A common genetic coding variant in the core autophagy gene ATG16L1 is associated with increased susceptibility to Crohn's disease (CD). The variant encodes an amino acid change in ATG16L1 such that the threonine at position 300 is substituted with an alanine (ATG16L1 T300A). How this variant contributes to increased risk of CD is not known, but studies with transfected cell lines and gene-targeted mice have demonstrated that ATG16L1 is required for autophagy, control of interleukin-1-β and autophagic clearance of intracellular microbes. In addition, studies with human cells expressing ATG16L1 T300A indicate that this variant reduces the autophagic clearance of intracellular microbes.
We demonstrate, using somatically gene-targeted human cells that the ATG16L1 T300A variant confers protection from cellular invasion by Salmonella. In addition, we show that ATG16L1-deficient cells are resistant to bacterial invasion.
These results suggest that cellular expression of ATG16L1 facilitates bacterial invasion and that the CD-associated ATG16L1 T300A variant may confer protection from bacterial infection.
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.
Genetics; Childhood-onset; Inflammatory bowel disease; Crohn’s disease; Genetic susceptibility; NOD2/CARD15; ATG16L1; IL23R; Polymorphisms
Numerous papers have addressed the association of mutations and polymorphisms of susceptibility genes with autoimmune inflammatory disorders. We investigated whether polymorphisms that confer susceptibility to Crohn's disease could be classified also as predisposing factors for the development of primary sclerosing cholangitis and primary biliary cirrhosis in Polish patients.
The study included 60 patients with CD, 77 patients with PSC, of which 61 exhibited IBD (40 UC, 8 CD, and 13 indeterminate colitis), and 144 patients with PBC. All the patients were screened against Crohn's disease associating genetic polymorphisms.
The polymorphisms were chosen according to previously confirmed evidence for association with Crohn's disease, including Pro268Ser, Arg702Trp, Gly908Arg and 1007fs in NOD2/CARD15, Leu503Phe/-207G>C in SLC22A4/OCTN1/SLC22A5/OCTN2, Arg30Gln in DLG5, Thr300Ala in ATG16L1, and Arg381Gln, His3Gln and exon-3'UTR in IL23R. Genotyping was carried out using TaqMan SNP genotyping assays.
We confirmed a strong association between three NOD2/CARD15 gene variants (Pro268Ser, OR = 2.52, 95% CI = 1.34 – 4.75); (Arg702Trp, OR = 6.65, 95% CI = 1.99 – 22.17); (1007fs, OR = 9.59, 95% CI = 3.94 – 23.29), and a weak association between both the protective OCTN1/OCTN2 CC haplotype (OR = 0.28, 95% CI = 0.08 – 0.94), and a variant of ATG16L1 gene (Thr300Ala, OR = 0.468, 95% CI = 0.24 – 0.90) with Crohn's disease. In contrast, none of the polymorphisms exhibited association with susceptibility to primary sclerosing cholangitis and primary biliary cirrhosis, including a group of primary sclerosing cholangitis patients with concurrent IBD.
Although the clinical data indicate non-random co-occurrence of inflammatory bowel disease and primary sclerosing cholangitis, consistently with the previously published studies, no genetic association was found between the genetic variants predisposing to Crohn's disease and hepatobiliary autoimmune disorders. However, since estimation of genetic variant disproportion is limited by sample size, these negative results may also indicate that eventually shared genetic predispositions are too little to be captured by small patient groups.
The polymorphism ATG16L1 T300A, associated with increased risk of Crohn’s disease, impairs pathogen defense mechanisms including selective autophagy, but specific pathway interactions altered by the risk allele remain unknown. Here, we use perturbational profiling of human peripheral blood cells to reveal that CLEC12A is regulated in an ATG16L1-T300A-dependent manner. Antibacterial autophagy is impaired in CLEC12A-deficient cells, and this effect is exacerbated in the presence of the ATG16L1∗300A risk allele. Clec12a−/− mice are more susceptible to Salmonella infection, supporting a role for CLEC12A in antibacterial defense pathways in vivo. CLEC12A is recruited to sites of bacterial entry, bacteria-autophagosome complexes, and sites of sterile membrane damage. Integrated genomics identified a functional interaction between CLEC12A and an E3-ubiquitin ligase complex that functions in antibacterial autophagy. These data identify CLEC12A as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease.
