1. IBD genetics in children across diverse populations Subra Kugathasan, MD Professor of Pediatrics and Human Genetics Emory University

2. The predictive power of genetic markers should be integrated into the management of IBD Biology (genetics, serology andmicrobial markers vary greatly across populations

3. US Population at Glance By Race: Foreign-Born by Race: Projected http://www.usc.edu/schools/price/futures/pdf/2011_Pitkin-Myers_Projections-Immigrant-Generations-and-Foreign-Born.pdf

4. White et al., Clinical Gastroenterology and Hepatology, 2008 N=138 N=1,406 IBD phenotypes in different populations Multi-center US populations

5. Most genetic studies of IBD to date have mainly been conducted in Caucasians. Finding of Caucasians (West Europeans) cannot be extrapolated to non-Caucasians. Most results found in Caucasians do not have prognostic utility in other minority population. Emerging data support the hypothesis that the prevalence and disease burden among African Americans (AA) is similar to that of Caucasians. The disease pathogenesis, disease course and treatment responses could vary with Race/Ethnicity genetics Why study IBD studies across populations?

6. Goal:  1500 African American IBD subjects from multiple sites  1150 African American control subjects Detailed phenotypic characterization Collection of whole blood for DNA and serum NIH/NIDDK Genetic consortium – ancillary R01 Progress to data Total CASES: 905 Total CONTROLS: 238

7. Genesis AA Enrollment by Site

8. IBD phenotypes in African Americans Our current data from over 900 subjects

9. Age of onset among African Americans Over 900 subjects, not population based

10. Prevalence of CD and UC in the US by Age

11. Age of Onset among Subjects AA enrolled at 18 years of age and older

12. Why study genetics across populations? Non-Africans and Non Europeans represent an admixed population, with founders from West Africa and Europe. They have a different genetic diversity and a various lenght LD block than their founders. This makes genetic studies ideal for identifying population specific disease variants. It is mandatory to perform GWAS in minority population to test the hypothesis that specific disease susceptibility SNPs exist either within the known IBD loci, or in undiscovered loci. Most results found in Caucasians do not have prognostic utility in other minority population Tishkoff SA et al. Science. 2009 May 22;324(5930):1035-44

13. Admixture African American = 80% African + 20% Caucasian loci Based on 1327 nuclear microsatellite and insertion/deletion markers used to asses the genetic ancestry of populations Modified from Tishkoff SA et al. Science. 2009 May 22;324(5930):1035-44 VS.

14. Ancestry Informative Markers can Confirm Self-reported Ethnicity Divers et al., BMC Genet. 2011 Mar 4;12:28 4 clusters distinguishes between 4 US populations

15. Populations Admixture vary within the US Modified from Tishkoff SA et al. Science. 2009 May 22;324(5930):1035-44 VS. http://en.wikipedia.org/wiki/Mexican_American African American = 80% African + 20% Caucasian ancestry

16. Sister 1 84% African, 13% European and 3% Asian Sister 2 78% African, 18% European and 4% Asian http://www.taneya- kalonji.com/genblog/category/23andme/page/2 Admixture can vary between individual of the same family Mother 93% African, 6% European and 1% Asian African Ancestry European Ancestry Asian Ancestry Not genotyped or too little data

17. An admixture mapping scan is typical Nature Reviews Genetics 6, 623-632 (August 2005) Percent Ancestry from Caucasian population Chromosome position Patients inherited high-risk alleles from Caucasian High proportion of ancestry from the Caucasian population

18. African Ancestry European Ancestry Asian Ancestry Not genotyped or too little data Representative AA admixture at NOD2 loci http://blog.openhelix.eu/?p=6906

19. Adeyanju et al. Inflamm Bowel Dis. 2012 Dec;18(12):2357-9

20. 100% European Ancestry 100% African Ancestry 80% African Ancestry 20% European Ancestry Adeyanju et al. Inflamm Bowel Dis. 2012 Dec;18(12):2357-9 Common NOD2 risk variants in AA with CD are due to recent Caucasian admixture Carrier of NOD2 variants (1000fs, R702W, G908R)

