Identifying Dysregulated Genes in Autoimmune Disease Chris Cotsapas - - PowerPoint PPT Presentation

Identifying Dysregulated Genes in Autoimmune Disease

Chris Cotsapas PhD Yale Neurology/Genetics Broad Institute cotsapas@broadinstitute.org

Causall Identifying Dysregulated Genes in Autoimmune Disease

Chris Cotsapas PhD Yale Neurology/Genetics Broad Institute cotsapas@broadinstitute.org

Multiple sclerosis GWAS

47 new ¡ hit

15,000

Immunochip 200,000 SNPs ¡ (targeted) ¡

10,000

25 new ¡ hits WTCCC2

8000

650,000 SNPs ¡

MS

6000

Subjects

Meta-­‑Analysis 3 new ¡ hits 2.6 million ¡SNPs ¡

4000

Meta v2.5 6 new ¡ hits 2.6 million ¡SNPs ¡ IMSGC GWAS 1 new ¡ hit

2000

345,000 SNPs ¡ ANZ ¡GWAS 2 hits 550,000 SNPs ¡

IMSGC NEJM 2007 De Jager et al. Nat Genet 2009

2007 2008 2009 2010 2011 2012

Rubio et al. Nat Genet 2009 IMSGC Nature 2011 Patsopoulos et al. Ann Neurol 2011

Date of comple/on ¡

Meta-­‑Analysis v3.0

… and that’s not all!

16K MS cases / 26K controls Replica;on 18K MS cases / 18K controls 100 new ¡hits 47 new ¡ hit

15,000

Immunochip 200,000 SNPs ¡ (targeted) ¡

10,000

25 new ¡ hits WTCCC2

8000

650,000 SNPs ¡

MS

6000

Subjects

Meta-­‑Analysis 3 new ¡ hits 2.6 million ¡SNPs ¡

4000

Meta v2.5 6 new ¡ hits 2.6 million ¡SNPs ¡ IMSGC GWAS 1 new ¡ hit

2000

345,000 SNPs ¡ ANZ ¡GWAS 2 hits 550,000 SNPs ¡

IMSGC NEJM 2007 De Jager et al. Nat Genet 2009

2007 2008 2009 2010 2011 2012

Rubio et al. Nat Genet 2009 IMSGC Nature 2011

Date of comple/on ¡

Patsopoulos et al. Ann Neurol 2011

GWAS signals are enriched in regulatory DNA

Maurano et al Science 2012

MS GWAS hits enriched in transcription factor binding sites

Farh et al Nature 2015

Plotted SNPs

− log10(p−value)

NFKB1 locus in MS GWAS

10 8 6 4 2 103.4 103.5 103.6 103.7 Position on chr4 (Mb)

IMSGC, Nat Genet, 2013 Housley et al STM 2015

20 40 60 80 100 Recombination rate (cM/Mb) rs7665090

• ●
• ●
• 0.2

0.4 0.6 0.8

r2

NFKB1 MANBA UBE2D3

72 A) Regional Association and B) Forest Plot Supplementary File Supplementary Figure 42. Discovery phase rs12946510. A B

IKZF3/ORMDL3 locus in MS GWAS

IMSGC, Nat Genet, 2013

Gene

Gene-DHS correlation posterior CP x PPA

DHS

Posterior probability

• f association

PPA

Approach

Total gene posterior GP = Σ(CPDHS,gene x PPADHS) Regulatory posterior RP = Σ(PPADHS) SNP Parisa Shooshtari