•Integrated genomics reveals risk-allele-specific autophagy pathway interactions•CLEC12A is important for antibacterial autophagy in epithelial and immune cells•CLEC12A knockdown amplifies antibacterial autophagy defects in ATG16L1
∗300A cells•Clec12a−/− mice are more susceptible to Salmonella infection in vivo
Although genome-wide association studies are valuable in identifying disease-associated loci, they produce only a partial view of pathogenesis. Using integrated, systems-level approaches to pinpoint genes that interact with the Crohn’s-disease-associated variant ATG16L1 T300A, Begun et al. identify CLEC12A as an innate defense gene that functions in antibacterial autophagy.
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.
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.
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.
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.
CARD15; inflammatory bowel disease; genetics; mucosal immunity
Inflammatory bowel diseases (IBD) are chronic diseases of the gastrointestinal tract. Although their pathogenesis is unclear, the combination of genetic predisposition and environmental components are believed to be the main cause of these diseases. Recently, many variants in interleukin 23 receptor (IL23R) and autophagy-related 16-like 1 (ATG16L1) genes have been associated with the disease. Our objective was to assess the frequency of ATG16L1 (T300A) and IL23R (L310P) variants in Moroccan IBD (Crohn’s disease and Ulcerative Colitis) patients and to evaluate a possible effect of these variants on disease’s phenotype and clinical course.
96 Moroccan IBD patients and 114 unrelated volunteers were genotyped for ATG16L1 (T300A) and IL23R (L310P) variants by PCR-restriction fragment length polymorphism.
This is the first report on the prevalence of ATG16L1 (T300A) and IL23R (L310P) variants in a Moroccan group. We found that IL23R (L310P) variant conferred a protective effect for crohn’s disease (CD) but not ulcerative colitis (UC) patients. The presence of ATG16L1 (T300A) mutated alleles was associated with CD type but not with disease onset. In addition, the carriage of T300A variant alleles conferred a protective effect in UC.
Our results showed that the prevalence of ATG16L1 and IL23R variants was not significantly different between patients and controls. However a possible role of ATG16L1 (T300A) on CD phenotype was suggested.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-570) contains supplementary material, which is available to authorized users.
IBD; ATG16L1; IL23R; Moroccan population
Multiple genes have been implicated by association studies in altering inflammatory bowel disease (IBD) predisposition. Paediatric patients often manifest more extensive disease and a particularly severe disease course. It is likely that genetic predisposition plays a more substantial role in this group.
To identify the spectrum of rare and novel variation in known IBD susceptibility genes using exome sequencing analysis in eight individual cases of childhood onset severe disease.
DNA samples from the eight patients underwent targeted exome capture and sequencing. Data were processed through an analytical pipeline to align sequence reads, conduct quality checks, and identify and annotate variants where patient sequence differed from the reference sequence. For each patient, the entire complement of rare variation within strongly associated candidate genes was catalogued.
Across the panel of 169 known IBD susceptibility genes, approximately 300 variants in 104 genes were found. Excluding splicing and HLA-class variants, 58 variants across 39 of these genes were classified as rare, with an alternative allele frequency of <5%, of which 17 were novel. Only two patients with early onset Crohn's disease exhibited rare deleterious variations within NOD2: the previously described R702W variant was the sole NOD2 variant in one patient, while the second patient also carried the L1007 frameshift insertion. Both patients harboured other potentially damaging mutations in the GSDMB, ERAP2 and SEC16A genes. The two patients severely affected with ulcerative colitis exhibited a distinct profile: both carried potentially detrimental variation in the BACH2 and IL10 genes not seen in other patients.
For each of the eight individuals studied, all non-synonymous, truncating and frameshift mutations across all known IBD genes were identified. A unique profile of rare and potentially damaging variants was evident for each patient with this complex disease.
IBD-genetics; inflammatory bowel disease; crohn's disease; paediatric gastroenterology; ulcerative colitis; zollinger ellison syndrome,
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.
Gene interaction; Interleukin-23 receptor; Autophagy-related 16-like 1; IBD5; Caspase recruitment domain-containing protein 15; Crohn’s disease; Inflammatory bowel disease
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.
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.
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).
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.
ATG16L1; Crohn’s disease; IL23R; Inflammatory bowel disease; Meta-analysis
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.
