Evaluation of the genetic impact on inflammatory bowel disease

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 ofIBD • 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 2nd – 4th decade with European prevalence of 100-150 per 100,000 Comparison of the clinical features of CD and UC

4. 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 intestinalimmunity • 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 variantshave 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 • 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 NOD2 loci IL23R locus c.1422G>A c.2104C>T c.2722G>C c.3020insC A tube NOD2 WT and IL23R variant primers B tube NOD2 variant and IL23R WT primers

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)