Marijn C. Visschedijk

Analysis of SNPs with an effect on gene expression identifies UBE2L3 and BCL3 as potential new risk genes for Crohn's disease

Genome-wide association studies (GWAS) for Crohns disease (CD) have identified loci explaining approximately 20% of the total genetic risk of CD. Part of the other genetic risk loci is probably partly hidden among signals discarded by the multiple testing correction needed in the analysis of GWAS data. Strategies for finding these hidden loci require large replication cohorts and are costly to perform. We adopted a strategy of selecting SNPs for follow-up that showed a correlation to gene expression [cis-expression quantitative trait loci (eQTLs)] since these have been shown more likely to be trait-associated. First we show that there is an overrepresentation of cis-eQTLs in the known CD-associated loci. Then SNPs were selected for follow-up by screening the top 500 SNP hits from a CD GWAS data set. We identified 10 cis-eQTL SNPs. These 10 SNPs were tested for association with CD in two independent cohorts of Dutch CD patients (1539) and healthy controls (2648). In a combined analysis, we identified two cis-eQTL SNPs that were associated with CD rs2298428 in UBE2L3 (P=5.22x10(-5)) and rs2927488 in BCL3 (P=2.94x10(-4)). After adding additional publicly available data from a previously reported meta-analysis, the association with rs2298428 almost reached genome-wide significance (P=2.40x10(-7)) and the association with rs2927488 was corroborated (P=6.46x10(-4)). We have identified UBE2L3 and BCL3 as likely novel risk genes for CD. UBE2L3 is also associated with other immune-mediated diseases. These results show that eQTL-based pre-selection for follow-up is a useful approach for identifying risk loci from a moderately sized GWAS.

Interplay of host genetics and gut microbiota underlying the onset and clinical presentation of inflammatory bowel disease

Objective Patients with IBD display substantial heterogeneity in clinical characteristics. We hypothesise that individual differences in the complex interaction of the host genome and the gut microbiota can explain the onset and the heterogeneous presentation of IBD. Therefore, we performed a case–control analysis of the gut microbiota, the host genome and the clinical phenotypes of IBD. Design Stool samples, peripheral blood and extensive phenotype data were collected from 313 patients with IBD and 582 truly healthy controls, selected from a population cohort. The gut microbiota composition was assessed by tag-sequencing the 16S rRNA gene. All participants were genotyped. We composed genetic risk scores from 11 functional genetic variants proven to be associated with IBD in genes that are directly involved in the bacterial handling in the gut: NOD2, CARD9, ATG16L1, IRGM and FUT2. Results Strikingly, we observed significant alterations of the gut microbiota of healthy individuals with a high genetic risk for IBD: the IBD genetic risk score was significantly associated with a decrease in the genus Roseburia in healthy controls (false discovery rate 0.017). Moreover, disease location was a major determinant of the gut microbiota: the gut microbiota of patients with colonic Crohns disease (CD) is different from that of patients with ileal CD, with a decrease in alpha diversity associated to ileal disease (p=3.28×10−13). Conclusions We show for the first time that genetic risk variants associated with IBD influence the gut microbiota in healthy individuals. Roseburia spp are acetate-to-butyrate converters, and a decrease has already been observed in patients with IBD.

Cell Specific eQTL Analysis without Sorting Cells

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn’s disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.