We sought to investigate whether variants in genes involved in bacterial sensing and autophagy (NOD2, TLR5, IRGM, ATG16L1) and the interleukin-23 signalling pathway (IL12B, IL23R, STAT3) were associated with development of antimicrobial antibodies in patients with Crohn’s disease (CD).
A cohort of 616 CD patients from a tertiary referral hospital (Mount Sinai Hospital, Toronto) was evaluated. DNA was tested for three CD-associated NOD2 variants (3020insC, G908R, R702W), variants in IRGM, ATG16L1, IL12B, IL23R, STAT3, and a TLR5-stop mutation. Serum was analyzed by ELISA for anti-Saccharomyces cervesiase (ASCA) IgG and IgA, anti-outer membrane protein C (anti-ompC), anti-Cbir1 flagellin, and anti-Pseudomonas fluorescens (anti-I2).
NOD2 3020insC was associated with cumulative seroreactivity by quartile sum (p=0.003) and number of positive antibodies (p=0.02). NOD2 G908R was also associated with quartile sum (p=0.05). Increased ASCA seropositivity was associated with NOD2 3020insC (odds ratio (OR)= 1.9, p=0.02) and G908R (OR=1.8, p=0.05), and ATG16L1 T300A (OR=1.4, p=0.01) variants; ASCA positive patients had an increased cumulative number of NOD2 3020insC and ATG16L1 T300A variants (p=0.007). TLR5-stop mutation abrogated development of anti-flagellin in a dominant-negative fashion (OR=0.5, p=0.009). The IRGM CD risk variant was associated with increased anti-flagellin seropositivity (OR=1.5, p=0.03). IL12B, IL23R, and STAT3 variants did not contribute to development of anti-microbial antibodies.
Variants in innate immune genes involved in pattern recognition and autophagy but not the IL-23 signaling pathway influence antimicrobial seroreactivity in CD. In particular, the additive effect of NOD2 3020insC and ATG16L1 T300A suggests a role for autophagy in development of ASCA.
Inflammatory bowel disease; biomarkers; ASCA; genetics; NOD2
Background & Aims
Recent studies reported a role for more than 70 genes or loci in the susceptibility to Crohn’s disease (CD). However, the impact of these associations in clinical practice remains to be defined. The aim of the study was to analyse the relationship between genotypes and phenotypes for the main 53 CD-associated polymorphisms.
A cohort of 798 CD patients with a median follow up of 7 years was recruited by tertiary adult and paediatric gastroenterological centres. A detailed phenotypic description of the disease was recorded, including clinical presentation, response to treatments and complications. The participants were genotyped for 53 CD-associated variants previously reported in the literature and correlations with clinical sub-phenotypes were searched for. A replication cohort consisting of 722 CD patients was used to further explore the putative associations.
The NOD2 rare variants were associated with an earlier age at diagnosis (p = 0.0001) and an ileal involvement (OR = 2.25[1.49–3.41] and 2.77 [1.71–4.50] for rs2066844 and rs2066847, respectively). Colonic lesions were positively associated with the risk alleles of IL23R rs11209026 (OR = 2.25 [1.13–4.51]) and 6q21 rs7746082 (OR = 1.60 [1.10–2.34] and negatively associated with the risk alleles of IRGM rs13361189 (OR = 0.29 [0.11–0.74]) and DEFB1 rs11362 (OR = 0.50 [0.30–0.80]). The ATG16L1 and IRGM variants were associated with a non-inflammatory behaviour (OR = 1.75 [1.22–2.53] and OR = 1.50 [1.04–2.16] respectively). However, these associations lost significance after multiple testing corrections. The protective effect of the IRGM risk allele on colonic lesions was the only association replicated in the second cohort (p = 0.03).
It is not recommended to genotype the studied polymorphisms in routine practice.
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.
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.
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%.
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.
