Predicting outcome in ulcerative colitis: clinical and genetic determinants of disease susceptibility and behaviour

Ulcerative colitis (UC) and Crohn's disease (CD), collectively known as inflammatory bowel disease (IBD) are common, chronic inflammatory disorders of the intestines. The pathogenesis and subsequent clinical course of IBD remain unclear; although, inter¬ relating factors such as environment triggers, dysregulated immune response, defective gut barrier defence, variability of drug response and genetic susceptibility all contribute. The current work described in this thesis involves a series of clinical and genetic studies investigating their respective roles in determining susceptibility and course in UC. Firstly, the importance of corticosteroid resistance/dependence as a co-factor in disease progression was studied in a detailed 5-year inception cohort (1998-2003). This study demonstrated remarkable concordance with older series and paediatric cohorts suggesting a defined phenomenon within the innate response to corticosteroids. Secondly, a risk score to stratify the likelihood of response to standard medical therapy (high dose corticosteroids) identifying 3 distinct risk groups - low, intermediate and high risk; based on 167 consecutive patients (largest series studied) with acute severe UC was developed. A novel robust model was formulated with a sensitivity/specificity of 85% and 75% respectively to predict failure of medical therapy. These initial studies have critically provided the assimilation of highly accurate phenotypic and follow-up data, permitting very detailed statistical analyses to be performed in subsequent genetic studies. The multidrug resistance gene (MDR1 gene-encoding P-glycoprotein 170, an epithelial efflux transporter protein) was studied in detail due to its potential role in conferring susceptibility (based on knock-out animal models and genomic position within IBD locus of susceptibility) and its function in corticosteroid resistance. In this study, allelic variations of the MDR1 gene were found to be implicated in disease susceptibility in UC, in particular extensive and severe disease subphenotype (p=0.003, OR 2.64 and T-allele, p=0.009, OR 1.70. In this study, bi-directional haplotypic contribution to susceptibility in UC (with protective and susceptible haplotypes) was observed. This led to a more rigorous analysis of this gene by the application of the novel 'gene-wide' haplotype tagging approach, confirming a more significant association with UC (p=4.22xl0"7) but not CD (p=0.22). The strongest association was with the sub-phenotype; extensive UC (p= 1.7 X 10"7), and critically dependent on one tSNP rs3789243 (p= 3.2 x 10"7- 3.6 x 10" 12). This catalysed further rational approaches to study the genetic variations of the genes involved in xenobiotic-metabolism and epithelial transport such as the ATP-binding cassette (ABC) efflux transporters and respective transcriptional regulators in our cohort. Further significant and replicable association of haplotypic variations of the ABCC3/MRP3 gene (also involved in epithelial barrier defence) with IBD (p=0.00004, 1200 IBD and 700 controls) was also shown. The characterisation of these genes in a hitherto unknown pathway regulated by key transcriptional regulators such as PregnaneX receptor (PXR) has now implicated this novel class of transport proteins as important regulators of mucosal defence and as critical in determining susceptibility to inflammatory bowel disease

Pathogenesis of Crohn's disease

Significant progress in our understanding of Crohn's disease (CD), an archetypal common, complex disease, has now been achieved. Our ability to interrogate the deep complexities of the biological processes involved in maintaining gut mucosal homeostasis is a major over-riding factor underpinning this rapid progress. Key studies now offer many novel and expansive insights into the interacting roles of genetic susceptibility, immune function, and the gut microbiota in CD. Here, we provide overviews of these recent advances and new mechanistic themes, and address the challenges and prospects for translation from concept to clinic

Advances in the understanding of mitochondrial DNA as a pathogenic factor in inflammatory diseases

Mitochondrial DNA (mtDNA) has many similarities with bacterial DNA because of their shared common ancestry. Increasing evidence demonstrates mtDNA to be a potent danger signal that is recognised by the innate immune system and can directly modulate the inflammatory response. In humans, elevated circulating mtDNA is found in conditions with significant tissue injury such as trauma and sepsis and increasingly in chronic organ-specific and systemic illnesses such as steatohepatitis and systemic lupus erythematosus. In this review, we examine our current understanding of mtDNA-mediated inflammation and how the mechanisms regulating mitochondrial homeostasis and mtDNA release represent exciting and previously under-recognised important factors in many human inflammatory diseases, offering many new translational opportunities

The contribution of OCTN1/2 variants within the IBD5 locus to disease susceptibility and severity in Crohn's disease

