1. State of the Art in IBD Genetics Judy H. Cho, M.D. Ward-Coleman Professor of Medicine and Genetics, Icahn School of Medicine at Mount Sinai

2. Central importance of human genetics  Germline DNA variants  disease susceptibility  Primary causality  Immediate molecular insight—genes, increased/decreased function  Humans as your experimental system—natural pertubagens  relevance for patients  Genome-wide genetic approaches are unbiased  Novel, unexpected insight—autophagy  Promise of new therapy development—major challenge for the field

3. Genetics: enormous impact on IBD research—autophagy & Paneth cells Cell 2010 Nature 2013 Critical role for the Paneth cell

4. Genetics impact: IL-23 pathway & treatment Science 2006 PNAS 2011 Nature 2013 Salt increases IL23R expression NEJM 2012

5. IBD Immunochip: 163 loci associated to IBD 50 Cumulative IBD loci 100 0 150 2000200220042006200820102012 Year MHC in UC NOD2 Single- center GWAS GWAS meta- analyses Immunochip 163 loci Nature, 2012

6. Major genetics concepts: functional variants and evolutionary selection  Overlap of major loci between related diseases— motivation for development of Immunochip  Most GWAS-identified variants are non-coding and affect gene expression (eQTLs)  Immune-mediated disease loci: evolved in response to historically significant pathogens  Population differences: may provide major insight  Common vs. rare variants

7. Crohn’s disease Ulcerative colitis  IL23R in both  MHC major in UC  Crohn’s disease- uniquely lacks a dominant MHC signal  Instead, innate immune defects: NOD2 & ATG16L1 Genetic architecture: Crohn’s disease vs. ulcerative colitis in European ancestry cohorts Science 2006;314:1461 Nature 2001;411:603 **Nat Genet. 2011;43:246 **Nat Genet. 2010;42:1118 Nat Genet. 2009;41:216 Nat Genet. 2011;43:1066 Arg381Gln

8. The Immunochip effort in IBD: international collaboration on a grand scale  38,565 cases & 37,747 controls  Combined 15 separate European ancestry IBD GWAS  25,075 SNPs with p < 0.01  Meta-analysis:  GWAS +  New cases genotyped on Immunochip  14,763 CD cases  10,920 UC cases  15,977 controls genotyped  71 new loci  163 genome-wide significant loci

9. Defining the genetic architecture of CD vs. UC IBD vs. control odds ratio >1.5 1.3 1.4 1.2 1.1 0.671.0>1.5 IL23R NOD2 PTPN22 CD vs. UC odds ratio 30 CD specific loci 23 UC specific loci 110 IBD loci MHC

10. Inflammatory bowel disease: 163 loci  genes & alleles  Annotation approaches for “hit SNPs”:  cSNPs: 24 loci (15%)  eQTLs: 64 loci (39%) **  Dapple (protein-protein interaction): 30 loci  Grail (literature mining): 87 loci  Bayesian network analysis: 43 loci  52 loci contain genes implicated by two or more annotation approaches

11. Striking overlap of IBD loci between diseases IBD loci Immune-mediated diseases 82 53 82 Primary immune deficiencies MSMD Mycobacterial disease Chronic mucocutaneous candidiasis (CMC): CARD9, STAT3 CMC

12. Striking overlap between IBD & mycobacterial susceptibility 163 IBD loci 6/7 7 leprosy GWAS loci 7/9 9 single gene mycobacterial (Tb) genes NOD2 RIPK2 TNFSF15 LRRK2 IL23R C13orf31 IL12B STAT1 IRF8 TYK2 STAT3 IFNGR2 IFNGR1 *

13. Why the specificity between IBD & mycobacterial infection?  NOD2 & glycolyl MDP: mycobacteria & Actinomycetes contain enzyme (NamH) which converts acetyl MDP to glycolyl MDP (Coulombe, JEM 2009)  TNF & IBD:  Over-expression of TNF  ileitis & arthritis (Kontoyiannis, Immunity 1999)  Anti-TNF highly effective in the treatment of Crohn’s disease & ulcerative colitis  Anti-TNF treatment  reactivation of latent Tb (Keane et al, NEJM 2001)  Ashkenazim, Crohn’s disease & mycobacterial susceptibility

14. Epidemiologic support for the Jewish-Tb hypothesis PopulationDeaths per 100,000 Mussulman Arabs1130 Europeans513 Jews75 Deaths from tuberculosis, London 1894-1900 PopulationNYBrooklyn African-American 774.21531.35 Ireland645.73452.79 Bohemia499.13347.22 Russia and Poland (mostly Jews) 98.2176.72 Scotland384.12269.24 Scandinavia357.00218.92 Canada352.32266.27 Germany328.80295.61 France394.98252.82 England and Wales322.50233.78 Italy233.85123.00 United States (White) 205.14180.79 Hungary (mostly Jews) 155.05120.77 Jacobs J. The Jewish Encyclopedia; a guide to its contents, an aid to its use. New York, London: Funk & Wagnalls company; 1906. NYC, 6 years before 1890 per 100,000

15. Tissue-based co-expression modules define genes with correlated gene expression Gene in IBD- associated locus Module with greatest enrichment for IBD genes: 523 module from adipose tissue NOD2 Highly correlated RNA expression between NOD2, IL10 & HCK (hematopoietic cell kinase) - HCK: key for differentiation of M2 macrophages

16. Unexpected relationship between abdominal fat and IBD Transmural disease  complications Creeping fat Adipose tissue an abundant source of TNF

17. Rare variants--less power to detect association, but greater effect sizes (i.e., odds ratios, OR) Magnitude of effect Frequency of genetic variation Uncommon variation of large effect (Mendelian) Common variation of small effects Not typically present Not identifiable 0.50 1 (baseline risk) RiskProtective GWAS Most associations with small effects, OR < 1.1 1% vs. 0.3%  OR ~3 Negative selection: deleterious alleles are low frequency

18. At least 3 of 4 components of Mendelian susceptibility to Mycobacterial diseases (MSMD) genes also associated to IBD Key components: 1.IL12/23 signaling 2.IFN  signaling 3.CD40-CD40L interaction 4.NADPH oxidase system * * * * * * * * * * * * * * * * IBD-associated gene Casanova et al., Immunity 2012 36: 515 * What about the NADPH oxidase system??

19. NADPH oxidase deficiency & IBD  Autosomal recessive mutations in NCF2 (p67phox) associated with chronic granulomatous disease  NCF4: nominal association to IBD by GWAS (association signal stronger in AJs)  NCF2 mutations at Arg38  Arg38Gln: 0.5% allele in very-early onset IBD with 24x increased risk (Muise et al, Gut 2012)  Ashkenazi Jewish exome sequencing: identified an AJ-specific, distinct mutation  Arg38Trp (0.51% allele, 4.4x increased risk)  Both mutations, Arg38Gln, Arg38Trp  impaired binding to RAC2  Implicates impaired NADPH oxidase function in adolescent/adult-onset IBD as well as very-early onset IBD

20. Conclusions & future directions  IBD genetics: foundation for many of the most impactful publications in IBD research  Genetic architecture of IBD shaped in response to mycobacterial infections—implications  Host-microbiome interactions  Can leverage the enormous existing biologic understanding of innate responses to mycobacteria  Leverage evolution and population differences  Rare mutations have higher effect sizes and may provide a more direct route to new therapies  Early onset  Population differences