[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies have highlighted a number of genes involved in autophagy, which are of potential importance in the pathogenesis of Crohn's disease (CD). The associated polymorphisms in ATG16L1 and IRGM have been confirmed, and functional studies have begun to shed light on how they link to CD pathogenesis. In this review we consider the most salient aspects of this rapidly expanding field.
[Show abstract][Hide abstract] ABSTRACT: Previous linkage studies have identified a region at 1p36 as the susceptibility locus (IBD7) of inflammatory bowel disease (IBD). The objective of this study was to investigate whether polymorphisms of caspase-9 (CASP9) gene and RUNX3 are associated with IBD susceptibility and clinical phenotypes. We studied 555 Crohn's disease (CD) and 651 ulcerative colitis (UC) patients recruited from a single UK center. A total of 964 healthy Caucasian subjects were recruited as controls from general practitioner well person clinics in Oxfordshire. Fourteen single nucleotide polymorphisms (SNPs) of CASP9 and 11 SNPs of RUNX3 were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (homogenous MassEXTEND, hME, Sequenom™, Sequenom Inc., San Diego, CA). Linkage disequilibrium (LD) and haplotype association analysis were performed using 2ld and phase v2.0 software. No association of individual SNPs of CASP9 or RUNX3 with UC or CD was identified. The rs1052571 of CASP9 was associated with severe UC [P = 0.0034, odds ratio (OR) = 1.957, 95% confidence interval (CI) = 1.240-3.088]. Significant haplotype associations between CASP9 and IBD were identified, while no association of RUNX3 haplotypes with either UC or CD was found. Our findings suggested that CASP9 gene might be another IBD susceptibility gene.
[Show abstract][Hide abstract] ABSTRACT: The first Crohn's disease (CD) susceptibility gene identified was CARD15, which is a member of the emerging NOD-like receptor (NLR) family. These function as intracellular cystosolic pattern recognition receptors (PRRs) and play a central role in the innate immune response. We studied other members of the NLR family using a gene-wide haplotype tagging approach in a well-characterised collection of 547 CD patients and 465 controls. Four single nucleotide polymorphisms (SNPs) in NLRP3 had P values < 0.05 and are in high linkage disequilibrium (LD) with each other (r(2) > 0.90 for all four SNPs). rs4925648 and rs10925019 were the most strongly associated with CD susceptibility (P = 0.001, odds ratio (OR) 1.62, 95% CI 1.2-2.18; and P = 6.5 x 10(-4), OR 1.65, 95% CI 1.23-2.19, respectively). rs1363758 located in NLRP11 was associated with CD susceptibility [P = 0.002 (1.64, 1.19-2.25)], which was weakly confirmed in an independent case-cohort collection on joint analysis [P = 0.05, (1.28, 1-1.64)]. On sub-phenotype analysis, an interesting association between NLRP1 and skin extra-intestinal manifestations and colonic, inflammatory CD was identified. None of these results was replicated in the Wellcome Trust Case Control Consortium study and therefore need replication in a further large cohort.
[Show abstract][Hide abstract] ABSTRACT: Nucleotide-binding oligomerization domain-containing-2 (NOD2) acts as a bacterial sensor in dendritic cells (DCs), but it is not clear how bacterial recognition links with antigen presentation after NOD2 stimulation. NOD2 variants are associated with Crohn's disease, where breakdown in self-recognition of commensal bacteria leads to gastrointestinal inflammation. Here we show NOD2 triggering by muramyldipeptide induces autophagy in DCs. This effect requires receptor-interacting serine-threonine kinase-2 (RIPK-2), autophagy-related protein-5 (ATG5), ATG7 and ATG16L1 but not NLR family, pyrin domain containing-3 (NALP3).We show that NOD2-mediated autophagy is required for both bacterial handling and generation of major histocompatibility complex (MHC) class II antigen-specific CD4(+) T cell responses in DCs. DCs from individuals with Crohn's disease expressing Crohn's disease-associated NOD2 or ATG16L1 risk variants are defective in autophagy induction, bacterial trafficking and antigen presentation. Our findings link two Crohn's disease-associated susceptibility genes in a single functional pathway and reveal defects in this pathway in Crohn's disease DCs that could lead to bacterial persistence via impaired lysosomal destruction and immune mediated clearance.
