Leif Törkvist

Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci

We undertook a meta-analysis of six Crohns disease genome-wide association studies (GWAS) comprising 6,333 affected individuals (cases) and 15,056 controls and followed up the top association signals in 15,694 cases, 14,026 controls and 414 parent-offspring trios. We identified 30 new susceptibility loci meeting genome-wide significance (P < 5 × 10−8). A series of in silico analyses highlighted particular genes within these loci and, together with manual curation, implicated functionally interesting candidate genes including SMAD3, ERAP2, IL10, IL2RA, TYK2, FUT2, DNMT3A, DENND1B, BACH2 and TAGAP. Combined with previously confirmed loci, these results identify 71 distinct loci with genome-wide significant evidence for association with Crohns disease.

Deep resequencing of GWAS loci identifies independent rare variants associated with inflammatory bowel disease

More than 1,000 susceptibility loci have been identified through genome-wide association studies (GWAS) of common variants; however, the specific genes and full allelic spectrum of causal variants underlying these findings have not yet been defined. Here we used pooled next-generation sequencing to study 56 genes from regions associated with Crohns disease in 350 cases and 350 controls. Through follow-up genotyping of 70 rare and low-frequency protein-altering variants in nine independent case-control series (16,054 Crohns disease cases, 12,153 ulcerative colitis cases and 17,575 healthy controls), we identified four additional independent risk factors in NOD2, two additional protective variants in IL23R, a highly significant association with a protective splice variant in CARD9 (P < 1 × 10−16, odds ratio ≈ 0.29) and additional associations with coding variants in IL18RAP, CUL2, C1orf106, PTPN22 and MUC19. We extend the results of successful GWAS by identifying new, rare and probably functional variants that could aid functional experiments and predictive models.

Genome-wide association identifies multiple ulcerative colitis susceptibility loci

Ulcerative colitis is a chronic, relapsing inflammatory condition of the gastrointestinal tract with a complex genetic and environmental etiology. In an effort to identify genetic variation underlying ulcerative colitis risk, we present two distinct genome-wide association studies of ulcerative colitis and their joint analysis with a previously published scan, comprising, in aggregate, 2,693 individuals with ulcerative colitis and 6,791 control subjects. Fifty-nine SNPs from 14 independent loci attained an association significance of P < 10−5. Seven of these loci exceeded genome-wide significance (P < 5 × 10−8). After testing an independent cohort of 2,009 cases of ulcerative colitis and 1,580 controls, we identified 13 loci that were significantly associated with ulcerative colitis (P < 5 × 10−8), including the immunoglobulin receptor gene FCGR2A, 5p15, 2p16 and ORMDL3 (orosomucoid1-like 3). We confirmed association with 14 previously identified ulcerative colitis susceptibility loci, and an analysis of acknowledged Crohns disease loci showed that roughly half of the known Crohns disease associations are shared with ulcerative colitis. These data implicate approximately 30 loci in ulcerative colitis, thereby providing insight into disease pathogenesis.

Combined Polymorphisms in Genes Encoding the Inflammasome Components NALP3 and CARD8 Confer Susceptibility to Crohn's Disease in Swedish Men

OBJECTIVES:Crohns disease (CD) is characterized by overproduction of proinflammatory cytokines like interleukin (IL)-1β. Production of mature IL-1β is dependent on a caspase-1-activatingprotein complex called the NALP3 inflammasome, composed of NALP3, ASC, and CARD8. NALP3 shares structural similarities with Nod2, and both of these proteins are required forbacteria-induced IL-1β secretion. The combination of the polymorphisms CARD8 (C10X)and NALP3 (Q705K) was recently shown to be associated with rheumatoid arthritis.Our aim was to investigate whether these combined polymorphisms play a role in the susceptibility to CD.METHODS:The study included 498 CD patients in two cohorts from different regions and 742 control individuals from a Swedish population. DNA was isolated from whole blood. Polymorphisms of (Q705K) NALP3 and (C10X) CARD8, as well as the Nod2 variants, R702W and G908R, were genotyped using the Taqman single nucleotide polymorphism assay. The Nod2 frameshift mutation, L1007fs, was detected by Megabace SNuPe genotyping.RESULTS:Our results show that men who have both the C10X and Q705K alleles in CARD8 and NALP3, and who express wild-type alleles of Nod2 are at an increased risk of developing CD (odds ratio, OR: 3.40 range: 1.32–8.76); P=0.011). No association with these polymorphisms was found in women (OR: 0.89 (range: 0.44–1.77); P=0.74).CONCLUSIONS:We suggest a role for combined polymorphisms in CARD8 and NALP3 in the development of CD in men, with obvious sex differences in the genetic susceptibility pattern. These findings give further support to the importance of innate immune responses in CD.

Association of TNFSF15 polymorphism with irritable bowel syndrome

Background Irritable bowel syndrome (IBS) is the most common gastrointestinal disorder, affecting more than 10% of the general population worldwide. Although a genetic component is suspected, unambiguous susceptibility genes have so far not been identified. This study tested the hypothesis that genes contributing to epithelial barrier integrity, control of mucosal immune responses and interactions with bacteria in the gut are associated with IBS. Methods Single nucleotide polymorphisms (SNPs) corresponding to top signals of association with Crohns disease at 30 known susceptibility loci were tested for their effect on IBS risk in 1992 individuals from two independent case–control cohorts from Sweden and the USA. Association tests included a conservative Bonferroni correction for multiple comparisons, and were also performed on specific subgroups of patients characterised by constipation (IBS-C), diarrhoea (IBS-D) or alternating constipation and diarrhoea (IBS-A). Results The Crohns disease risk allele rs4263839 G in the TNFSF15 gene was significantly associated with an increased risk of both IBS (p=2.2×10−5; OR 1.37) and more pronouncedly, IBS-C (p=8.7×10−7; OR 1.79) in the entire sample. Similar associations and risk effects of the same magnitude were observed in the two cohorts analysed separately. A correlation between rs4263839 genotype and TNFSF15 mRNA expression was detected both in peripheral blood and in rectal mucosal biopsies from healthy individuals (combined p=0.0033). Conclusions TNFSF15 is a susceptibility gene for IBS and IBS constipation. As TL1A, the protein encoded by TNFSF15, contributes to the modulation of inflammatory responses, the results support a role of immune activation in IBS.