Background and Aims: Recent data suggest that polymorphisms in the organic cation transporter (OCTN) genes OCTN1 (SLC22A4) and OCTN2 (SLC22A5) represent disease-causing mutations within the IBD5 locus (chromosome 5q31). We investigated associations with disease susceptibility, phenotype, and evidence for epistasis with CARD15 in 679 patients with Crohn’s disease (CD) or ulcerative colitis (UC). Methods: A total of 374 patients with CD, 305 patients with UC, and 294 healthy controls (HCs) were studied. Genotyping for single nucleotide polymorphisms IGR2096, IGR2198, and IGR2230, OCTN1 variant (SLC22A4 1672C→T), and OCTN2 variant (SLC22A5 −207G→C) was performed using the TaqMan system. Results: The IBD5 OCTN1 and OCTN2 polymorphisms were in strong linkage disequilibrium (D′, >0.959). IGR2198 variant allele frequency (49.1% vs 40.8%; P = .0046) and homozygosity (21% vs 14.8%; P = .044) were associated with CD versus HCs. Variant allelic frequency of OCTN1 (53.6% vs 43%; P = .0008) and OCTN2 (56.1% vs 48.4%; P = .0092) polymorphisms and homozygosity for the OCTN1/2-TC haplotype (28.4% vs 16%; P = .0042) were associated with CD versus HCs. IGR2198 homozygosity and TC homozygosity were associated with stricturing/penetrating disease at follow-up (P = .011 and P = .011, respectively) and disease progression (P = .038 and P = .049, respectively) on univariate analysis and with need for surgery on multivariate analysis (P = .016 and P = .004, respectively). In the absence of the IBD5 risk haplotype, no association of OCTN1/2 variants with CD was detected. No associations were seen with UC. Conclusions: The IBD5 locus influences susceptibility, progression, and need for surgery in CD. However, the contribution of OCTN1/2 variants is not independent of the IBD5 haplotype; a causative role for these genes remains plausible but is not yet proven. Further genetic, functional, and expression data are now required. </p

Serum Calprotectin - A novel diagnostic and prognostic marker in Inflammatory Bowel Diseases

OBJECTIVES: There is an unmet need for novel blood-based biomarkers that offer timely and accurate diagnostic and prognostic testing in inflammatory bowel diseases (IBD). We aimed to investigate the diagnostic and prognostic utility of serum calprotectin (SC) in IBD. METHODS: A total of 171 patients (n=96 IBD, n=75 non-IBD) were prospectively recruited. A multi-biomarker model was derived using multivariable logistic regression analysis. Cox proportional hazards model was derived to assess the contribution of each variable to disease outcomes. RESULTS: SC correlated strongly with current biomarkers, including fecal calprotectin (FC) (n=50, ρ=0.50, P=1.6 × 10−4). SC was the strongest individual predictor of IBD diagnosis (odds ratio (OR): 9.37 (95% confidence interval (CI): 2.82–34.68), P=4.00 × 10−4) compared with other markers (C-reactive protein (CRP): OR 8.52 (95% CI: 2.75–28.63), P=2.80 × 10−4); albumin: OR 6.12 (95% CI: 1.82–22.16), P=0.004). In a subset of 50 patients with paired SC and FC, the area under receiver operating characteristic discriminating IBD from controls was better for FC than for SC (0.99, (95% CI 0.87–1.00) and 0.87 (95% CI:0.78–0.97), respectively; P=0.01). At follow-up (median 342 days; interquartile range: 88–563), SC predicted treatment escalation and/or surgery in IBD (hazard ratio (HR) 2.7, 95% CI: 1.1–4.9), in particular Crohn’s disease (CD) (HR 4.2, 95% CI 1.2–15.3). A model incorporating SC and either CRP or albumin has a positive likelihood ratio of 24.14 for IBD. At 1 year, our prognostic model can predict treatment escalation in IBD in 65% of cases (95% CI: 43–79%) and 80% (95% CI: 31–94%) in CD if ≥2 blood marker criteria are met. CONCLUSIONS: A diagnostic and prognostic model that combines SC and other blood-based biomarkers accurately predicts the inflammatory burden in IBD and has the potential to predict disease and its outcomes. Our data warrant further detailed exploration and validation in large multicenter cohorts

Altered DNA methylation within DNMT3A, AHRR, LTA/TNF loci mediates the effect of smoking on inflammatory bowel disease

This work aims to investigate how smoking exerts effect on the development of inflammatory bowel disease (IBD). A prospective cohort study and a Mendelian randomization study are first conducted to evaluate the association between smoking behaviors, smoking-related DNA methylation and the risks of Crohn’s disease (CD) and ulcerative colitis (UC). We then perform both genome-wide methylation analysis and co-localization analysis to validate the observed associations. Compared to never smoking, current and previous smoking habits are associated with increased CD (P = 7.09 × 10−10) and UC (P &lt; 2 × 10−16) risk, respectively. DNA methylation alteration at cg17742416 [DNMT3A] is linked to both CD (P = 7.30 × 10−8) and UC (P = 1.04 × 10−4) risk, while cg03599224 [LTA/TNF] is associated with CD risk (P = 1.91 × 10−6), and cg14647125 [AHRR] and cg23916896 [AHRR] are linked to UC risk (P = 0.001 and 0.002, respectively). Our study identifies biological mechanisms and pathways involved in the effects of smoking on the pathogenesis of IBD