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The inflammatory bowel diseases (IBDs) are a group of chronic conditions affecting the gastrointestinal tract, often presenting with non-specific clinical features such as abdominal pain, weight loss and diarrhoea. Approximately 25% of patients are diagnosed with IBD in childhood. For epidemiological studies, previously collected (1990-1995) and original (2003-2008) Scottish incidence data were used to determine national trends in newly diagnosed paediatric IBD (PIBD). A smaller, geographically defined, prospective 14-year cohort (1997- 2011) in South-East Scotland (SES) was used to assess regional trends in incidence, point prevalence, disease extent, medication use and PIBD surgery rates in 326 children. For the detailed analysis of the role of ICOSLG and CRP in Scottish children with PIBD, haplotype-tagging of both genes in 448 children (and their parents) registered on the Paediatric Inflammatory bowel disease Cohort and Treatment Study (PICTS) database was performed. Further clinical information from this database and previously gathered adult mRNA microarray data were also used to inform the analysis. For the faecal calprotectin (FC) case-control study, all PIBD patients diagnosed in SES between 01.01.05 and 31.12.10 (aged 1- 17yrs) with a FC performed during initial workup were identified; controls were matched non- IBD patients who had similarly undergone endoscopy with a referral FC level available. The systematic review and meta-analysis of FC case-control studies was performed with keywords relating to IBD and calprotectin in electronic resources from 1946 to May 2012. Inclusion criteria were studies that reported FC levels prior to the endoscopic investigation of IBD in children less than 18 years old. Laboratory work used newly derived HEK293 and HCT116 cell lines stably expressing wild-type NOD2 and the CD-associated NOD2 frameshift mutant, as well as utilising previously derived HEK293 and HCT116 cells stably expressing green fluorescent-labelled protein LC3 during the assessment of autophagy. Western blot, immunofluorescent microscopy and flow cytometry were used for analysis. There was a significant rise in PIBD incidence in Scotland since the early 1990s, with 260 new cases between 1990-1995 (4.45/100,000/year) and 436 in the 2003-2008 epoch (7.82/100,000/year) (p<0.001). A five-fold increase in Crohn's disease (CD) in the last 40 years was also demonstrated. SES was shown to have the highest recorded PIBD incidence rate in the UK for the six-year epoch from 2006-2011 (9.50/100,000/year) with a significant rise in ulcerative colitis (UC) to 2.67/100,000/year (p=0.010). Point prevalence rates for PIBD in SES had also risen significantly to 41.2/100,000 between the 2000-2005 and 2006-2011 epochs (p=0.016). With a follow up of 1577 patient years, the severe phenotype in children with PIBD was confirmed; 34% of children with CD presented with pan-enteric disease (44% at follow up), and 76% of children with UC had pancolonic disease at diagnosis (81% at follow up). 26% of patients required methotrexate and 18% were exposed to infliximab/adalimumab, with the time to first exposure of both significantly lower in children diagnosed between 2006-2011 (p=0.001 and p<0.001 respectively). A total of 70% of children were exposed to azathioprine and 20% underwent IBD-related surgery. Using a haplotype-tagging approach and transmission disequilibrium testing (TDT) in 230 PIBD case-parent trios there was significant overtransmission of the rs8126734-A single nucleotide polymorphism (SNP) in ICOSLG following correction (p=0.0467). In the CD TDT analysis the same SNP was overtransmitted (p=0.0084). The strongest susceptibility signal was evident across the two marker haplotype rs762421-A / rs8126734-G (p=0.0072), suggesting that the 3-prime untranslated region in ICOSLG may be targeted for deep sequencing. mRNA microarray data from adult patients showed downregulation of ICOSLG expression in the ascending colon (p=0.023) and upregulation in the descending colon (p=0.0351) in uninflamed biopsies from CD patients and non-IBD controls; no difference in gene expression was shown in UC patients. Using a similar approach, the A allele of two SNPs tagging CRP showed significant over-transmission to affected IBD patients after correction (rs1417938, p=0.006; rs1130864, p=0.015). The six-marker haplotype (ACACAC) showed significant distortion of transmission to affected individuals (p=8x10-4). CD and UC patients demonstrated differences in rs1205 genotype (p=0.0085) and CRP haplotype (p=0.0024), with the influence of the rs1205 SNP on response to anti-tumour necrosis factor-alpha therapy also shown (p=0.021). During the FC case-control study significantly elevated FC levels at diagnosis were demonstrated compared to controls (1265 μg/g vs 65 μg/g; p<0.001). FC also outperformed commonly used blood parameters (e.g. CRP, ESR, platelets), with an area under the curve of 0.93 (95% CI 0.89-0.97) and good sensitivity (0.93 [95% CI 0.86-0.98]) and specificity (0.74 [95% CI 0.64-0.82]) when values above 200μg/g were used. FC levels were not influenced by disease location in CD or UC. The systematic review and meta-analysis highlighted the often poor methodological quality of previous studies and concluded that across all studies FC had a pooled sensitivity of 0.98 (95% CI 0.95-1.00) and pooled specificity of 0.68 (95% CI 0.50-0.86) for PIBD at diagnosis. Characterisation of cells stably-expressing wild-type NOD2 or the CD-associated NOD2 frameshift mutation demonstrated increased cell proliferation compared to empty vector, and an accentuated apoptotic response to serum starvation. The NOD2 frameshift protein had a shorter half-life (at 11 hours) than the wild-type protein, with degradation of the NOD2 protein shown to be mediated through a proteasome-dependent pathway, possibly through lysine residues on the CARD domain. Following the establishment of a robust method of assessing autophagy in a cell culture system, experimental work showed that muramyl dipeptide-induced autophagy is unlikely to signal through the mammalian target of rapamycin, with the intermediate filament vimentin shown to be intimately involved in this pathway; the vimentin gene (Vim) was also shown to be a candidate susceptibility gene for CD. Using a panel of PIBD drugs azathioprine was shown to induce autophagy in a dose-dependent manner through an mTOR-dependent, ERK-independent pathway. It can be seen that with the increasing incidence and prevalence of PIBD in Scotland that a greater understanding of epidemiological trends, the role of genetic susceptibility, the optimal use of biomarkers and translational functional biology are all needed to understand further the aetiopathogenesis of PIBD. This future work will undoubtedly help to inform service design and the clinical care pathways utilised to provide the best care for children in addition to targeting pathways for potential drug development, with these measures helping to prepare for the increasing disease burden generated by PIBD.

