1. Understanding the Next Steps in Determining the Role of the Genome in IBD Judy H. Cho, M.D. Ward-Coleman Professor of Translational Genetics and Medicine, Icahn School of Medicine at Mount Sinai December 6th, 2014

2. Questions  What naturally occurring genetic polymorphisms are associated with IBD?

3. What naturally occurring genetic polymorphisms are associated with IBD?  Value: molecular insight  profoundly shaped the landscape of IBD research  What’s worked and what hasn’t  Family-based: linkage  NOD2. Otherwise, family-based studies have not worked—re-vist with microbiome-based studies & genetic counseling  Case-control:  163 loci and counting  Surprises & advances  Magnitude of sample sizes required unexpected: NOD2 (Nature 2001) involved 416 CD samples -  > 30,000 cases  Number of significant loci larger than expected: 163 and counting  Advances: autophagy, IL-23 pathway, M1-M2 macrophage subsets  Challenges: long journey from genes  biology  drug targeting  new drugs Assessment

4. Questions  What naturally occurring genetic polymorphisms are associated with IBD?  What are the functional consequences of IBD- associated polymorphisms?

5. What are the functional consequences of IBD- associated polymorphisms?  Altered cytokine responses to microbial stimulation : NOD2, XIAP (direct); many indirect effects  Genotype-dependent variable bacterial clearance: autophagy, NADPH oxidase  Direct ex vivo analyses  IL23R and Th17/Tc17 cells (Sarin et al., PNAS 2011)  NOD2 & Paneth cell morphologies (Van Dussen Gastroenterology 2014)  Altered regulation of gene expression

6. What are the functional consequences of IBD- associated polymorphisms?  Altered cytokine responses to microbial stimulation : NOD2, XIAP  Genotype-dependent variable bacterial clearance: autophagy, NADPH oxidase  Direct ex vivo analyses  IL23R and Th17/Tc17 cells (Sarin et al., PNAS 2011)  NOD2 & Paneth cell morphologies (Van Dussen Gastroenterology 2014)  Altered regulation of gene expression

7. Expression quantitative trait loci (eQTL) mapping  mRNA expression as a continous trait  Heritable  Mappable to specific SNPs  Cell lines, tissues and context- specificity  Presently defined eQTLs likely only a subset of genuine eQTLs  LPS- & IFN  stimulated monocytes define more eQTLs  80% of transcripts with eQTLs Morley, et al., Nature 2004; 430: 743 Dixon, et al., Nature Genetics 3007; 39: 1202 Fairfax et al., Science 2014

8. Fine-mapping in autoimmunity  Fine-mapped autoimmune loci  90% are non-coding  60% map to immune enhancers  Histone marks: greatest enrichment seen for H3K27ac—active/stimulated enhancers  Disease-associated SNPs in enhancers are near, but not within consensus transcription factor binding sites Farh et al., Nature 2014

9. Enrichment of CD loci genes in open chromatin regions of Th17 cells, but not monocytes  Apples to oranges: Th17 cells vs. monocytes  Pending: tissue-specific enhancer landscape of organ- and context-specific tissue macrophages

10. From co-expression to (genetic) causal networks Gene in IBD- associated locus Highly correlated RNA expression between NOD2, IL10 & HCK (hematopoietic cell kinase)  HCK: key for differentiation of M2 macrophages  Cis eQTL in HCK: variable HCK expression is driving variable NOD2 & IL10 expression aka NRAMP Jostins et al, Nature 2012 Eric Schadt

11. Primary value of direct ex-vivo analyses: pathogenic & protective cells  High dimensional analyses needs to be matched to deep clinical information  Basic science questions  Cellular plasticity and diffentiation  DNA-RNA-protein Defining innate cell hierarchies by high-dimensional Cytof analysis

12. What are the functional consequences of IBD- associated polymorphisms?  Altered cytokine responses to microbial stimulation : NOD2, XIAP  Genotype-dependent variable bacterial clearance: autophagy, NADPH oxidase  Direct ex vivo analyses  IL23R and Th17/Tc17 cells (Sarin et al., PNAS 2011)  NOD2 & Paneth cell morphologies (Van Dussen Gastroenterology 2014)  Altered regulation of gene expression Acceleration of in vitro & in vivo studies with CRISPR/CAS9 technologies: refined genetic & molecular definition will improve modeling

13. Questions  What naturally occurring genetic polymorphisms are associated with IBD?  What are the functional consequences of IBD- associated polymorphisms?  Why do Ashkenazi Jewish populations have a higher IBD prevalence?  Rare variants  Common variants

14. Rare variants are “less rare” in AJs  128 whole genome sequenced in AJs  Rare variants are “less rare” in AJs compared to Flemish  Profound population bottleneck in AJs Derived allele frequency AJ Flemish Carmi et al., Nature Commun 2014

15. IBD Genetic Burden Score Ashkenazi Jews with a higher composite burden score than non-Jewish European ancestry Developing integrative models of common variant risk?

16. Striking overlap between IBD & mycobacterial susceptibility 163 IBD loci 6/7 7 multigenic leprosy GWAS loci 7/9 9 single gene mycobacterial (Tb) genes NOD2 RIPK2 **TNFSF15 LRRK2 IL23R C13orf31 IL12B STAT1 IRF8 TYK2 STAT3 IFNGR2 IFNGR1 Anti-TNF treatment of IBD associated with re-activation of latent mycobacterial disease NEJM 2001; 345: 1098

17. 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 Polygenic adaptation: positive selection at scores to hundreds of genes that confer selective advantage

18. Questions  What naturally occurring genetic polymorphisms are associated with IBD?  What are the functional consequences of IBD- associated polymorphisms?  Why do Ashkenazi Jewish populations have a higher IBD prevalence?  What factors are genetic and what are non- genetic/stochastic/developmental?  Better modeling of genetic contributions  More frequent modeling of environmental/stochastic factors

19. Systematic analysis of rare variants via enteroids Bank of rare variants broadly amenable for study Stem cells Biopsy Add growth factors Stem cells > Ascertaining by genotypes > CRISPR-CAS9

20. IgA-coating enriches for colitogenic bacteria  Stool-based collections: IgA enrichment may identify functionally important bacteria—reducing microbial complexity  Frequent sampling Palm et al., Cell 2014

21. Questions  What naturally occurring genetic polymorphisms are associated with IBD?  What are the functional consequences of IBD- associated polymorphisms?  Why do Ashkenazi Jewish populations have a higher IBD prevalence?  What factors are genetic and what are non- genetic/stochastic/developmental?  UC: limited vs. extensive disease  CD: ileal post-op—modeling first steps of disease recurrence  Age-dependent effects: disease severity & age of onset

22. Acknowledgements  Cho lab  Ken Hui  Monica Bowen  Kyle Gettler  Kaida Ning  Nai-Yun Hsu  Felix Chuang  Yashoda Sharma  Mount Sinai collaborators  Inga Peter  Eric Schadt  Miriam Merad  Bruce Sands  Jean-Fred Colombel  NIDDK IBD Genetics Consortium  Steve Brant  Richard Duerr  Dermot McGovern  John Rioux  Mark Silverberg  Mark Daly  Phil Schumm  Thad Stappenbeck-Wash U: enteroids  Yale University  Clara Abraham  Richard Flavell  RISK Pediatric Consortium  Subra Kugathasan  Ted Denson