An investigation of the genetic determinants of succeptibility and disease behavoir in early onset Inflammatory Bowel Disease in Scottish children.

Abstract

A series of investigations examining the importance of genetic factors in the development of the inflammatory bowel diseases (IBD) namely Crohn’s disease (CD), Ulcerative Colitis (UC) and Indeterminate Colitis (IC) has been undertaken in Scottish children. This has been performed by collection of clinical details and DNA from children with IBD, then analysing the contribution of various candidate genes to both disease susceptibility and disease phenotype. In order to carry out these studies the presenting features of a large cohort of children from across Scotland with IBD diagnosed at less than 16 years were collected, both by examination of hospital case records and by patient interview and questionnaire. For each patient a detailed analysis was made of disease phenotype at presentation including detailed examination of disease location, disease behaviour and growth parameters. A repository of clinical material (DNA, plasma and lymphocytes) was collected from children to accompany the detailed clinical parameters allowing genotype-phenotype analysis at a later stage. Additionally, DNA was also collected from parents where possible to facilitate family based association analysis of candidate genes by transmission disequilibrium testing. A previous DNA repository of healthy Scottish controls had been collected previously and the data generated was available for use in this study. The phenotypic data was collected using an established phenotypic classification (the Vienna classification) used in adult studies as well as a personally devised paediatric phenotypic classification designed for use in this thesis. Firstly, the contribution of the three common mutations within the NOD2/CARD15 gene (R702W, G908R and Leu1007finsC) was analysed in 247 children with IBD. The Leu1007finsC variant was associated with Crohn’s disease by case-control (p = 0.01) and TDT analysis (p = 0.006). Genotype phenotype analysis demonstrated NOD2/CARD15 variants were strongly associated with several markers of disease severity in CD most notably with an increased need for surgery on multifactorial analysis. Then to examine the further contribution of other mutations within the whole NOD2/CARD15 gene, the 12 exons of the gene were sequenced in 24 paediatric CD patients, to identify any additional SNPs that may have conferred an increased susceptibility to CD. Two mutations (V955I, M863V) identified in xii sequencing were genotyped in a large patient cohort, but were not found to confer increased disease susceptibility. Next, the contribution of IBD5 locus was analysed in 299 children with IBD studying 5 SNPs, including mutations in the proposed candidate genes OCTN 1 and 2. Allele frequencies of OCTN1/2 variants were significantly higher in IBD/CD cases (p<0.04). The homozygous mutant OCTN1/2 haplotype was increased in IBD and UC patients (p = 0.02 for both) compared to healthy controls. OCTN1/2 variants however were not independent of the background IBD5 risk haplotype in conferring disease susceptibility. Genotype- phenotype analysis demonstrated association of the risk haplotype with both lower weight and body mass index centiles at diagnosis as analysed by multifactorial analysis. The contribution of the 113 G/A mutation within the discs, large homolog 5 (DLG5) gene was examined in 296 children with IBD. TDT analysis demonstrated a significant association with IBD (p<0.05). Genotype-phenotype analysis demonstrated associations with higher social class, male sex and taller children. Finally, the Anti-Saccharomyces cerevisiae antibodies (ASCA) status of 301 IBD patients was determined. CD patients had a higher prevalence of ASCA antibodies compared to UC patients and healthy controls (p<0.001 for both). A positive ASCA antibody was more common in CD patients with markers of more severe disease and on multifactorial analysis in patients with CD involvement of the oral cavity (p = 0.001). In summary, the candidate genes examined thus far in children with IBD in Scotland have demonstrated a relatively minor contribution to disease susceptibility but have been demonstrated to be associated with specific disease phenotypes in patients with Crohn’s disease. The use of a novel paediatric phenotypic classification in this thesis has allowed description of these novel genotype-phenotype associations.