HNF4α and CDH1 are associated with ulcerative colitis in a Dutch cohort

Background: Inflammatory bowel diseases (IBDs), consisting of ulcerative colitis (UC) and Crohns disease (CD), are complex disorders with multiple genes contributing to disease pathogenesis. A recent genome‐wide association scan identified three novel susceptibility loci for UC: HNF4&agr;, CDH1, and LAMB1. We performed an analysis of these three loci in an independent cohort. Methods: In all, 821 UC patients and 1260 healthy controls of central European Caucasian descent were genotyped for single nucleotide polymorphisms (SNPs): rs6017342 (HNF4&agr;), rs1728785 (CDH1), and rs6949033 (LAMB1). Differences in allele and genotype distribution in cases and controls were tested for significance with the χ2 test. Results: Allelic association analysis showed that SNP rs6017342 in the HNF4&agr; locus was strongly associated with UC (P = 1,04 × 10−11, odds ratio [OR] = 1.56, 95% confidence interval [CI] = 1.37–1.77) and SNP rs1728785 (CDH1) was associated with P = 0.01 (OR = 1.23, 95% CI = 1.05–1.44). SNP rs6949033 in LAMB1 was not associated in our cohort (P = 0.12, OR = 1.11, 95% CI = 0.97–1.26). We found an association for SNP rs6949033 (LAMB1) for disease limited to the rectum (P = 0.02). However, this association was lost after correcting for multiple testing. No further specific subphenotype associations were identified. Conclusions: This is the first independent study to replicate the HNF4&agr; and CDH1 loci as susceptibility loci for UC. The main candidate genes in these risk loci play important roles in the maintenance of the integrity of the epithelial barrier, highlighting the importance of the mucosal barrier function for UC pathogenesis. (Inflamm Bowel Dis 2010)

A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis

Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10−7, odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain.

Down the line from genome-wide association studies in inflammatory bowel disease: the resulting clinical benefits and the outlook for the future

Inflammatory bowel disease (IBD), consisting of Crohn’s disease and ulcerative colitis, is a chronic inflammatory disease of the gut. The etiology of IBD is complex, involving genetic as well as environmental factors. Genetic studies have identified 163 genetic risk loci for IBD, which have led to new insights into the biological mechanisms of the disease. The currently known IBD risk loci show an almost 75% overlap with genetic risk loci for other immune mediated diseases. Current studies are focused on the translation of the identified risk loci to clinical practice. The first steps towards this translation are being taken with the identification of genetic risk factors for drugs toxicity, specific disease course and response to therapy. In this review we will discuss how the IBD genetic risk loci were identified and how this knowledge can be translated towards clinical practice.

A large variety of clinical features and concomitant disorders in celiac disease – A cohort study in the Netherlands

BACKGROUND AND AIMS Celiac disease (CeD) is a gluten triggered, immune-mediated disease of the small intestine. Few clinical cohort descriptions are available, despite the diverse clinical picture. This study provides an overview of a large Dutch CeD cohort focusing on presenting symptoms, co-occurrence of immune mediated diseases (IMD) and malignancies. METHODS We performed a retrospective study in a Dutch university and a non-university medical hospital and included only biopsy proven (≥Marsh type 2 classification) CeD patients. RESULTS 412 patients were included, selected from 9468 small-bowel biopsy pathology reports and financial codes. Classical symptoms were present in approximately one third of the cohort (diarrhea (37.4%), fatigue (35.0%), weight loss (31.6%), abdominal pain (33.3%)). Atypical symptoms as constipation (10.4%) and reflux (12.4%) were reported as well. 11.7% was diagnosed without reported symptoms. In 25.2% concomitant IMD occurred (most prevalent: type 1 diabetes mellitus (4.9%), microscopic colitis (4.9%), immune mediated-thyroid disease (4.1%)). CeD patients with a concomitant IMD were diagnosed at a significantly higher age compared to those without (P=0.002). Malignancies occurred in 53 cases (12.9%), including eight Enteropathy Associated T-cell Lymphomas. CONCLUSION This is the first study describing a CeD cohort in such detail in the Netherlands and highlights the clinical heterogeneity and importance of screening for concomitant diseases in CeD.

