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

Questions What naturally occurring genetic polymorphisms are

associated with IBD?

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

Questions What naturally occurring genetic polymorphisms are

associated with IBD? What are the functional consequences of IBD-

associated polymorphisms?

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

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

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 g stimulated monocytes define more eQTLs 80% of transcripts with eQTLs

Morley, et al., Nature 2004; 430: 743Dixon, et al., Nature Genetics 3007; 39: 1202Fairfax et al., Science 2014

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

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

Category (N) CD loci

(#gene=1328) non CD loci

(#gene=23,134) log OR

(95% CI) p-value

Yes (14,267) 945 (71.2%) 13,322 (57.6%) No (10,195) 383 (28.8%) 9812 (42.4%) Yes (16,317) 862 (64.9%) 15,455 (66.8%)

No (8145) 466 (35.1%) 7679 (33.2%)

Th17 cell DNase I HS

site Monocyte DNase I HS

site

-0.08 (-0.20, 0.032)

0.6 (0.48, 0.72)

5.90E-22

0.15

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

NOD2

HCK

IL10VDR

SLC11A1

DOK3

CARD9

LGALS9

aka NRAMP

Jostins et al, Nature 2012

Eric Schadt

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

Uninflamed Inflamed

CD14

HLADR

BDCA3

Defining innate cell hierarchies by high-dimensional Cytof analysis

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

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

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

IBD Genetic Burden Score

Ashkenazi Jews with a higher composite burden score than non-Jewish European ancestry

Developing integrative models of common variant risk?

Striking overlap between IBD & mycobacterial susceptibility

163 IBD loci

6/7

7 multigenic leprosy

GWAS loci

7/9

9 single gene mycobacterial (Tb) genes

NOD2RIPK2

**TNFSF15LRRK2IL23R

C13orf31

IL12B STAT1 IRF8 TYK2 STAT3

IFNGR2IFNGR1

Anti-TNF treatment of IBD associated with re-activation of latent mycobacterial disease

NEJM 2001; 345: 1098

Epidemiologic support for the Jewish-Tb hypothesis

Population Deaths per 100,000

Mussulman Arabs 1130

Europeans 513

Jews 75

Deaths from tuberculosis, London 1894-1900 Population NY Brooklyn

African-American 774.21 531.35

Ireland 645.73 452.79

Bohemia 499.13 347.22

Russia and Poland (mostly Jews) 98.21 76.72

Scotland 384.12 269.24

Scandinavia 357.00 218.92

Canada 352.32 266.27

Germany 328.80 295.61

France 394.98 252.82

England and Wales 322.50 233.78

Italy 233.85 123.00

United States (White) 205.14 180.79

Hungary (mostly Jews) 155.05 120.77

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

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

Systematic analysis of rare variants via enteroids

Bank of rare variants broadly amenable for study

Stem cells

Biopsy

Add growth factorsStem cells

> Ascertaining by genotypes> CRISPR-CAS9

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

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

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