Nature medicine 12/2009; 16(1):90-7. · 27.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Linkage in families and association in population case-control investigations have clearly shown that genes within the major histocompatibility complex region on chromosome 6p are relevant to the susceptibility and pathogenesis of ulcerative colitis (UC) and Crohn's disease. However, identifying the causative variants by fine mapping has not been conclusive. In this study using 58 single nucleotide polymorphisms (SNPs) with 616 UC cases, there was significant association with SNP rs2294881 of the (butyrophilin-like 2) BTNL2 gene with odds ratio (OR) = 2.80, confidence interval (CI) = 1.62-4.84 and P = 5.69 x 10(-4) (P(Bonferroni) = 3.3 x 10(-2)) and replication of SNP rs9268480. The missense SNP rs2076523 (K196E) showed novel association with a subset of UC cases with colectomy (n = 126), OR = 0.25, CI = 0.11-0.58 and P = 4.42 x 10(-4) (P(Bonferroni) = 2.56 x 10(-2)). These three associated variants within the BTNL2 gene were neither in linkage disequilibrium with each other nor correlated with the SNPs tagging the human leukocyte antigen (HLA)-DRB1*1502 and HLA-DRB1*0301 alleles.
[Show abstract][Hide abstract] ABSTRACT: Genetic variation in myosin IXB (MYO9B) was found to be associated with ulcerative colitis (UC) in a recent collaborative study. A nonsynonymous single nucleotide polymorphism (SNP) rs1545620 at the 3' end of the gene was found to be significantly associated with UC and weakly associated with Crohn's disease (CD). The aim of our current study was to replicate these findings in an independent UC cohort and to investigate association with CD. We also investigated subphenotype association and interactions with CARD15, IL23R, ATG16L1, and the IBD5 risk haplotype.
In all, 652 CD patients, 650 UC patients, and 1190 controls were genotyped for 8 MYO9B SNPs. Haplotype testing, epistasis testing with known polymorphisms, and subphenotype analysis were performed.
An intronic SNP rs2305767 in the MYO9B gene was associated with inflammatory bowel disease (IBD) overall (corrected P-value 0.002, odds ratio [OR] 0.76, 95% confidence interval [CI] 0.67-0.86). On individual disease analysis an association was found with CD (corrected P-value 0.001, OR 0.62, 95% CI 0.53-0.73) but not with UC. Analysis of the common MYO9B haplotypes showed significant association for CD and UC alone and IBD overall. No subphenotypic association was found. These data support an association between CD and SNPs in MYO9B independent of the established effects of SNPs in CARD15, IL23R, ATG16L1, and the IBD5 haplotype. There was no evidence of epistasis between SNPs in MYO9B and these established genes.
MYO9B variants may be involved in IBD pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Reduced expression of Paneth cell antimicrobial alpha-defensins, human defensin (HD)-5 and -6, characterizes Crohn's disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathway transcription factor, orchestrates Paneth cell differentiation, directly regulates the expression of HD-5 and -6, and was previously associated with the decrease of these antimicrobial peptides in a subset of ileal CD. To investigate a potential genetic association of TCF-4 with ileal CD, we sequenced 2.1 kb of the 5' flanking region of TCF-4 in a small group of ileal CD patients and controls (n = 10 each). We identified eight single nucleotide polymorphisms (SNPs), of which three (rs3814570, rs10885394, rs10885395) were in linkage disequilibrium and found more frequently in patients; one (rs3814570) was thereby located in a predicted regulatory region. We carried out high-throughput analysis of this SNP in three cohorts of inflammatory bowel disease (IBD) patients and controls. Overall 1399 healthy individuals, 785 ulcerative colitis (UC) patients, 225 CD patients with colonic disease only and 784 CD patients with ileal involvement were used to determine frequency distributions. We found an association of rs3814570 with ileal CD but neither with colonic CD or UC, in a combined analysis (allele positivity: OR 1.27, 95% CI 1.07 to 1.52, p = 0.00737), which was the strongest in ileal CD patients with stricturing behaviour (allele frequency: OR 1.32, 95% CI 1.08 to1.62, p = 0.00686) or an additional involvement of the upper GIT (allele frequency: OR 1.38, 95% CI 1.03 to1.84, p = 0.02882). The newly identified genetic association of TCF-4 with ileal CD provides evidence that the decrease in Paneth cell alpha-defensins is a primary factor in disease pathogenesis.
