1. Evaluation of the genetic impact on inflammatory bowel disease Natalie Bibb Trainee Project KGC

2. Presentation aims Overview of inflammatory bowel disease (IBD) Purpose of project Genome-wide association studies (GWAS) and identification of IBD susceptibility genes Pathogenesis of IBD Assay design for detection of common variants Results Conclusions and further work

3. Overview of IBD Complex polygenic disorders influenced by genetic, environmental and immunological factors Includes Crohn’s disease (CD) and ulcerative colitis (UC) both characterised by chronic relapsing intestinal inflammation Peak age of onset 2 nd – 4 th decade with European prevalence of 100-150 per 100,000 UCCD Clinical features Extra-intestinal manifestationsCommon HaematochesiaCommonRare Passage of mucus/pusCommonRare Fistulas, skip lesions and perianal disease NoYes Transmural mucosal inflammation NoYes Comparison of the clinical features of CD and UC

5. Purpose of project Grant proposal - Multidisciplinary study of 4000 IBD patients and case controls –Immunological profiling - dendritic cell function –Microbiological profiling - quantify faecal and gut microbiota –Genetic profiling - Identify variants most significantly associated with IBD Current treatments follow non-targeted approach - steroids and surgery can have adverse side effects To assess if combined or individual factors indicate requirement for specific treatments To provide more effective and individualised treatments for IBD

6. Genetic factors of IBD IBD results from a defective immune response to intestinal microbes in a genetically susceptible host Evidence for genetic contribution –Familial aggregation - higher incidence of IBD in individuals with a positive family history –Higher disease concordance in monozygotic twins than dizygotic twins –These observations are more evident in CD than UC –Racial and ethnic differences - specific variants observed more frequently in Ashkenazi Jews –Ethnic heterogeneity - different genetic variants observed in different ethnic populations

7. Genome wide association studies (GWAS) GWAS identified significant linkage disequilibrium in variant allele frequencies between IBD cases and controls Associating 9 genomic regions known as IBD locus with susceptibility to disease Implicated 30+ genes many with roles in intestinal immunity – CD and UC share some genetic susceptibilty loci; which is evident from the co-occurrence in IBD families; but differ at others – IBD variants are present in the normal healthy population so are not sufficient or necessary to cause disease

8. NOD2 NOD2 (nucleotide-binding oligomerisation domain 2) consists of 2 CARD domains, a central NBD and an LRR domain LRR domain recognizes bacterial muramyl dipeptide (MDP) which regulates NF  activation and the production of pro-inflammatory cytokines 3 variants in the LRR domain of NOD2 show significant association with CD, but no association with UC

9. Role of NOD2 in IBD pathogenesis Exact mechanism unclear - proposed loss or gain of function Carriers of one NOD2 high risk variants have a 2-4 fold increase risk of developing CD Carriers of two NOD2 high risk variants have a 20-40 fold increased risk NOD2 high risk variant carriers also show association with ileal disease

10. IL23R IL23R (interleukin 23 receptor) encodes a subunit of the receptor for the pro- inflammatory cytokine IL23 IL23 binds to receptor (IL23R/IL-12RB1) inducing a pro-inflammatory response A rare variant c.1442G>A (p.Arg381Gln) confers a strong protective effect against CD and UC The mechanism by which this variant confers a protective effect is not yet understood

11. ATG16L1 ATG16L1 has a role in the autophagy pathway The variant c.898A>G, p.Thr300Ala is associated with CD and not UC Two recent studies have shown evidence of NOD2 and ATG16L1 functioning in a single pathway

12. Assay design Ethics approval for testing 4000 patients not yet received Genotype 25 healthy controls and 25 IBD patients for variants with most significant association Multiplex PCR - detects 3 NOD2 variants and single variant in IL23R Touchdown PCR method Allele specific fluorescent primer design using primer 3 program One fluorescent tagged common primer and two untagged allele specific primers per variant

13. Results Genotype NC (/25)IBD (/25) NOD2 variants No variants identified21/25 (84%)22/25 (88%) c.2104 het1/25 (4%)0 c.2722 het01/25 (4%) c.3020 het1/25 (4%)0 c.3020hom1/25 (4%)0 c.2104/c.3020 compound het01/25 (4%) c.2722/c.3020 compound het01/25 (4%) NOD2/ IL23R variantsc.3020/c.14421/25 (4%)0 NOD2 variants only account for ~20% genetic contribution to IBD Presence of variants in healthy controls is expected Identification of IL23R variant in healthy control supports its reduced frequency in IBD

14. Electropherogram analysis Electropherograms display a peak for each of the 3 NOD2 and single IL23R loci A tube NOD2 WT and IL23R variant primers B tube NOD2 variant and IL23R WT primers c.2104C>Tc.2722G>Cc.3020insC c.1422G>A NOD2 lociIL23R locus

15. NOD2 c.2104C>T (p.Arg702Trp) heterozygote

16. NOD2 c.3020insC (p.Leu1007fs) heterozygote

17. NOD2 c.3020insC (p.Leu1007fs)/IL23R c.1442G>A (p.Arg381Gln) compound heterozygote

18. Conclusion A multiplex PCR assay has been designed to detect IBD associated variants No significant associations can be concluded from this small pilot cohort Multidisciplinary approach may provide better understanding of disease type, severity and requirement for treatment Pharmacogenomics - Genotyping could contribute to providing targeted therapies and treatment according to variant carrier status

19. Further work Development of high throughput variant detection using pyrosequencing Assay expansion - incorporate further known variants and include other genetic associations as identified Testing of 4000 IBD patients once ethics approval received

20. Acknowledgments Stewart Payne Lindsey Sutherland Marta Pereira Dr Alisa Hart (St Mark’s Hospital, London) David Walker (St Mary’s Hospital, London) Dr Andrew Milestone (Division of Medicine, Imperial College London)