Despite many years of effort, linkage and candidate gene association studies have yielded disappointingly few risk loci for common human diseases such as diabetes, auto-immune disorders and cancers. Large sample sizes, increased understanding of the patterns of correlation in genetic variation, and plunging genotyping costs have enabled genome-wide association studies, which have good power to detect common risk alleles of modest effect. I present an evaluation of SNP choice in study design and show that overall, despite substantial differences in genotyping technologies, marker selection strategies and number of markers assayed, the first generation platforms all offer good levels of genome coverage (∼ 70%). I next describe the largest such project undertaken to date, the Wellcome Trust Case Control Consortium, which consisted of 2000 cases from each of seven common diseases and 3000 shared controls. It identified nearly two dozen new associations. I demonstrate the importance of careful data quality control, including both standard and unorthodox analyses. I next focus on the association results therein for Crohn’s disease. I present a replication experiment in over 1000 additional Crohn’s patients which unambiguously confirmed six previously published loci and four new loci. Next I describe, in a general context, several issues impeding the combination of genome-wide scans, including data annotation, population structure and differences in genotyping platform. Each of these problems is shown to be tractable with available methods, provided that these methods are applied prudently. I present the results of a meta-analysis of three genome-wide scans for Crohn’s disease. The data showed a striking excess of significant associations, and a replication experiment involving over 4000 independent Crohn’s patients verified twenty new risk loci. Finally, I discuss the early success of genome-wide association and its consequences for further understanding the biology of human disease.

Immune-mediated inflammatory diseases (IMID) represent a group of ostensibly unrelated, chronic and highly disabling diseases that preferentially affect different organ systems. IMID are assumed to manifest as a result of the accumulation of genetic, environmental and immunological factors. A fundamental commonality between IMID is the idiopathic nature of disease, and moreover, substantial similarities are apparent in disease etiopathogenesis. The complex assemblage of microbes and their genes that exists within and on the human body, collectively known as the microbiome has emerged as a critical factor in human health and, altered microbial populations within the gastrointestinal tract lumen and mucosa have been linked to several IMID. Accordingly, we conducted several studies investigating the association of the gut microbiome with IMID. Our main study investigated differences in the microbial profile and functional potential of multiple IMID utilizing 16S rDNA amplicon sequencing and analysis of stool. We also investigated the mucosal-associated microbiome in IBD to characterize the microbial populations and their functions residing in distinct gastrointestinal compartments from inflamed and noninflamed mucosa. We also explored a potential environmental factor; specifically assessing whether microbes present in drinking water in low or high incidence areas of IBD might contribute to disease etiology. The findings of these studies are manifold. First, we show important differences of the stool microbial profile in IMID. In doing so, we were able to identify distinct states of gut dysbiosis and have revealed numerous microbes that are consistently or uniquely disproportionate between IMID. Second, we have shown the microbial profile associated with inflamed and noninflamed mucosa and have reported that a localized dysbiosis is not observed in the presence of inflammation. Third, we have revealed that distinct gastrointestinal compartments are comprised of similar microbial communities. Lastly, we have reported the drinking water microbiome to differ between low and high incidence areas of IBD, thus suggesting a potential role in IBD etiology. Understanding the role of the gut microbiome in human disease will enable the development and application of more appropriate therapeutic strategies that specifically target microbes within the gut. / May 2017

