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

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

US Population at Glance By Race: Foreign-Born by Race: Projected

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

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?

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

Genesis AA Enrollment by Site

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

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

Prevalence of CD and UC in the US by Age

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

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 May 22;324(5930):

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 May 22;324(5930): VS.

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

Populations Admixture vary within the US Modified from Tishkoff SA et al. Science May 22;324(5930): VS. African American = 80% African + 20% Caucasian ancestry

Sister 1 84% African, 13% European and 3% Asian Sister 2 78% African, 18% European and 4% Asian 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

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

African Ancestry European Ancestry Asian Ancestry Not genotyped or too little data Representative AA admixture at NOD2 loci

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

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

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 Total (39.6%) 1069 (60.4%) 767 (43.3%) 251 (14.2%) Global YRI (%)83.0 ± ± ± ± ± 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

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

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.

Minor Allele Frequencies of Selected SNPs Loci Risk Allele Frequency (RAF) CaucasiansAfrican Americans SNP*Gene* Cases*Controls (Hapmap CEU)CasesControls rs NOD rs ATG16L rs IL23R rs STAT rs FUT rs TNFSF rs SMAD rs IL rs CARD rs IRF rs IL12B rs JAK rs MST *Jostins L et al. Nature Nov 1;491(7422):119-24

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)

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

Acknowledgements to our partners NIH/NIDDK Genetic consortium