Crohn’s disease; IL23R; ATG16L1; IBD5; genetics
Human dendritic cells (DCs) play an important role in induction and progression of Crohn’s disease (CD). Accumulating evidence suggests that viral infection is required to trigger CD pathogenesis in genetically predisposed individuals. NOD2 and ATG16L1 are among the major CD susceptibility genes implicated in impaired immune response to bacterial infection. In this study, we investigated gene expression and allelic imbalance (AI) of NOD2 and ATG16L1 using common variants in human monocyte-derived DCs. Significant AI was observed in ~40% and ~70% of NOD2 and ATG16L1 heterozygotes, respectively (p < 0.05). AI of NOD2 was inversely associated with its expression level (p = 0.015). No correlation was detected between gene expression and AI for ATG16L1. When infected with Newcastle Disease Virus (NDV), NOD2 expression in DCs was induced about four-fold (p < 0.001), whereas ATG16L1 expression was not affected (p = 0.88). In addition, NDV infection tended to lower the variance in AI among DC populations for the NOD2 gene (p = 0.05), but not the ATG16L1 gene (p = 0.32). Findings of a simulation study, aimed to verify whether the observed variation in gene expression and AI is a result of sample-to-sample variability or experimental measurement error, suggested that NOD2 AI is likely to result from a deterministic event at a single cell level. Overall, our results present initial evidence that AI of the NOD2 and ATG16L1 genes exists in populations of human DCs. In addition, our findings suggest that viral infection may regulate NOD2 expression.
Allelic imbalance; NOD2; ATG16L1; Human dendritic cells; NDV; Crohn’s disease
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.
ATG16L1; Inflammatory bowel diseases; Crohn’s disease; Ulcerative colitis; Meta-analysis
Background & Aims
African Americans (AA) 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. 354 AA CD cases and 354 ethnicity-matched controls were genotyped for total 21 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 cases (211 to 227) stratified by phenotypic sub-classifications 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 (L503F [10.6% 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 significantly contribute to AA CD risk. Established NOD2, SLC22A4-A5, and ATG16L1 variants show increased CD risk, with IBD5 in recessive. Although CD is more common in whites, European admixture is similar among AA cases and controls.
genetics; epidemiology; Crohn's disease
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.
NOD2; IL23R; ATG16L1; Single nucleotide polymorphisms; Crohn’s disease; Ulcerative colitis; Lithuania
Genome-wide association studies identified PTPN2 (protein tyrosine phosphatase, non-receptor type 2) as susceptibility gene for inflammatory bowel diseases (IBD). However, the exact role of PTPN2 in Crohn's disease (CD) and ulcerative colitis (UC) and its phenotypic effect are unclear. We therefore performed a detailed genotype-phenotype and epistasis analysis of PTPN2 gene variants.
Genomic DNA from 2131 individuals of Caucasian origin (905 patients with CD, 318 patients with UC, and 908 healthy, unrelated controls) was analyzed for two SNPs in the PTPN2 region (rs2542151, rs7234029) for which associations with IBD were found in previous studies in other cohorts. Our analysis revealed a significant association of PTPN2 SNP rs2542151 with both susceptibility to CD (p = 1.95×10−5; OR 1.49 [1.34–1.79]) and UC (p = 3.87×10−2, OR 1.31 [1.02–1.68]). Moreover, PTPN2 SNP rs7234029 demonstrated a significant association with susceptibility to CD (p = 1.30×10−3; OR 1.35 [1.13–1.62]) and a trend towards association with UC (p = 7.53×10−2; OR 1.26 [0.98–1.62]). Genotype-phenotype analysis revealed an association of PTPN2 SNP rs7234029 with a stricturing disease phenotype (B2) in CD patients (p = 6.62×10−3). Epistasis analysis showed weak epistasis between the ATG16L1 SNP rs2241879 and PTPN2 SNP rs2542151 (p = 0.024) in CD and between ATG16L1 SNP rs4663396 and PTPN2 SNP rs7234029 (p = 4.68×10−3) in UC. There was no evidence of epistasis between PTPN2 and NOD2 and PTPN2 and IL23R. In silico analysis revealed that the SNP rs7234029 modulates potentially the binding sites of several transcription factors involved in inflammation including GATA-3, NF-κB, C/EBP, and E4BP4.
Our data confirm the association of PTPN2 variants with susceptibility to both CD and UC, suggesting a common disease pathomechanism for these diseases. Given recent evidence that PTPN2 regulates autophagosome formation in intestinal epithelial cells, the potential link between PTPN2 and ATG16L1 should be further investigated.