21. First Collaborative AA Immunochip 2,236 AA IBD 192,402 SNPs 1770 AA IBD 136,497 SNPs QC filtering Cases + Controls Healthy Controls All IBD Cases CD Cases UC Cases Total1770 701 (39.6%) 1069 (60.4%) 767 (43.3%) 251 (14.2%) Global YRI (%)83.0 ± 8.382.9 ± 8.483.1 ± 8.283.2 ± 8.082.9 ± 8.5  Allele frequencies of Yoruba's (YRI) and Caucasians (CEU) HapMap used to estimate ancestry.  Joint admixture/association tests (logistic regressions) were implemented: 1.Estimate test burdens for admixture and association mapping 2.Genotypic association stratified by local ancestry 3.Combined regression coefficient in each strata and calculated pooled p-values 4.Converted pooled p-values into posterior probability (pp) by using pp from admixture mapping as prior and test burden from association mapping. Removed SNPs with low genotyping quality or errors in duplicates/parent-offspring trios Cedars Sinai Emory University John Hopkins

22. Second Collaborative AA Immunochip Cedars Sinai Emory University John HopkinsTOTAL Cases2817469982025 Controls4928316912023 TOTAL330102926894048 CDUCIBD-U Affected (no disease record) Control non- IBD TOTAL14724608013199429 2025 Cases2023 Controls ~ 500 pediatric cases (2 to 17 years); include ~200 early onset (2 to 10 years)

23. Second Collaborative AA ichip QC Process 4,048 Samples (2025 cases and 2023 controls) All genotypes jointly called with Illumina tools Samples with <98.5% call rate temporarily removed Remaining samples (n=~3,100) reclustered 4,048 samples re-called using clusters from best performing samples Final call rate >97.7%. Only 85 samples having call rates <92%. Mendelian inconsistencies and gender mismatches still being resolved.

24. Minor Allele Frequencies of Selected SNPs Loci Risk Allele Frequency (RAF) CaucasiansAfrican Americans SNP*Gene* Cases*Controls (Hapmap CEU)CasesControls rs5743289 NOD2 0.024 0.2260.0560.033 rs12994997 ATG16L1 0.523 0.5750.3520.311 rs11209026 IL23R 0.933 0.9590.9890.986 rs12942547 STAT3 0.580 0.5450.5970.582 rs516246 FUT2 0.483 0.5310.5170.495 rs4246905 TNFSF15 0.709 0.6760.9280.932 rs17293632 SMAD3 0.235 0.2300.0770.068 rs3024505 IL10 0.160 0.1810.0650.048 rs10781499 CARD9 0.412 0.4870.3190.284 rs4728142 IRF5 0.444 0.3980.2880.287 rs6871626 IL12B 0.337 0.3670.2320.191 rs10758669 JAK2 0.349 0.3660.230.206 rs3197999 MST1 0.296 0.2590.250.241 *Jostins L et al. Nature. 2012 Nov 1;491(7422):119-24

25. Exome Sequencing –A pilot project Sample size: 27 CD patients  20 samples with severe perianal disease phenotype  7 samples with of early onset disease 17 novel functional variants in 16 genes could be implicated in neutrophil dysfunction (validation in progress)

26. Through collaborations, we have assembled a well powered, well phenotyped a understudied US population (AA, both adults and children) for genetic studies Immunochip studies, GWAS and sequencing (exome & whole genome) studies in AA are underway in both adult and children. Admixture analysis is a powerful way to narrow down the causatice loci when admixed population like AA is studied. We will be able to these results in risk stratification and prognostic utility in minority population directly rather than extrapolating the Caucasian found results. Conclusions & Future directions

27. Acknowledgements to our partners NIH/NIDDK Genetic consortium