Problem 1: DHS-gene correlations

CD 3 CD 14 CD 34 Thymus Kidney Lung Heart Brain

Gene 1 Gene 2

CD 3 CD 14 CD 34 Thymus Kidney Lung Heart Brain

Parisa Shooshtari

Aligning DHSs Over Samples

Parisa Shooshtari

Identify detectable DHS clusters

Scenario 2 Align over 57 tissue replicates from REP

C1 1 C1 2

Tissues Hotspot peaks

C2 C3 1 1 C2 C3 2 1

T1-Rep1

C4 C4

T1-Rep2

2

C5 C5

T2-Rep1 T2-Rep2

1

C6 1 C6 2

T3-Rep1 T3-Rep2

2

Unreliable Cluster Reliable Cluster

T4-Rep1 T4-Rep2

2

T5-Rep1

1 1,079,138/1,994,675 (54.1%) clusters pass Cover 8% of genome (cf. 14% of all DHS)

T5-Rep2 T6-Rep1

2

T6-Rep2

NB singletons, low power Scenario 1 1 1 1 1 1 1 1 1 1 1 Parisa Shooshtari

0.0 0.2 0.4

Proportion of Heritability

QC+ DHS clusters capture most MS heritability

Caveat DHS clusters are

Status DHS_Peaks_Hotspot DHS_Clusters Penis_Foreskin_Melanocyte_Primary_Cells Penis_Foreskin_Keratinocyte_Primary_Cells Penis_Foreskin_Fibroblast_Primary_Cells Pancreas Mobilized_CD4_Primary_Cells Mobilized_CD34_Primary_Cells Mobilized_CD3_Primary_Cells IMR90 H9 H1_Derived_Mesenchymal_Stem_Cells H1_BMP4_Derived_Trophoblast_Cultured_Cells H1_BMP4_Derived_Mesendoderm_Cultured_Cells H1 Gastric Fibroblasts_Fetal_Skin_Upper_Back Fibroblasts_Fetal_Skin_Scalp Fibroblasts_Fetal_Skin_Quadriceps_Right Fibroblasts_Fetal_Skin_Quadriceps_Left Fibroblasts_Fetal_Skin_Biceps_Right Fibroblasts_Fetal_Skin_Biceps_Left Fibroblasts_Fetal_Skin_Back Fibroblasts_Fetal_Skin_Abdomen Fetal_Thymus Fetal_Testes Fetal_Stomach Fetal_Spinal_Cord Fetal_Renal_Pelvis_Right Fetal_Renal_Pelvis_Left Fetal_Renal_Pelvis Fetal_Renal_Cortex_Right Fetal_Renal_Cortex_Left Fetal_Renal_Cortex Fetal_Placenta Fetal_Muscle_Trunk Fetal_Muscle_Leg Fetal_Muscle_Back Fetal_Muscle_Arm Fetal_Lung_Right Fetal_Lung_Left Fetal_Lung Fetal_Kidney_Right Fetal_Kidney_Left Fetal_Kidney Fetal_Intestine_Small Fetal_Intestine_Large Fetal_Heart Fetal_Brain Fetal_Adrenal_Gland CD8_Primary_Cells CD56_Primary_Cells CD4_Primary_Cells CD3_Primary_Cells CD19_Primary_Cells CD14_Primary_Cells Breast_vHMEC

Cell

wider than DHS peaks (250-400bp vs 150bp Parisa Shooshtari Hilary Finucane Alkes Price

Proportion of h2g

Correction

Challenge 2: Gene expression correlation

QQ plot for P Value of Correlation Between One DHS and 14000 Genes Correlation Structure of the Gene Expression Data Before Correction After

Parisa Shooshtari

Gene

Gene-DHS correlation posterior CP x PPA

DHS

Posterior probability

• f association

PPA

Approach

Total gene posterior GP = Σ(CPDHS,gene x PPADHS) Regulatory posterior RP = Σ(PPADHS) SNP Parisa Shooshtari

Application to MS GWAS

Chr 6 90.5-91.5Mb RP = 0.945 Gene MDN1 0.555 BACH2 0.162 GABRR2 0.106 RRAGD 0.065 GJA10 0.029 MAP3K7 0.028 GP Parisa Shooshtari IMSGC NG 2013