Ileal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensins are exclusively produced in Paneth cells (PCs) at small intestinal crypt bases. Different ileal CD–associated genes including NOD2, ATG16L1, and recently the β-catenin–dependant Wnt transcription factor TCF7L2 have been linked to impaired PC antimicrobial function. The Wnt pathway influences gut mucosal homeostasis and PC maturation, besides directly controlling HD-5/6 gene expression. The herein reported candidate gene study focuses on another crucial Wnt factor, the co-receptor low density lipoprotein receptor-related protein 6 (LRP6). We analysed exonic single nucleotide polymorphisms (SNPs) in a large cohort (Oxford: n = 1,893) and prospectively tested 2 additional European sample sets (Leuven: n = 688, Vienna: n = 1,628). We revealed an association of a non-synonymous SNP (rs2302685; Ile1062Val) with early onset ileal CD (OR 1.8; p = 0.00034; for homozygous carriers: OR 4.1; p = 0.00004) and additionally with penetrating ileal CD behaviour (OR 1.3; p = 0.00917). In contrast, it was not linked to adult onset ileal CD, colonic CD, or ulcerative colitis. Since the rare variant is known to impair LRP6 activity, we investigated its role in patient mucosa. Overall, LRP6 mRNA was diminished in patients independently from the genotype. Analysing the mRNA levels of PC product in biopsies from genotyped individuals (15 controls, 32 ileal, and 12 exclusively colonic CD), we found particularly low defensin levels in ileal CD patients who were carrying the variant. In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection. Taken together, we identified LRP6 as a new candidate gene in ileal CD. Impairments in Wnt signalling and Paneth cell biology seem to represent pathophysiological hallmarks in small intestinal inflammation and should therefore be considered as interesting targets for new therapeutic approaches.
Crohn's Disease (CD) is to date incurable and is characterized by severe, reoccurring inflammations that can affect different intestinal locations. The complicated and multifactorial pathogenesis is not completely understood but involves disturbed epithelial barriers and immune reactions against the commensal flora in genetically predisposed individuals. Some inherited disease mechanisms are specific for small intestinal CD and are often connected to impaired Paneth cells. These specialized cells are critical for epithelial defences in the small intestine, and their most abundant antimicrobials (HD-5 and HD-6) are primarily diminished in patients who suffer from CD at this location. In this context, we previously identified a primary role of the Wnt pathway factor TCF7L2, which regulates the expression of both these antimicrobial peptides. We have now studied a functional mutation in the Wnt co-receptor LRP6 that was more frequent in early onset ileal CD. It was also associated with severe penetrating behaviour and linked to especially low HD-5 expression in ileal CD patient biopsies. Independently, we found that a reduced epithelial expression likely represents an additional impairment of the Wnt co-receptor in the disorder. LRP6 is a new player in small intestinal CD and underlines the importance of Paneth cell antimicrobial defences in the disease pathogenesis.
Polymorphisms in NOD2, encoding an intracellular pattern recognition receptor, contribute the largest fraction of genetic risk for Crohn’s disease among the >40 risk loci identified so far. Autophagy plays a prominent role in the innate immune response towards intracellular bacteria. The discovery of the autophagy genes ATG16L1 and IRGM as risk factors for Crohn’s disease turned autophagy into the spotlight in inflammatory bowel disease (IBD). Remarkably, NOD2 has recently been identified as a potent autophagy inducer. A physical interaction of NOD2 and ATG16L1 appears to be required for autophagic clearance of intracellular pathogens. Moreover, Crohn’s disease-associated NOD2 and ATG16L1 variants exhibit a defect in the induction of an autophagic response and hence predict autophagy as a key converging mechanism that leads to Crohn’s disease. Another pathway that is closely intertwined with autophagy and mutually cross-regulated is the unfolded protein response (UPR), which is induced by endoplasmic reticulum (ER) stress. Genes involved in the UPR (XBP1, ORMDL3) have also been genetically associated with Crohn’s disease and ulcerative colitis. Moreover, the intestinal epithelium at the interface between host and microbe appears particularly affected by IBD-associated hypomorphic function of autophagy and the UPR. The functional convergence of main genetic risk factors for IBD on these innate immune pathways has hence important implications for the host’s interaction with the microbiota. Moreover, the genetic convergence on these molecular mechanisms may open novel therapeutic options for IBD that deserve further exploration.
