Identification of novel genetic determinants in the high prevalence early-onset inflammatory bowel disease population in Scotland

Grant no. 072789/Z/03/ZBackground & aims: The inflammatory bowel diseases (IBD), Crohn‟s disease (CD) and ulcerative colitis (UC), are common causes of chronic gastrointestinal morbidity, affecting up to 1 in 250 of the general population in Northern Europe. Up to 25% of IBD is diagnosed during childhood or adolescence. The aims for this thesis were to study the epidemiology, natural history and novel genetic determinants of childhood onset IBD in Scotland. Methods: The existing repository of childhood onset and adult onset IBD patients, established at the Western General Hospital in Edinburgh, was used and expanded. Thus, anatomical location and behaviour of disease were assessed in 416 childhood onset (276 CD, 99 UC, 41 IBDU diagnosed before 17th birthday) and 1297 adult patients (596 CD, 701 UC) using the Montreal classification. Additional phenotypic (at diagnosis and at regular follow-up intervals) and epidemiological data were gathered. In this cohort, genotyping of germline variants in putative susceptibility genes (NOD1/CARD4, IL23R, ATG16L1, IRGM, FLG) was performed to enable single variant and haplotype-tagging association studies. Genotypic data of population-matched healthy controls were obtained locally (n=342) and from the Wellcome Trust Case Control Consortium (n=2937). Results: Compared with adults, childhood-onset CD was characterized by a more extensive, “panenteric” phenotype (ileocolonic plus upper GI; p<0.0001 OR23.3; 95% CI (13.4–40.6) with less isolated ileal (p<0.0001 OR 0.06 (0.03–0.1) or colonic disease (p<0.0001, OR 0.3 (0.2–0.5)). In 39%, the anatomic extent increased within 2 years. UC was also more extensive in children at diagnosis vs adults (p0.05 after Bonferroni correction). We found that the allelic frequency of rs11209026*A located within the IL23R gene, differed significantly between IBD / CD cases and controls (p=0.01 OR 0.51(0.3-0.9) and p=0.04 OR 0.5 (0.3-0.98)). Using a gene-wide haplotype-tagging strategy, we demonstrated that the multiple association signals of the IL23R locus are independent of rs11209026 in childhood onset IBD and CD. In Scottish children, the effect of germline variation of ATG16L1 and IRGM on CD susceptibility was relatively small (OR< 1.4), and appeared less than in adult disease. Genotype–phenotype analysis demonstrated an association of pure ileal disease with the ATG16L1 rs2241880G-allele (p=0.02 OR 1.3 (1.03–1.7)). Using binary logistic regression analysis, we confirmed the effect of rs2241880 genotype (GG) on ileal disease versus colonic disease (p=0.03 OR 2.4 (1.05–5.6)). Null alleles of the epithelial barrier protein FLG have no important effect on IBD susceptibility (p>0.4), but contribute to the high prevalence of atopy, notably co-existent eczema and food allergy (p=0.0003 OR 3.3 (1.7–6.6) and p=0.0001 OR 4.5 (2.0–10.0), respectively). Conclusion: Childhood onset IBD is characterised by extensive intestinal involvement and progression of disease after diagnosis. Genetic association studies in childhood and adult IBD have provided evidence for a large number of new genomic loci. These loci encode genes involved in a number of homeostatic mechanisms: innate pattern recognition receptors, the differentiation of Th17-lymphocytes, autophagy, maintenance of epithelial barrier integrity and the orchestration of the secondary immune response

Diagnostic Delay Is Associated with Complicated Disease and Growth Impairment in Paediatric Crohn\u27s Disease

Background: Paediatric data on the association between diagnostic delay and inflammatory bowel disease [IBD] complications are lacking. We aimed to determine the effect of diagnostic delay on stricturing/fistulising complications, surgery, and growth impairment in a large paediatric cohort, and to identify predictors of diagnostic delay. Methods: We conducted a national, prospective, multicentre IBD inception cohort study including 1399 children. Diagnostic delay was defined as time from symptom onset to diagnosis \u3e75th percentile. Multivariable proportional hazards [PH] regression was used to examine the association between diagnostic delay and stricturing/fistulising complications and surgery, and multivariable linear regression to examine the association between diagnostic delay and growth. Predictors of diagnostic delay were identified using Cox PH regression. Results: Overall (64% Crohn\u27s disease [CD]; 36% ulcerative colitis/IBD unclassified [UC/IBD-U]; 57% male]), median time to diagnosis was 4.2 (interquartile range [IQR] 2.0-9.2) months. For the overall cohort, diagnostic delay was \u3e9.2 months; in CD, \u3e10.8 months and in UC/IBD-U, \u3e6.6 months. In CD, diagnostic delay was associated with a 2.5-fold higher rate of strictures/internal fistulae (hazard ratio [HR] 2.53, 95% confidence interval [CI] 1.41-4.56). Every additional month of diagnostic delay was associated with a decrease in height-for-age z-score of 0.013 standard deviations [95% CI 0.005-0.021]. Associations persisted after adjusting for disease location and therapy. No independent association was observed between diagnostic delay and surgery in CD or UC/IBD-U. Diagnostic delay was more common in CD, particularly small bowel CD. Abdominal pain, including isolated abdominal pain in CD, was associated with diagnostic delay. Conclusions: Diagnostic delay represents a risk factor for stricturing/internal fistulising complications and growth impairment in paediatric CD

