Genome-wide pathway analysis highlight the role of
Wnt signaling in endometriosis SEUD 9 May 2015, Paris
Outi Uimari*1 N. Rahmioglu*1, D.R. Nyholt2, C.M. Becker3,
A.P. Morris1, G.W. Montgomery4, K.T. Zondervan1,3.
1University of Oxford, The Wellcome Trust Centre for Human Genetics, Oxford, UK.
2QIMR Berghofer Medical Research Institute, Neurogenetics, Brisbane, Australia. 3University of Oxford, Nuffield Department of Obstetrics and Gynaecology, Oxford,
UK. 4QIMR Berghofer Medical Research Institute, Molecular Epidemiology, Brisbane,
Australia.
estrogen
progesterone
inflammation immunology
angiogenesis
ENDOMETRIOSIS
ENDOMETRIOSIS
Complex disease: genetic and environmental
factors Genetic component ~ 52% heritability (Treloar 1999)
HYPOTHESIS DRIVEN
RESEARCH
HYPOTHESIS FREE RESEARCH
CANDIDATE GENE ASSOCIATION
STUDIES
GENOME-WIDE ASSOCIATION STUDIES
122 CANDIDATE GENES (Rahmioglu 2012)
9 GENETIC VARIANTS BY GWAS (Rahmioglu 2014)
BIOLOGICAL PATHWAY
= series of actions among molecules in a cell that lead to a certain product or change in the cell
GENE SET SIZE: 100 2099 ESR1 2100 ESR2 3320 HSP90AA1 3326 HSP90AB1 7184 HSP90B1 2288 FKBP5 3312 HSPA8 3303 HSPA1A 3306 HSPA2 3305 HSPA1L 3304 HSPA1B 3310 HSPA6 2353 FOS 3725 JUN … … … 51806 CALML5 4846 NOS3 2911 GRM1
Pathway example: ESTROGEN SIGNALING KEGG Pathway Database
PATHWAY ANALYSIS
PRINCIPLE
SNP1
I SNP2
I SNP3
I SNP4
I SNP5
I SNP6
I
GENE A GENE B GENE C
BIOLOGICAL PATHWAY
I I I I I I
CASES AND CONTROLS
I
I I I I I
STUDY AIMS
Overall aim Identify causal genetic pathways underlying endometriosis through pathway analysis using GWAS data to test for genetic enrichment. Two-staged analysis I. Hypothesis-driven selection of set of pathways
(1) 122 candidate genes (2) Studied biological categories
II. Hypothesis-free non-limited pathway selection
GWAS STUDY MATERIAL
Genotype data: 1000 Genomes Phase 3 Panel, coverage 77.8 million variant SNP’s
PATHWAY DATABASES
TOTAL number of pathways: MSigDB 1320 PANTHER 176 Downloaded from www.pantherdb.org (14/8/2014) www.broadinstitute.org (24/9/2014)
STUDY DESIGN
122 Candidate
Genes recognised until
Sep 2014
Most Studied Biological Categories
Literature search until Jul 2014 Search terms: endometriosis/
pathophysiology/pathway
PANTHER 176
pathways
15 pathways
317 pathways
21 pathways
MAGENTA Software
MAGENTA algorithm: STEP1 – map SNP’s onto genes, STEP2 – score genes based on regional SNP p-values, STEP3 – correct for gene score confounders, STEP4 – estimate gene set enrichment p-value for gene sets of interestb
International EndoGene Consortium, IEC GWA-data: 3,194 cases and 7,060 controls. Cases staged according to rAFS classification system.
“All” stA stB
PANTHER 176
pathways
MSig C2a BioCarta/KEGG/PID
Reactome/SA/SIG/STa 1320 canonical pathways
All 1320 pathways
All 176 pathways
Panther 176
pathways
MSigC2 1320
canonical pathways
HYPOTHESIS DRIVEN ANALYSIS HYPOTHESIS FREE ANALYSIS
HYPOTHESIS
PATHWAY DATABASES
IDENTIFIED PATHWAYS
ANALYSIS SOFTWARE
DATA
RESULTS
a"IPA,"Ingenuity"Pathway"Analysis;"MSig"C2,"Molecular"Signatures"Curated"Gene"sets;"KEGG,"Kyoto"Encyclopedia"of"Genes"and"Genomes;"PID,"Pathway"InteracBon"Database;"SA,"SigmaAldrich;"SIG,"Signaling"Gateway;"ST,"Signal"TransducBon"b"Segre"et"al."PLoSGeneBcs"2010."