Crohn’s disease (CD), a chronic inflammatory condition of the gut affecting 1:1000 of western populations, is thought to arise from a dysregulated immune response in a genetically susceptible individual. Polymorphisms in the ligand recognition domain of an intracellular pattern recognition receptor (PRR), NOD2, remain the strongest genetic risk factor for the development of CD. NOD2 directs autophagy in human DCs to facilitate bacterial destruction and antigen presentation; the CD-variant-NOD2 shows defects in this pathway. Recent work in the laboratory has demonstrated NOD2 signals to control expression induction of microRNA-29, which is impaired in cells from CD patients expressing CD NOD2-variants, and among other immunoregulatory effects, microRNA-29 suppresses IL-12p40/IL-23. Thus NOD2 directs key anti-microbe and immunoregulatory functions whose breakdown in the presence of CD-variant-NOD2 could act as a trigger for inflammation in this disease. In comparison with other PRRs, relatively little is understood of the hardwiring of NOD2 signalling, the mechanism of NOD2-mediated autophagy induction, the means by which NOD2 recruits a signalling platform within the cytosol and the mechanism of synergy with other PRRs and the inflammasome. In this work I used quantitative proteomics to map the NOD2 signalling cascade and its cross-talk with TLR2, demonstrating novel mediators: LCP1, a plastin, reduced phagocytosis of bacteria but did not alter bacterial killing, and aided control of the release of MCP1 and may be involved in IL-12p40 release. SHP1, a phospho-tyrosine phosphatase, is required for the propagation of signalling cascades via p38, p44/42 MAPK and NF-κB. It controls release of MIP1β and IL-12p40. HMGB1, an alarmin, is dephosphorylated on NOD2 stimulation and would result in changes to cellular location of HMGB1. Lastly, DAPK1, a serine/threonine kinase, is associated with HLA-DM loading compartment on NOD2 triggering, but does not alter CLIP levels on the surface of the cells. Thus, defining the hard wiring of NOD2 signalling in healthy donors, in comparison with CD donors expressing variant NOD2, is important to define targets amenable to drug design within this pathway.

In diploma thesis we deal with inflammatory bowel diseases. The theoretical part describes etiology, pathogenesis, clinical symptoms, out-of-body manifestation and drug therapy. Current knowledge about the relationship between nutrition and disease is described. Since inflammatory bowel diseases are incurable, therapy pays attention to keep the patient in remission and preventing relapse. Nutrition therapy plays an important role in achieving these goals. In the practical part, we deal with the level of nutrition awareness of patients, in the form of a questionnaire and a non- standardized interview. We also study the influence of education on vitamin levels in patients. We determine whether they know the importance of eating and eating nutrients in their illnesses. From the background, it can be appreciated that information resources are insufficient for patients. Patients would also welcome more detailed education, and therefore part of the practical part of the educational material for patients with Crohn's disease. Key words: Crohn's disease, ulcerative colitis, nutrition, education, diet

For over two decades, the study of genetics has been making significant progress towards understanding the causes of common disease. Across a wide range of complex disorders there have been hundreds of associated loci identified, largely driven by common genetic variation. Now, with the advent of next-generation sequencing technology, we are able to interrogate rare and low frequency variation in a high throughput manner for the first time. This provides an exciting opportunity to investigate the role of rarer variation in complex disease risk on a genome-wide scale, potentially o↵ering novel insights into the biological mechanisms underlying disease pathogenesis. In this thesis I will assess the potential of this technology to further our understanding of the genetics of complex disease, using inflammatory bowel disease (IBD) as an example. After first reviewing the history of genetic studies into IBD, I will describe the analytical challenges that can occur when using sequencing to perform case-control association testing at scale, and the methods that can be used to overcome these. I then test for novel IBD associations in a low coverage whole genome sequencing dataset, and uncover a significant burden of rare, damaging missense variation in the gene NOD2, as well as a more general burden of such variation amongst known inflammatory bowel disease risk genes. Through imputation into both new and existing genotyped cohorts, I also describe the discovery of 26 novel IBD-associated loci, including a low frequency missense variant in ADCY7 that approximately doubles the risk of ulcerative colitis. I resolve biological associations underlying several of these novel associations, including a number of signals associated with monocyte-specific changes in integrin gene expression following immune stimulation. These results reveal important insights into the genetic architecture of inflammatory bowel disease, and suggest that a combination of continued array-based genome- wide association studies, imputed using substantial new reference panels, and large scale deep sequencing projects will be required in order to fully understand the genetic basis of complex diseases like IBD.