Successful dietary therapy in paediatric Crohn’s disease is associated with shifts in bacterial dysbiosis and inflammatory metabotype towards healthy controls

Background and aims: Nutritional therapy with the Crohn’s Disease Exclusion Diet + Partial Enteral Nutrition [CDED+PEN] or Exclusive Enteral Nutrition [EEN] induces remission and reduces inflammation in mild-to-moderate paediatric Crohn’s disease [CD]. We aimed to assess if reaching remission with nutritional therapy is mediated by correcting compositional or functional dysbiosis. Methods: We assessed metagenome sequences, short chain fatty acids [SCFA] and bile acids [BA] in 54 paediatric CD patients reaching remission after nutritional therapy [with CDED + PEN or EEN] [NCT01728870], compared to 26 paediatric healthy controls. Results: Successful dietary therapy decreased the relative abundance of Proteobacteria and increased Firmicutes towards healthy controls. CD patients possessed a mixture of two metabotypes [M1 and M2], whereas all healthy controls had metabotype M1. M1 was characterised by high Bacteroidetes and Firmicutes, low Proteobacteria, and higher SCFA synthesis pathways, and M2 was associated with high Proteobacteria and genes involved in SCFA degradation. M1 contribution increased during diet: 48%, 63%, up to 74% [Weeks 0, 6, 12, respectively.]. By Week 12, genera from Proteobacteria reached relative abundance levels of healthy controls with the exception of E. coli. Despite an increase in SCFA synthesis pathways, remission was not associated with increased SCFAs. Primary BA decreased with EEN but not with CDED+PEN, and secondary BA did not change during diet. Conclusion: Successful dietary therapy induced correction of both compositional and functional dysbiosis. However, 12 weeks of diet was not enough to achieve complete correction of dysbiosis. Our data suggests that composition and metabotype are important and change quickly during the early clinical response to dietary intervention. Correction of dysbiosis may therefore be an important future treatment goal for CD

The Role of Carrageenan and Carboxymethylcellulose in the Development of Intestinal Inflammation

Although the exact pathophysiology remains unknown, the development of inflammatory bowel disease (IBD) is influenced by the interplay between genetics, the immune system, and environmental factors such as diet. The commonly used food additives, carrageenan and carboxymethylcellulose (CMC), are used to develop intestinal inflammation in animal models. These food additives are excluded from current dietary approaches to induce disease remission in Crohn’s disease such as exclusive enteral nutrition (EEN) using a polymeric formula. By reviewing the existing scientific literature, this review aims to discuss the role that carrageenan and CMC may play in the development of IBD. Animal studies consistently report that carrageenan and CMC induce histopathological features that are typical of IBD while altering the microbiome, disrupting the intestinal epithelial barrier, inhibiting proteins that provide protection against microorganisms, and stimulating the elaboration of pro-inflammatory cytokines. Similar trials directly assessing the influence of carrageenan and CMC in humans are of course unethical to conduct, but recent studies of human epithelial cells and the human microbiome support the findings from animal studies. Carrageenan and CMC may trigger or magnify an inflammatory response in the human intestine but are unlikely to be identified as the sole environmental factor involved in the development of IBD or in disease recurrence after treatment. However, the widespread use of carrageenan and CMC in foods consumed by the pediatric population in a “Western” diet is on the rise alongside a corresponding increase in IBD incidence, and questions are being raised about the safety of frequent usage of these food additives. Therefore, further research is warranted to elucidate the role of carrageenan and CMC in intestinal inflammation, which may help identify novel nutritional strategies that hinder the development of the disease or prevent disease relapse post-EEN treatment

The Role of Carrageenan and Carboxymethylcellulose in the Development of Intestinal Inflammation

Although the exact pathophysiology remains unknown, the development of inflammatory bowel disease (IBD) is influenced by the interplay between genetics, the immune system, and environmental factors such as diet. The commonly used food additives, carrageenan and carboxymethylcellulose (CMC), are used to develop intestinal inflammation in animal models. These food additives are excluded from current dietary approaches to induce disease remission in Crohn’s disease such as exclusive enteral nutrition (EEN) using a polymeric formula. By reviewing the existing scientific literature, this review aims to discuss the role that carrageenan and CMC may play in the development of IBD. Animal studies consistently report that carrageenan and CMC induce histopathological features that are typical of IBD while altering the microbiome, disrupting the intestinal epithelial barrier, inhibiting proteins that provide protection against microorganisms, and stimulating the elaboration of pro-inflammatory cytokines. Similar trials directly assessing the influence of carrageenan and CMC in humans are of course unethical to conduct, but recent studies of human epithelial cells and the human microbiome support the findings from animal studies. Carrageenan and CMC may trigger or magnify an inflammatory response in the human intestine but are unlikely to be identified as the sole environmental factor involved in the development of IBD or in disease recurrence after treatment. However, the widespread use of carrageenan and CMC in foods consumed by the pediatric population in a “Western” diet is on the rise alongside a corresponding increase in IBD incidence, and questions are being raised about the safety of frequent usage of these food additives. Therefore, further research is warranted to elucidate the role of carrageenan and CMC in intestinal inflammation, which may help identify novel nutritional strategies that hinder the development of the disease or prevent disease relapse post-EEN treatment