Abnormal host-microbe interactions are implicated in the pathogenesis of inflammatory bowel diseases. Previous 16S rRNA sequence analysis of intestinal tissues demonstrated that a subset of Crohn’s disease (CD) and ulcerative colitis (UC) samples exhibited altered intestinal associated microbial compositions characterized by depletion of Bacteroidetes and Firmicutes (particularly Clostridium taxa). We hypothesize that NOD2 and ATG16L1 risk alleles may be associated with these alterations.
To test this hypothesis, we genotyped 178 specimens collected from 35 CD, 35 UC and 54 control patients for the three major NOD2 risk alleles (Leu 1007fs, R702W and G908R) and the ATG16L1T300A risk allele, that had undergone previous 16S rRNA sequence analysis. Our statistical models incorporated the following independent variables:1.) disease phenotype (CD, UC, Non-IBD Control); 2.) NOD2 composite genotype (NOD2R = at least one risk allele, NOD2NR = no risk alleles); 3.) ATG16L1T300A genotype (ATG16L1R/R, ATG16L1R/NR, ATG16L1NR/NR); 4.) patient age at time of surgery and all first order interactions. The dependent variable(s) were the relative frequencies of bacterial taxa classified by applying the RDP 2.1 classifier to previously reported 16S rRNA sequence data.
Disease phenotype, NOD2 composite genotype and ATG16L1 genotype were significantly associated with shifts in microbial compositions by nonparametric MANCOVA. Shifts in the relative frequencies of Faecalibacterium and Escherichia taxa were significantly associated with disease phenotype by nonparametric ANCOVA.
These results support the concept that disease phenotype and genotype are associated with compositional changes in intestinal associated microbiota.
NOD2; ATG16L1; inflammatory bowel diseases; microbiota
BACKGROUND & AIMS
Variants in genes that regulate autophagy have been associated with Crohn’s disease (CD). Defects in autophagy-mediated removal of pathogenic microbes could contribute to pathogenesis of CD. We investigated the role of the micro-RNAs (miRs) MIR106B and MIR93 in induction of autophagy and bacterial clearance in human cell lines, and the correlation between MIR106B and autophagy-related gene 16L1 (ATG16L1) expression in tissues from patients with CD.
We studied the ability of MIR106B and MIR93 to regulate ATG transcripts in human cancer cell lines (HCT116, SW480, HeLa, and U2OS) using luciferase report assays and bioinformatics analyses; MIR106B and MIR93 mimics and antagonists were transfected into cells to modify levels of miRs. Cells were infected with LF82, a CD-associated adherent-invasive strain of Escherichia coli, and monitored by confocal microscopy and for colony-forming units. Colon tissues from 41 healthy individuals (controls), 22 with active CD, 16 with inactive CD, and 7 with chronic inflammation were assessed for levels of MIR106B and ATG16L1 by in situ hybridization and immunohistochemistry.
Silencing Dicer 1, an essential processor of miRs, increased levels of ATG protein and formation of autophagosomes in cells, indicating that miRs regulate autophagy. Luciferase reporter assays indicated that MIR106B and MIR93 targeted ATG16L1 mRNA. MIR106B and MIR93 reduced levels of ATG16L1 and autophagy; these increased following expression of ectopic ATG16L1. In contrast, MIR106B and MIR93 antagonists increased formation of autophagosomes. Levels of MIR106B were increased in intestinal epithelia from patients with active CD, whereas levels of ATG16L1 were reduced, compared with controls. Levels of CMYC were also increased in intestinal epithelia of patients with active CD, compared with controls. These alterations could impair removal of CD-associated bacteria by autophagy.
In human cell lines, MIR106B and MIR93 reduce levels of ATG16L1 and autophagy, and prevent autophagy-dependent eradication of intracellular bacteria. This process also appears to be altered in colon tissues from patients with active CD.
inflammatory bowel disease; microRNA; cell biology; infection
Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Genetic polymorphisms can confer CD risk and influence disease phenotype. Indoleamine 2,3 dioxygenase-1 (IDO1) is one of the most over-expressed genes in CD and mediates potent anti-inflammatory effects via tryptophan metabolism along the kynurenine pathway. We aimed to determine whether non-synonymous polymorphisms in IDO1 or IDO2 (a gene paralog) are important either as CD risk alleles or as modifiers of CD phenotype.
