Mauro D'Amato

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.

Detection of Celiac Disease and Lymphocytic Enteropathy by Parallel Serology and Histopathology in a Population-Based Study

BACKGROUND & AIMS Although serologic analysis is used in diagnosis of celiac disease, histopathology is considered most reliable. We performed a prospective study to determine the clinical, pathologic, and serologic spectrum of celiac disease in a general population (Kalixanda study). METHODS A random sample of an adult general population (n = 1000) was analyzed by upper endoscopy, duodenal biopsy, and serologic analysis of tissue transglutaminase (tTg) levels; endomysial antibody (EMA) levels were analyzed in samples that were tTg+. The cut off values for diagnosis of celiac disease were villous atrophy with 40 intraepithelial lymphocytes (IELs)/100 enterocytes (ECs). RESULTS Samples from 33 subjects were tTg+, and 16 were EMA+. Histologic analysis identified 7 of 1000 subjects (0.7%) with celiac disease; all were tTg+, and 6 of 7 were EMA+. Another 26 subjects were tTg+ (7/26 EMA+). This was addressed by a second quantitative pathology study (nested case control design) using a threshold of 25 IELS/100 ECs. In this analysis, all 13 samples that were tTg+ and EMA+ had > or =25 IELs/100 ECs. In total, 16 subjects (1.6%) had serologic and histologic evidence of gluten-sensitive enteropathy. IELs were quantified in duodenal biopsy samples from seronegative individuals (n = 500); 19 (3.8%) had >25 IELs and lymphocytic duodenosis. CONCLUSIONS Measurement of > or =25 IELs/100 ECs correlated with serologic indicators of celiac disease; a higher IEL threshold could miss 50% of cases. Quantification of tTg is a sensitive test for celiac disease; diagnosis can be confirmed by observation of > or =25 IELs/100ECs in duodenal biopsy specimens. Lymphocytic enteropathy (celiac disease and lymphocytic duodenosis) is common in the population (5.4%).

Genome-wide association analysis identifies variation in vitamin D receptor and other host factors influencing the gut microbiota

Human gut microbiota is an important determinant for health and disease, and recent studies emphasize the numerous factors shaping its diversity. Here we performed a genome-wide association study (GWAS) of the gut microbiota using two cohorts from northern Germany totaling 1,812 individuals. Comprehensively controlling for diet and non-genetic parameters, we identify genome-wide significant associations for overall microbial variation and individual taxa at multiple genetic loci, including the VDR gene (encoding vitamin D receptor). We observe significant shifts in the microbiota of Vdr−/− mice relative to control mice and correlations between the microbiota and serum measurements of selected bile and fatty acids in humans, including known ligands and downstream metabolites of VDR. Genome-wide significant (P < 5 × 10−8) associations at multiple additional loci identify other important points of host–microbe intersection, notably several disease susceptibility genes and sterol metabolism pathway components. Non-genetic and genetic factors each account for approximately 10% of the variation in gut microbiota, whereby individual effects are relatively small.

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.

Semliki Forest Virus Nonstructural Protein 2 Is Involved in Suppression of the Type I Interferon Response

ABSTRACT The type I interferons (IFNs) are potent mediators of antiviral immunity, and many viruses have developed means to block their expression or their effects. Semliki Forest virus (SFV) infection induces rapid and profound silencing of host cell gene expression, a process believed to be important for the inhibition of the IFN response. In SFV-infected cells, a large proportion of the nonstructural protein nsp2 is found in the nucleus, but a role for this localization has not been described. In this work we demonstrate that a viral mutant, SFV4-RDR, in which the nuclear localization sequence of nsp2 has been rendered inactive, induces a significantly more robust IFN response in infected cells. This mutant virus replicates at a rate similar to that of the parental SFV4 strain and also shuts off host cell gene expression to similar levels, indicating that the general cellular shutoff is not responsible for the inhibition of IFN expression. Further, the rate of virus-induced nuclear translocation of early IFN transcription factors was not found to differ between the wild-type and mutant viruses, indicating that the effect of nsp2 is at a later stage. These results provide novel information about the mode of action of this viral IFN antagonist.

Association Between Variants of PRDM1 and NDP52 and Crohn's Disease, Based on Exome Sequencing and Functional Studies

BACKGROUND & AIMS Genome-wide association studies (GWAS) have identified 140 Crohns disease (CD) susceptibility loci. For most loci, the variants that cause disease are not known and the genes affected by these variants have not been identified. We aimed to identify variants that cause CD through detailed sequencing, genetic association, expression, and functional studies. METHODS We sequenced whole exomes of 42 unrelated subjects with CD and 5 healthy subjects (controls) and then filtered single nucleotide variants by incorporating association results from meta-analyses of CD GWAS and in silico mutation effect prediction algorithms. We then genotyped 9348 subjects with CD, 2868 subjects with ulcerative colitis, and 14,567 control subjects and associated variants analyzed in functional studies using materials from subjects and controls and in vitro model systems. RESULTS We identified rare missense mutations in PR domain-containing 1 (PRDM1) and associated these with CD. These mutations increased proliferation of T cells and secretion of cytokines on activation and increased expression of the adhesion molecule L-selectin. A common CD risk allele, identified in GWAS, correlated with reduced expression of PRDM1 in ileal biopsy specimens and peripheral blood mononuclear cells (combined P = 1.6 × 10(-8)). We identified an association between CD and a common missense variant, Val248Ala, in nuclear domain 10 protein 52 (NDP52) (P = 4.83 × 10(-9)). We found that this variant impairs the regulatory functions of NDP52 to inhibit nuclear factor κB activation of genes that regulate inflammation and affect the stability of proteins in Toll-like receptor pathways. CONCLUSIONS We have extended the results of GWAS and provide evidence that variants in PRDM1 and NDP52 determine susceptibility to CD. PRDM1 maps adjacent to a CD interval identified in GWAS and encodes a transcription factor expressed by T and B cells. NDP52 is an adaptor protein that functions in selective autophagy of intracellular bacteria and signaling molecules, supporting the role of autophagy in the pathogenesis of CD.

Dynamics of the human gut microbiome in inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterized by flares of inflammation with a periodic need for increased medication and sometimes even surgery. The aetiology of IBD is partly attributed to a deregulated immune response to gut microbiome dysbiosis. Cross-sectional studies have revealed microbial signatures for different IBD subtypes, including ulcerative colitis, colonic Crohns disease and ileal Crohns disease. Although IBD is dynamic, microbiome studies have primarily focused on single time points or a few individuals. Here, we dissect the long-term dynamic behaviour of the gut microbiome in IBD and differentiate this from normal variation. Microbiomes of IBD subjects fluctuate more than those of healthy individuals, based on deviation from a newly defined healthy plane (HP). Ileal Crohns disease subjects deviated most from the HP, especially subjects with surgical resection. Intriguingly, the microbiomes of some IBD subjects periodically visited the HP then deviated away from it. Inflammation was not directly correlated with distance to the healthy plane, but there was some correlation between observed dramatic fluctuations in the gut microbiome and intensified medication due to a flare of the disease. These results will help guide therapies that will redirect the gut microbiome towards a healthy state and maintain remission in IBD.