency of G allele of the rs2241880 ATG16L1 polymorphism was increased in both paediatric and adult CD patients compared to controls (P = 0.017 and P = 0.001, respectively), no difference was observed between the childhood and the adult cohort. The rare Q allele of IL23R rs11209026 polymorphism was underrepresented in both paediatric and adult CD cases (P = 0.0018 and P = 0.04, respectively) and no difference was observed between the childhood and the adult cohort. The presence of the rs2241880 ATG16L1 and rs11209026 IL23R polymorphisms did not influence disease phenotype.

CONCLUSION: Polymorphism 3020insC in NOD2/CARD15 occurs statistically significantly more often in patients with childhood-onset CD than in patients with adult-onset CD. The ATG16L1 and IL23R variants are associated with susceptibility to CD, but not early-onset disease.

SUMMARY

Surfactant protein D (SP-D) is a mucosal collectin that functions in the innate immune response to microorganisms in the pulmonary tract, and possibly the gastrointestinal tract. We studied the genetic association of the two non-synonymous SP-D SNPs rs721917 (C/T Met11Thr) and rs2243639 (G/A Ala160Thr) in 256 IBD cases (123 CD and 133 UC) and 376 unrelated healthy individuals from an inflammatory bowel disease (IBD) population from Central Pennsylvania. Case-control analysis revealed a significant association of rs2243639 with susceptibility to Crohn’s disease (CD) (p=0.0036), but not ulcerative colitis (UC) (p=0.883), and no association of rs721917 with CD (p=0.328) or UC (p=0.218). Using intestinal tissues from 19 individuals heterozygous for each SNP, we compared allele expression of these two SNPs between diseased and matched normal tissues. rs2243639 exhibited balanced biallele expression; while rs721917 exhibited differential allele expression (balanced biallelic 37%, imbalanced biallelic 45%, and dominant monoallelic 18%). Comparison of allelic expression pattern between diseased and matched normal tissues, 13 out 19 individuals (14 UC, 5 CD) showed a similar pattern. The six patients exhibiting a different pattern were all UC patients. The results suggest that differential allelic expression may affect penetrance of the SNP rs721917 disease-susceptibility allele in IBD. The potential impact of SP-D monoallelic expression on incomplete penetrance is discussed.