There is increasing evidence that psychological stress and associated mood disorders are linked with, and can adversely affect the course of inflammatory bowel disease (IBD). Stress is perceived to be relieved by smokers, and this, like a lack of knowledge about its adverse effects, and nicotine dependence, could contribute to continued smoking by patients with Crohn’s disease (CD). Stress has previously been shown to influence disease course in patients with inactive ulcerative colitis (UC) but its influence in acute severe UC is not known. Emerging trial evidence supports the suggestion that psychologically-orientated therapy may ameliorate IBD-associated mood disorders, but there is no strong data yet to indicate that stress management has a beneficial effect on the activity or course of IBD. In addition gut-focussed hypnotherapy has been successfully used in the setting of functional bowel disorders. The 4 main hypotheses tested in thesis are: 1. In patients with IBD: (1) poor knowledge of the effects of smoking on their disease and/or (2) high nicotine dependence explain the higher prevalence of smoking in CD than UC 2. Anxiety, depression and stress are more common and worsen outcome in patients with acute severe UC. 3. Stress management in the form of psychotherapy given by a counsellor has a beneficial effect on the activity and course of IBD. 4. Gut-focussed hypnotherapy reduces the relapse rate in patients with UC. The major findings are as follows: 1. Despite more patients with CD being smokers, they were better informed about the effects of smoking on their own disease than UC patients. Nicotine dependence was no higher in patients with CD than UC. In IBD patients as a whole, nicotine dependence was lower than in smokers’ clinic clients and comparable to that of the general population, suggesting that most IBD patients could be weaned off smoking successfully in the IBD clinic.

Detection of evolutionarily conserved pathogen motifs by pattern recognition receptors (PRRs), particularly on dendritic cells (DCs), is crucial for adequate immune responses. Defects in DC function are known to be associated with inflammatory bowel disease (IBD). The endocannabinoid system (ECS) is the system through which exocannabinoids such as Δ⁹-tetrahydrocannabinol and cannabidiol signal. Regarding inflammation, cannabinoids generally exert anti-inflammatory effects, including on experimental colitis. However, most work has been performed in animal models and less is known about the function of this system in human immune cells, particularly DCs. Monoacylglycerol lipase (MGLL) is the key enzyme for hydrolysis of the endocannabinoid 2-arachidonoylglycerol, and a member of the serine hydrolase enzyme superfamily. This thesis defines the activity of serine hydrolase enzymes for the first time in human DCs upon stimulation by NOD2/TLR2 ligands using activity-based protein profiling (ABPP). MGLL is shown to be ubiquitously upregulated upon stimulation of DCs and in monocyte-derived macrophages. Through pharmacological inhibition studies, MGLL is demonstrated to regulate cellular and secreted lipids, not limited to endocannabinoids. However, overall DC function is independent of this enzyme suggesting that the effects of lipid modulation may be on bystander cells. Challenging the current literature, MGLL inhibition with a novel inhibitor worsens murine Citrobacter rodentium colitis. Finally, ABPP demonstrates a rich serine hydrolome in colonic tissue from human IBD with many enzymes previously undefined in this disease. Gene expression of ECS components suggests the enzymes ABHD12 and DAGLα/β may be potential markers of field change in IBD.