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.

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.

Thesis (Ph.D.) - University of Glasgow, 2009. / Ph.D. thesis submitted to the Division of Developmental Medicine, Faculty of Medicine, University of Glasgow, 2009. Includes bibliographical references. Print version also available.

Background: Inflammatory Bowel Disease (IBD) is a group of chronic gastrointestinal diseases with a relapsing nature. The two main types are Crohn’s disease (CD) and ulcerative colitis (UC). Both CD and UC patients experience very similar and distressing symptoms: acute abdominal pain, vomiting, malnutrition, fever, fatigue, diarrhoea and rectal bleeding. These symptoms are disabling and have a severe impact on physical and psychosocial wellbeing. Around 30% of patients suffer from moderate to severe psychological distress and have difficulties coping with the illness even in remission. However, it appears that mental health is overlooked by clinicians who often focus on physical gastrointestinal symptoms only. Mindfulness-Based Cognitive Therapy (MBCT) is evidence based, group psychological intervention that has been successful in reducing depression and anxiety scores in patients with depression while improving overall quality of life. However, MBCT has never been tested in the IBD population before. PhD question: Can MBCT be used as an adjunct therapy to IBD symptom management, for improving IBD patients' general well-being and quality of life? Aims and objectives: The overall aim of the thesis was to develop and collate the evidence for a definitive randomised controlled trial (RCT) testing the effectiveness of MBCT for patients with inflammatory bowel disease (IBD). The thesis brings together six publications. The six publications were integrated into four objectives that collectively contributed in answering the overall PhD question. Results: The findings from the first three publications highlighted the disease-related concerns and psychological needs for patients with IBD. The findings from the last three publications highlighted how feasible it is to use MBCT in IBD and emphasised the IBD patients’ perspectives about MBCT. Conclusion: The thesis concluded that a definitive RCT of MBCT for IBD patients is both feasible and acceptable.

Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/T_H17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.