PLoS ONE 02/2009; 4(2):e4496. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Twin studies and large-scale population studies have confirmed an increased sibling risk for both Crohn's disease (CD) and ulcerative colitis (UC). Unlike single gene disorders, CD and UC are thought to result from a complex interplay of multiple genes and environmental factors. The confirmation of CARD15/NOD2 as a CD susceptibility gene in the late 1990s caused much excitement in the field of complex diseases in general and since then, the rapid rate of progress in molecular genetics, with the advent of large-scale affordable genotyping techniques, has resulted in large collaborations and the identification of over 30 inflammatory bowel disease (IBD)-associated genes. In particular, the importance of the innate immune system has been reaffirmed with the identification of IRGM and ATG16L1 genes in the autophagy pathway as CD susceptibility genes. Disturbance in the adaptive immune system, in particular the IL-23/Th17 axis, has also shown to be of importance for IBD overall. In this era of genome-wide association studies it may be possible to, at last, identify the multiple genes involved in IBD and thus improve our understanding of the genotype-phenotype correlation and improve treatment.
[Show abstract][Hide abstract] ABSTRACT: Clinical, serological, and molecular data support the existence of discrete subsets of Crohn's disease (CD) defined by location of disease. Little is known about the epidemiology and natural history of isolated CD of the colon (Montreal Classification L2) because most studies have not accurately distinguished it from ileocolonic disease. Our objectives were to describe the clinical features and natural history of isolated colonic CD in a rigorously characterized patient cohort and to investigate the association of polymorphisms in a number of genes with colonic location of disease and disease behavior.
Patients with L2 disease were identified from a database of 675 CD patients. Only patients with a normal small bowel enema (70%), ileoscopy alone (30%), or both (20%) were included. Genotyping was performed using PCR-SSP or the iPLEX platform.
In all, 135 patients were classified with L2 disease. L2 disease was more common in women (74.0% versus 58.0%; P = 0.0004; odds ratio [OR] = 2.11, 95% confidence interval [CI] 1.36-3.26) and in never smokers (48.9% versus 36.9%; P = 0.008; OR = 1.64, 95% CI 1.09-2.45); 20.7% underwent colonic resection for severe disease. We confirmed that carriage of the HLA-DRB1*0103 allele is strongly associated with isolated colonic CD (14.9% versus 4.0%; P = 0.000016; OR 4.6, 95% CI 2.25-9.47) and report the novel association of this allele with time to first surgical event (log rank P = 0.001). There was no association with any of the known CD susceptibility loci (NOD2, IBD5, NOD1, IL23R, ATG16L1) and isolated colonic CD. A nonsynonymous polymorphism in MEKK1 (rs832582) was associated with CD susceptibility overall (15% versus 19%; P = 0.0083; OR = 1.28, 95% CI 1.07-1.54). The association was strongest in those patients not carrying a NOD2 mutation and had no effect on disease location.
This study describes the clinical features of isolated colonic CD and demonstrates the importance of the HLA region in determining the molecular basis of colonic inflammation.
[Show abstract][Hide abstract] ABSTRACT: Genomewide linkage studies identified chromosome 3p21 as an IBD locus. Genomewide association studies have supported this locus and the Wellcome Trust Case Control Consortium (WTCCC) study narrowed it to a 0.6 Mb region. Our objectives were to perform a 2-stage candidate gene association study of the 3p locus and to identify linkage disequilibrium (LD) between significant single-nucleotide polymorphisms (SNPs) and an Oxfordshire subset (n = 282) of the WTCCC as well as the HapMap SNPs.
A total of 197 SNPs in 53 genes from the 3p locus were genotyped on the Illumina platform in a screening cohort of 469 Crohn's disease (CD) patients and 461 controls. Significant associations were then genotyped on the iPLEX platform in the original as well as a second cohort of 139 CD patients, 670 ulcerative colitis (UC) patients, and 1131 controls. All cases and controls were Caucasian and from the Oxfordshire region of the UK.
An intronic SNP rs1128535 in the TRAIP gene was associated with CD in the screening and validation cohorts (combined [n = 608] P = 0.0004 [corrected 0.002], odds ratio [OR] 0.77, 95% confidence interval [CI], 0.67-0.89]). No association was seen for UC. Epistasis was seen with the common CARD15 mutations (P = 0.00003 [corrected 0.0006], OR 0.48, 95% CI, 0.34-0.68). No LD was demonstrated with the WTCCC SNPs. Strong LD was demonstrated with 2 nonsynonymous HapMap SNPs in the MST1R gene in an adjacent LD block to the peak WTCCC association, suggesting a distinct association signal.