Epigenetic alterations including DNA methylation and microRNAs may provide important insights into gene-environment interaction in complex immune diseases such as inflammatory bowel disease (IBD). An integrative genome-wide approach was used to analyse whole blood genetic, DNA methylation and gene expression data in 240 newly diagnosed IBD patients and 190 controls. Using the Illumina 450k array, differences in whole blood DNA methylation were observed in IBD cases versus controls including 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs). The top DMP (RPS6KA2, discovery Holm adjusted p=1.22×10-16, replication p=1×10-9) and DMRs (VMP1, ITGB2, TXK) were replicated in an independent cohort using pyrosequencing. Paired genetic and epigenetic data allowed the identification of methylation quantitative trait loci (meQTL); two of the five DMRs (VMP1, ITGB2) demonstrated significant association with genetic polymorphisms. Methylation in the VMP1/microRNA-21 region was significantly associated with two single nucleotide polymorphisms (cg18942579 -rs10853015 [meQTL FDR adjusted p=9.4 × 10-5], cg16936953 - rs8078424 [meQTL FDR adjusted p=8.8 × 10-5]), both of which are in linkage disequilibrium with a known IBD susceptibility variant (rs1292053). Separated leukocyte methylation data highlight the cell type of origin of epigenetic signals seen in whole blood. IBD-associated hypermethylation within the TXK gene transcription start-site negatively correlated with gene expression in whole blood and CD8+ T-cells, but not other cell types, highlighting that cell-specificity and gene location-specificity of DNA methylation change is critical when associating methylation and gene expression. These data offer significant translational potential as diagnostic biomarkers. Least absolute shrinkage and selection operator (lasso) modelling identified 30 methylation probes can be used to accurately discriminate IBD cases from controls (Area under receiver operating characteristic curve = 0.898, sensitivity = 90.6%, specificity = 84.7%). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. MiRNAs have been increasingly implicated in many of the important IBD pathogenic pathways including autophagy, intestinal epithelial barrier integrity and the Th17 pathway. In common with all epigenetic mechanisms, miRNA expression is dynamic and cell-specific. Small RNA sequencing (RNA-seq) was performed on RNA extracted from CD14+, CD4+ and CD8+ cells isolated from 8 newly diagnosed cases of ileal or ileocolonic CD and 8 age and sex matched controls. There was a median of 2.4 million reads per sample (range 132,800-12.8 million reads per sample). One microRNA was differentially expressed in CD compared with controls (hsa-miR-503-5p log fold change = 0.7, FDR adjusted p = 9.1 × 10-5) in CD4+ lymphocytes, however this finding did not remain significant when alternative normalisation methods were used. The small number of cases used in microRNA analyses raises the possibility of both type I and II error, and limits the ability to draw firm conclusion from this series of experiments. Site-specific differences in DNA methylation in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression. This is the most detailed characterisation of the epigenome carried out in IBD to date. The findings strongly validate this approach in complex disease, are replicable, and provide clear translational opportunities.

Background The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) are chronic relapsing inflammatory disorders which have a rising incidence and cause significant morbidity. There are currently several treatment options with many more in the drug pipeline, but there are a lack of accurate biomarkers for decisions on treatment choice, assessment of disease activity and prognostication. There is a growing interest and desire for personalised or 'precision' medicine in IBD where novel biomarkers may help individualise IBD care in terms of diagnosis, choice of therapy, monitoring of response and detection of relapse. One class of functionally active biomarkers which have yet to be thoroughly investigated in IBD is damage-associated molecular patterns (DAMPs) including mitochondrial DNA (mtDNA). It has been recently shown that gut mitochondrial dysfunction can result in loss of epithelial barrier function and the development of colitis. Mitochondrial DAMPs have recently been described as elevated in several inflammatory diseases. Hypothesis The primary hypothesis of this thesis is that circulating levels of mtDNA is elevated in IBD. Secondary hypotheses are: (a) levels of other mitochondrial DAMPs are elevated in IBD, (b) circulating mtDNA can be used as a novel biomarker in IBD and (c) mtDNA is released locally at sites of inflammation in IBD. Methods Plasma and serum were collected prospectively from recruited IBD patients and non-IBD controls. Faeces and colonic tissue were collected from a subset of these patients. mtDNA in serum, plasma and faeces was measured using qPCR (amplifying COXIII/ND2 genes). Mass spectrometry was used to detect mitochondrial formylated peptides in the plasma of a subset of patients. IBD tissue was assessed for (a) mitochondrial damage using transmission electron microscopy (TEM) and (b) TLR9 expression, the target for mtDNA. Results 97 patients with IBD (67 UC and 30 CD), and 40 non-IBD controls were recruited. Plasma mtDNA levels were increased in UC and CD (both p < 0.0001) compared to non-IBD controls; with significant correlations with blood (CRP, albumin, white cell count), clinical and endoscopic markers of severity; and disease activity. In active UC, we detected significantly higher circulating mitochondrial formylated peptides and faecal mtDNA levels (vs. non-IBD controls [p < 0.01 and < 0.0001 respectively]) with demonstrable TEM evidence of intestinal mucosal mitochondrial damage. In active IBD, TLR9+ lamina propria inflammatory cells were significantly higher in UC/CD compared to controls (both p < 0.05). Conclusions Taken together, the findings suggest mtDNA is released during active inflammation in inflammatory bowel disease and is a potential novel mechanistic biomarker.