Utilizing a prospectively collected database, clinically phenotyped CD patients (n = 734) and non-IBD controls (n = 354) were genotyped for established IDO1 and IDO2 non-synonymous single nucleotide polymorphisms (SNPs) and novel genetic variants elucidated in the literature. Allelic frequencies between CD and non-IBD controls were compared. Genotype-phenotype analysis was conducted. IDO1 enzyme activity was assessed by calculating the serum kynurenine to tryptophan ratio (K/T).
IDO1 SNPs were rare (1.7% non-IBD vs 1.1% CD; p = NS) and not linked to Crohn's disease diagnosis in this population. IDO1 SNPs did however associate with a severe clinical course, presence of perianal disease, extraintestinal manifestations and a reduced serum K/T ratio during active disease suggesting lower IDO1 function. IDO2 minor allele variants were common and one of them, rs45003083, associated with reduced risk of Crohn's disease (p = 0.025). No IDO2 SNPs associated with a particular Crohn's disease clinical phenotype.
This work highlights the functional importance of IDO enzymes in human Crohn's disease and establishes relative rates of IDO genetic variants in a US population.
Polymorphisms in the essential autophagy gene Atg16L1 have been linked with susceptibility to Crohn’s disease, a major type of inflammatory bowel disease (IBD). Although the inability to control intestinal bacteria is thought to underlie IBD, the role of Atg16L1 during extracellular intestinal bacterial infections has not been sufficiently examined and compared to the function of other IBD susceptibility genes such as Nod2, which encodes a cytosolic bacterial sensor. We find that Atg16L1 mutant mice are resistant to intestinal disease induced by the model bacterial pathogen Citrobacter rodentium. An Atg16L1 deficiency alters the intestinal environment to mediate an enhanced immune response that is dependent on monocytic cells, but this hyper-immune phenotype and protective effects are lost in Atg16L1/Nod2 double mutant mice. These results reveal an immuno-suppressive function of Atg16L1, and suggest that gene variants affecting the autophagy pathway may have been evolutionarily maintained to protect against certain life-threatening infections.
Genome-wide association studies and follow-up meta-analyses in Crohn's disease (CD) and ulcerative colitis (UC) have recently identified 163 disease-associated loci that meet genome-wide significance for these two inflammatory bowel diseases (IBD). These discoveries have already had a tremendous impact on our understanding of the genetic architecture of these diseases and have directed functional studies that have revealed some of the biological functions that are important to IBD (e.g. autophagy). Nonetheless, these loci can only explain a small proportion of disease variance (∼14% in CD and 7.5% in UC), suggesting that not only are additional loci to be found but that the known loci may contain high effect rare risk variants that have gone undetected by GWAS. To test this, we have used a targeted sequencing approach in 200 UC cases and 150 healthy controls (HC), all of French Canadian descent, to study 55 genes in regions associated with UC. We performed follow-up genotyping of 42 rare non-synonymous variants in independent case-control cohorts (totaling 14,435 UC cases and 20,204 HC). Our results confirmed significant association to rare non-synonymous coding variants in both IL23R and CARD9, previously identified from sequencing of CD loci, as well as identified a novel association in RNF186. With the exception of CARD9 (OR = 0.39), the rare non-synonymous variants identified were of moderate effect (OR = 1.49 for RNF186 and OR = 0.79 for IL23R). RNF186 encodes a protein with a RING domain having predicted E3 ubiquitin-protein ligase activity and two transmembrane domains. Importantly, the disease-coding variant is located in the ubiquitin ligase domain. Finally, our results suggest that rare variants in genes identified by genome-wide association in UC are unlikely to contribute significantly to the overall variance for the disease. Rather, these are expected to help focus functional studies of the corresponding disease loci.
Genetic studies of common diseases have seen tremendous progress in the last half-decade primarily due to recent technologies that enable a systematic examination of genetic markers across the entire genome in large numbers of patients and healthy controls. The studies, while identifying genomic regions that influence a person's risk for developing disease, often do not pinpoint the actual gene or gene variants that account for this risk (called a causal gene/variant). A prime example of this can be seen with the 163 genetic risk factors that have recently been associated with the chronic inflammatory bowel diseases known as Crohn's disease and ulcerative colitis. For less than a handful of these 163 is the causative change in the genetic code known. The current study used an approach to directly look at the genetic code for a subset of these and identified a causative change in the genetic code for eight risk factors for ulcerative colitis. This finding is particularly important because it directs biological studies to understand the mechanisms that lead to this chronic life-long inflammatory disease.