The LD with these functional MST1R variants implicate this gene as having a possible role in CD pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: A North American genome-wide single nucleotide polymorphism (SNP) association study identified IL23R as a novel inflammatory bowel disease (IBD) susceptibility gene. Association was reported with multiple risk variants in the centromeric portion of IL23R in 3 large independent cohorts. The aims of this study were to replicate the association of IL23R with Crohn's disease (CD), examine subphenotype relationships, and look for evidence of epistasis with the known CD susceptibility gene CARD15 and susceptibility haplotype IBD5 in a large collection of CD patients. We further investigated the relationship between IL23R and ulcerative colitis (UC).
In all, 604 CD and 647 UC patients who had been rigorously phenotyped and who had been recruited from a single UK center were used in this study. Controls were either spouses of patients (141) or were recruited from well-person clinics (993). Eight SNPs were genotyped using MassArray (Sequenom). All 8 SNPs genotyped were significantly associated with CD.
The association with the nonsynonymous SNP rs11209026 was confirmed (P=6.65x10(-6), odds ratio [OR], 0.43, 95% confidence interval [CI]: 0.29-0.64). The most significant SNP in our study was rs7517847 (P=4.9x10(-9), OR 0.65, 0.56-0.75), which is statistically independent of rs11209026. Preliminary evidence suggests an epistatic interaction with the IBD5 risk haplotype. The effects of mutations in this IL23R appear weaker in UC (P=0.008, OR 0.63, 0.45-0.89 and 0.005 OR, 0.81, 0.71-0.94, respectively). No subphenotype associations were identified.
We confirmed the findings that IL23R is a susceptibility gene for IBD with suggestive epistasis with the IBD5 locus in the CD population.
[Show abstract][Hide abstract] ABSTRACT: A German genome-wide nonsynonymous single nucleotide polymorphism (nsSNP) association study identified ATG16L1 as a Crohn's disease (CD) susceptibility gene. The association appeared to be confined to the nsSNP rs2,241,880 and was confirmed in 2 German independent case-control collections (combined P = 4.0 x 10(-8), odds ratio [OR] 1.45; 95% confidence interval [CI]: 1.21-1.74), a CD transmission disequilibrium test (TDT) collection, and an independent UK cohort. A weak statistical interaction with CARD15 was demonstrated. No association with ulcerative colitis (UC) was demonstrated. The aims of the study were to replicate the association with CD, examine subphenotype associations and statistical interactions with CARD15, IL23R, and the IBD5 risk haplotype, as well as explore the association with UC.
The study included 645 CD and 676 UC rigorously phenotyped patients recruited from a single UK center. Unaffected controls comprised either spouses of patients (141) or individuals recruited from well-person clinics (1,049). The nsSNP rs2,241,880 was genotyped using MassArray (Sequenom).
A strong association with CD was demonstrated (P = 2.33 x 10(-7), OR 1.45 [1.25-1.67]), but no significant association was demonstrated with any subphenotype. We failed to replicate the reported interaction between rs2,241,880 and the CARD15 low-risk haplotypes dd and Dd. No significant statistical interaction with the 3 known CD susceptibility genes was seen. No association with UC susceptibility (P = 0.37, OR 1.06 [0.93-1.22]), or any UC subphenotype was identified.
We confirmed the findings that ATG16L1 is a CD susceptibility gene and found no evidence of interaction with CARD15, IL23R, or IBD5.
[Show abstract][Hide abstract] ABSTRACT: Clinical trials on novel drug therapies require clear criteria for patient selection and agreed definitions of disease remission. This principle has been successfully applied in the field of rheumatology where agreed disease scoring systems have allowed multi-centre collaborations and facilitated audit across treatment centres. Unfortunately in ulcerative colitis this consensus is lacking. Thirteen scoring systems have been developed but none have been properly validated. Most trials choose different endpoints and activity indices, making comparison of results from different trials extremely difficult. International consensus on endoscopic, clinical and histological scoring systems is essential as these are the key components used to determine entry criteria and outcome measurements in clinical trials on ulcerative colitis. With multiple new therapies under development, there is a pressing need for consensus to be reached.