Deep Resequencing of GWAS Loci Identifies Rare Variants in CARD9, IL23R and RNF186 That Are Associated with Ulcerative Colitis

Genome-wide association studies and follow-up meta-analyses in Crohns 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.

IL23R in the Swedish, Finnish, Hungarian and Italian populations: association with IBD and psoriasis, and linkage to celiac disease

BackgroundAssociation of the interleukin-23 receptor (IL23R) with inflammatory bowel disease (IBD) has been confirmed in several populations. IL23R also associates with psoriasis, suggesting that the gene may be an important candidate for many chronic inflammatory diseases.MethodsWe studied association of single-nucleotide variants in IL23R with IBD in Swedish patients, in both Crohns disease (CD) and ulcerative colitis (UC) subsets. The same genetic variants were also studied in Finnish patients with psoriasis or celiac disease, and in Hungarian and Italian patients with celiac disease.ResultsAssociation of IL23R with IBD was replicated in our Swedish patients, and linkage and association of the IL23R region with psoriasis was found in the Finnish population. The IL23R region was also linked to celiac disease in Finnish families, but no association of IL23R variants with celiac disease was found in the Finnish, Hungarian or Italian samples.ConclusionOur study is the first to demonstrate association of IL23R with CD and UC in Swedish patients with IBD. It is also the first study to report linkage and association of the IL23R region with psoriasis in the Finnish population. Importantly, this is the first report of linkage of the IL23R region to celiac disease, a chronic inflammatory condition in which IL23R has not been previously implicated.

CD98 expression modulates intestinal homeostasis, inflammation, and colitis-associated cancer in mice

Expression of the transmembrane glycoprotein CD98 (encoded by SLC3A2) is increased in intestinal inflammatory conditions, such as inflammatory bowel disease (IBD), and in various carcinomas, yet its pathogenetic role remains unknown. By generating gain- and loss-of-function mouse models with genetically manipulated CD98 expression specifically in intestinal epithelial cells (IECs), we explored the role of CD98 in intestinal homeostasis, inflammation, and colitis-associated tumorigenesis. IEC-specific CD98 overexpression induced gut homeostatic defects and increased inflammatory responses to DSS-induced colitis, promoting colitis-associated tumorigenesis in mice. Further analysis indicated that the ability of IEC-specific CD98 overexpression to induce tumorigenesis was linked to its capacity to induce barrier dysfunction and to stimulate cell proliferation and production of proinflammatory mediators. To validate these results, we constructed mice carrying conditional floxed Slc3a2 alleles and crossed them with Villin-Cre mice such that CD98 was downregulated only in IECs. These mice exhibited attenuated inflammatory responses and resistance to both DSS-induced colitis and colitis-associated tumorigenesis. Together, our data show that intestinal CD98 expression has a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in IECs therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as IBD and colitis-associated cancer.

Analysis of germline GLI1 variation implicates hedgehog signalling in the regulation of intestinal inflammatory pathways.

Background Ulcerative colitis (UC) and Crohns disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis. Methods and Findings Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C→G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09–1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1+/lacZ mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1+/lacZ mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model. Conclusions HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.

PepT1 oligopeptide transporter (SLC15A1) gene polymorphism in inflammatory bowel disease

Background: Human polymorphisms affecting gut epithelial barrier and interactions with bacteria predispose to the inflammatory bowel diseases (IBD) Crohns disease (CD) and ulcerative colitis (UC). The intestinal transporter PepT1, encoded by the SLC15A1 gene, mediates intracellular uptake of bacterial products that can induce inflammation and NF‐&kgr;B activation upon binding to NOD2, a protein often mutated in CD. Hence, we tested SLC15A1 polymorphisms for association with IBD. Methods: Twelve SLC15A1 single nucleotide polymorphisms (SNPs) were genotyped in 1783 individuals from 2 cohorts of Swedish and Finnish IBD patients and controls. An in vitro system was set up to evaluate the potential impact of SLC15A1 polymorphism on PepT1 transporter function by quantification of NOD2‐mediated activation of NF‐&kgr;B. Results: The common allele (C) of a coding polymorphism (rs2297322, Ser117Asn) was associated with CD susceptibility both in Sweden and in Finland, but with genetic effects in opposite directions (risk and protection, respectively). The best evidence of association was found in both populations when the analysis was performed on individuals not carrying NOD2 common risk alleles (Sweden allelic P = 0.0007, OR 1.97, 95% confidence interval [CI] 1.34–2.92; Finland genotype P = 0.0013, OR 0.63, 95% CI 0.44–0.90). The PepT1 variant encoded by the C allele (PepT1‐Ser117) was associated with reduced signaling downstream of NOD2 (P < 0.0001 compared to Pept1‐Asn117). Conclusions: A functional polymorphism in the SLC15A1 gene might be of relevance to inflammation and antibacterial responses in IBD. Whether this polymorphism truly contributes to disease susceptibility needs to be further addressed, and should stimulate additional studies in other populations. Inflamm Bowel Dis 2009