Pooled Resequencing of 122 Ulcerative Colitis Genes in a Large Dutch Cohort Suggests Population-Specific Associations of Rare Variants in MUC2

Genome-wide association studies have revealed several common genetic risk variants for ulcerative colitis (UC). However, little is known about the contribution of rare, large effect genetic variants to UC susceptibility. In this study, we performed a deep targeted re-sequencing of 122 genes in Dutch UC patients in order to investigate the contribution of rare variants to the genetic susceptibility to UC. The selection of genes consists of 111 established human UC susceptibility genes and 11 genes that lead to spontaneous colitis when knocked-out in mice. In addition, we sequenced the promoter regions of 45 genes where known variants exert cis-eQTL-effects. Targeted pooled re-sequencing was performed on DNA of 790 Dutch UC cases. The Genome of the Netherlands project provided sequence data of 500 healthy controls. After quality control and prioritization based on allele frequency and pathogenicity probability, follow-up genotyping of 171 rare variants was performed on 1021 Dutch UC cases and 1166 Dutch controls. Single-variant association and gene-based analyses identified an association of rare variants in the MUC2 gene with UC. The associated variants in the Dutch population could not be replicated in a German replication cohort (1026 UC cases, 3532 controls). In conclusion, this study has identified a putative role for MUC2 on UC susceptibility in the Dutch population and suggests a population-specific contribution of rare variants to UC.

IBD risk loci are enriched in multigenic regulatory modules encompassing putative causative genes

GWAS have identified >200 risk loci for Inflammatory Bowel Disease (IBD). The majority of disease associations are known to be driven by regulatory variants. To identify the putative causative genes that are perturbed by these variants, we generate a large transcriptome data set (nine disease-relevant cell types) and identify 23,650 cis-eQTL. We show that these are determined by ∼9720 regulatory modules, of which ∼3000 operate in multiple tissues and ∼970 on multiple genes. We identify regulatory modules that drive the disease association for 63 of the 200 risk loci, and show that these are enriched in multigenic modules. Based on these analyses, we resequence 45 of the corresponding 100 candidate genes in 6600 Crohn disease (CD) cases and 5500 controls, and show with burden tests that they include likely causative genes. Our analyses indicate that ≥10-fold larger sample sizes will be required to demonstrate the causality of individual genes using this approach.Most of the more than 200 known genetic risk loci for inflammatory bowel disease (IBD) reside in regulatory regions. Here, the authors provide eQTL datasets for six circulating immune cell types and ileal, colonic and rectal biopsies to map regulatory modules and identify potential causative genes for IBD.

Colon ischemia: Right-sided colon involvement has a different presentation, etiology and worse outcome. A large retrospective cohort study in histology proven patients

BACKGROUND Colon ischemia (CI), is generally considered a non-occlusive mesenteric ischemia disorder that usually runs a benign course, but right-sided involvement (RCI) has been associated with worse outcome. The poor outcome of RCI has been associated with comorbidity, but more recently also with occlusions of the mesenteric arteries. We performed a retrospective analysis of a large cohort of CI-patients to assess differences in presentation, etiology, and comorbidity between right-sided colon ischemia (RCI) and non-right-sided colon ischemia (NRCI), and their relation to outcome. METHODS We performed a retrospective cohort study in two centers from 2000 to 2011 for CI and analyzed clinical presentation, etiology, treatment and outcome. Diagnosis was based on full colonoscopy and/or surgical findings and confirmed by histopathology. RESULTS 239 patients were included (mean age 69, 52% female). RCI was found in 48% and NRCI in 52%. Patients with NRCI presented more often with rectal bleeding (87% vs. 45%; p<0.001). In RCI more nausea (58% vs. 39%; p=0.013), weight loss (56% vs. 19%; p<0.001), paralytic ileus (32% vs. 18%; p=0.018) and peritoneal signs (27% vs. 7%; p<0.001) was observed compared to NRCI. The cause of CI was more often idiopathic in NRCI (46% vs. 26%; p=0.002); an occlusive cause was seen more often in RCI (26.3 vs 2.4%, p<0.0001). RCI patients had longer hospital stay (15 vs. 8 days, p<0.001), need for surgery (61% vs. 34%, p<0.001), and trend toward higher 30-day in-hospital mortality (20% vs. 12%, p=0.084). CONCLUSIONS RCI ischemia has different etiology, presentation, and outcome. The series shows a high proportion of - treatable - vessel occlusion. It reinforces the advice to perform CT angiography in RCI as means to improve its poor outcome.