72 B A) Regional Association and B) Forest Plot Supplementary File Supplementary Figure 42. Discovery phase rs12946510. A

IKZF3/ORMDL3 locus

Gene GP Chr 17 ORMDL3 0.029 34.5-35.5Mb PIP4K2B 0.022 RP = 0.295 IGFBP4 0.018 IKZF3 0.015 GSDMB 0.014 SMARCE1 0.013 CCR7 0.013 TNS4 0.01 ZPBP2 0.009 MED1 0.009 MED24 0.009 KRT24 0.009 PNMT 0.008 CDK12 0.007 RPL23 0.007 PSMD3 0.007 PLXDC1 0.006 TOP2A 0.006 RARA 0.006

MS GWAS hits enriched in transcription factor binding sites

Farh et al Nature 2015

MS GWAS risk effect: NFKB1 locus

MS patients show altered NFκB signaling in CD4+ T cells

Figure 1. Naïve CD4 cells from patients with MS exhibit increased phospho-p65 NFκB. Flow cytometry of PBMCs from age-matched healthy control (HC) and relapsing-remitting MS (RRMS) patients stained for CD4, CD45RA, CD45RO, and pS529 p65 NFκB. MFI of p65 results are shown gated on naïve CD4+CD45RA+CD45RO- T-cells.

CD4+ T cells from MS patients proliferate more rapidly after stimulus (Kofler et al JCI 2014) ex vivo CD4+ T cells show higher p-p65 (Housley et al, STM 2015)

15 30 10 20 30 Minutes Nuclear localization GG AA

p= 0.05

p50 NFkB 10 20 30 GG AA

p= 0.037 20 40 60 80 100 % IκBα p= 0.0091 p= 0.019 GG AA AG AA GG TNF-α No Stim PMA AG AA GG AG 500 1000 1500 2000 pNFκB p= 0.029 p= 0.027 GG AG AA GG AG AA TNF-α PMA

a b

rs228614

Housley et al, STM 2015

MS risk effect near NFKB1 alters signaling in CD4+ cells

Housley, unpublished

MS variant in TNFRSF1A alters TNFα-dependent NFκB signaling

GWAS loci harbor many NFκB genes

Housley, unpublished

Model: NFκB signaling variation

p50 P-p50 External stimulus p65 *NFκB Activation Proliferation Survival p65

*NFκB

Activation Proliferation Survival Broader phenotype? p50 P-p50

GV in NFκB pathway GV in NFκB TFBS New gene activation patterns by NFκB

Systematic dissection I

CD4+ T cells Timing Event exposure TNFα 15m signaling Phospho-flow NFκB 30m translocation NFκB CHiP-seq 2h gene activation H3K27Ac-seq RNA-seq Brad Bernstein Chris Cotsapas David Hafler Will Housley 3d cell phenotype αCD3/CD28 CyTOF 25 NFKB1 risk variant homozygotes 25 NFKB1 non-risk variant homozygotes

Acknowledgements

• IMSGC

– David Hafler – Phil De Jager – Steve Hauser – Adrian Ivinson – Nikos Patsopoulos – Many, many others

• Partners

– David Hafler – Phil De Jager – Brad Bernstein – John Stamatoyannopoulos

• Yale labs

– Parisa Shooshtari – Mitja Mitrovic – Alex Casparino – Will Housley

8.6e-09 1.6e-08 3.2e-09 1.5e-08 1.5e-08

Genes Burdened DHSs

DHS1 DHS2 DHS3 DHS4 DHS5

• ●
• ●
• ●
• ●
• ●
• ●
• ●
• ●
• ●
• ●
• 2

4 6 8 42.5 43.0 43.5 44.0 Chromosome 1 position (Mb) −log10(P)

In credible interval

• No

Yes

THAD A PLEKHH2 LRPP RC DYNC2LI1 ABCG8 ABCG5 ZFP36L2 KCNG3 OXER1 EML4 MTA3 HAAO

Position on Chromosome 2