La maladie de Crohn (MC) est caractérisée par une inflammation intestinale chronique affectant potentiellement n'importe quel segment du tube digestif. L’étiologie de la maladie reste encore inconnue, cependant, la théorie la plus largement acceptée repose sur une réponse inflammatoire anormale dirigée contre le microbiote intestinal chez un hôte génétiquement prédisposé. Plusieurs études ont démontré que la muqueuse iléale de patients atteints de MC est anormalement colonisée par des souches de Escherichia coli adhérentes et invasives (AIEC). Toutefois, à ce jour, au Brésil, aucune étude ne démontre la présence de telles souches d’E. coli chez les patients atteints de MC. Le but de cette étude était d'isoler et de caractériser les souches de E. coli chez les patients atteints de MC au Brésil. Les biopsies ont été réalisées sur 35 sujets, 24 atteints de MC et 11 contrôles. La colonisation par des entérobactéries associées à la muqueuse iléale de patients atteints de MC a été montré élevée par rapport au groupe contrôle. Parmi les 270 souches isolées, 241 ont été identifiées comme étant des E. coli : 183 à partir de patients atteints de MC et 58 des contrôles. La recherche de différents groupes phylogénétiques de E. coli a été réalisée par PCR. Il n'y a pas de différence significative entre la répartition des groupes phylogénétiques des souches de E. coli isolées dans le groupe témoin et les patients MC. Les capacités d'adhésion et d’invasion des souches aux cellules épithéliales intestinales humaines I-407 ont été analysées, aussi bien que sa capacité à survivre et se multiplier en macrophages humains THP-1. L'analyse moléculaire par PCR a également été réalisée pour la détection des facteurs de virulence et la présence de polymorphismes génétiques associées à des souches AIEC. Dans cette étude, seuls quelques- uns des isolats de E. coli présentaient des propriétés invasives et la capacité de survivre dans les macrophages. En outre, l’analyse de la séquence fimH des souches de E. coli isolées dans cette étude n'a pas révélé la sélection de polymorphismes dans l’adhésine FimH, comme décrit pour la collection de souches AIEC isolées chez des patients européens. Ces résultats ont donc permis de montrer que les souches isolées chez les patients atteints de MC brésiliens n’ont probablement pas encore co-évolué avec leur hôte pour développer un phénotype adhérent-invasif fort, mais il sera essentiel de suivre à l'avenir l'évolution des ces souches dans la population brésilienne pour comprendre la sélection et l'évolution du phénotype AIEC. / Crohn's disease (CD) is characterized by chronic intestinal inflammation potentially affecting any segment of the digestive tract. The etiology of the disease is still unknown, however, the most widely accepted theory relies on an abnormal inflammatory response directed against the gut microbiota in a genetically predisposed host. Several studies have shown that the ileal mucosa of patients with CD is abnormally colonized by adherent and invasive Escherichia coli strains (AIEC). However, to date, in Brazil, no study has demonstrated the presence of such E. coli strains. coli in patients with CD. The purpose of this study was to isolate and characterize E. coli strains in patients with CD in Brazil. Biopsies were performed on 35 subjects, 24 with MC and 11 controls. Colonization with enterobacteria associated with the ileal mucosa of MC patients was shown to be elevated relative to the control group. Of the 270 strains isolated, 241 were identified as E. coli: 183 from CD patients and 58 controls. The search for different phylogenetic groups of E. coli was performed by PCR. There is no significant difference between the distribution of phylogenetic groups of E. coli strains isolated in the control and MC patients. The adhesion and invasion abilities of strains to human intestinal epithelial cells I-407 were analyzed, as well as its ability to survive and multiply into human macrophages THP-1. PCR molecular analysis was also performed for the detection of virulence factors and the presence of genetic polymorphisms associated with AIEC strains. In this study, only a few of the E. coli isolates had invasive properties and the ability to survive in macrophages. In addition, analysis of the fimH sequence of E. coli strains isolated in this study did not reveal the selection of polymorphisms in the FimH adhesin, as described for the collection of AIEC strains isolated from European patients. These results have thus shown that strains isolated from patients with Brazilian CD probably have not yet co-evolved with their host to develop a strong adherent-invasive phenotype, but it will be essential to monitor the future evolution of these strains in the Brazilian population to understand the selection and evolution of the AIEC phenotype.