Figure I. Study design.
“All” stA stB “All” stA stB
HYPOTHESIS DRIVEN ANALYSIS
PATHWAY SELECTION BY 122 candidate genes
PATHWAY SELECTION BY Most studied biological categories
STUDY DESIGN
122 Candidate
Genes recognised until
Sep 2014
Most Studied Biological Categories
Literature search until Jul 2014 Search terms: endometriosis/
pathophysiology/pathway
PANTHER 176
pathways
15 pathways
317 pathways
21 pathways
MAGENTA Software
MAGENTA algorithm: STEP1 – map SNP’s onto genes, STEP2 – score genes based on regional SNP p-values, STEP3 – correct for gene score confounders, STEP4 – estimate gene set enrichment p-value for gene sets of interestb
International EndoGene Consortium, IEC GWA-data: 3,194 cases and 7,060 controls. Cases staged according to rAFS classification system.
“All” stA stB
PANTHER 176
pathways
MSig C2a BioCarta/KEGG/PID
Reactome/SA/SIG/STa 1320 canonical pathways
All 1320 pathways
All 176 pathways
Panther 176
pathways
MSigC2 1320
canonical pathways
HYPOTHESIS DRIVEN ANALYSIS HYPOTHESIS FREE ANALYSIS
HYPOTHESIS
PATHWAY DATABASES
IDENTIFIED PATHWAYS
ANALYSIS SOFTWARE
DATA
RESULTS
a"IPA,"Ingenuity"Pathway"Analysis;"MSig"C2,"Molecular"Signatures"Curated"Gene"sets;"KEGG,"Kyoto"Encyclopedia"of"Genes"and"Genomes;"PID,"Pathway"InteracBon"Database;"SA,"SigmaAldrich;"SIG,"Signaling"Gateway;"ST,"Signal"TransducBon"b"Segre"et"al."PLoSGeneBcs"2010."
Figure I. Study design.
“All” stA stB “All” stA stB
MAGENTA Meta-Analysis Gene-set Enrichment of variaNT
Associations
INPUT for analysis: the SNP association p-value with chromosomal positions from GWA study of interest
MAGENTA Software
MAGENTA algorithm: STEP1 – map SNP’s onto genes, STEP2 – score genes based on regional SNP p-values, STEP3 – correct for gene score confounders, STEP4 – estimate gene set enrichment p-value for gene sets of interestb
NULL HYPOTHESIS: Tested genes are randomly distributed within the predefined pathway. ALTERNATIVE HYPOTHESIS: Enrichment of genes in the predefined pathway with a p-value below the cut-off.
DATABASE BIOLOGICAL PATHWAY
GENE SET SIZE
NOMINAL P-VALUE
95% CUTOFF FDR
95% CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES ABOVE
95% CUTOFF 1 BIOCARTA PTEN PATHWAY 18 0.0024 0.0652 1 5 2 PANTHER FGF SIGNALING PATHWAY 98 0.0095 0.0739 5 11 3 PANTHER CHOLESTEROL BIOSYNTHESIS 11 0.01.73 0.0802 1 3 4 SA B CELL RECEPTOR COMPLEXES 24 0.0281 0.0931 1 4 5 BIOCARTA IL3 PATHWAY 13 0.0213 0.110 1 3 6 BIOCARTA IGF1 PATHWAY 21 0.0197 0.114 1 4 7 BIOCARTA INSULIN PATHWAY 22 0.00360 0.115 1 5 8 BIOCARTA IL2 PATHWAY 20 0.0160 0.126 1 4 9 BIOCARTA IL6 PATHWAY 22 2.19E-02 0.136 1 4
10 BIOCARTA EPO PATHWAY 18 1.12E-02 0.140 1 4
Example of results
STUDY DESIGN
122 Candidate
Genes recognised until
Sep 2014
Most Studied Biological Categories
Literature search until Jul 2014 Search terms: endometriosis/
pathophysiology/pathway
PANTHER 176
pathways
15 pathways
317 pathways
21 pathways
MAGENTA Software
MAGENTA algorithm: STEP1 – map SNP’s onto genes, STEP2 – score genes based on regional SNP p-values, STEP3 – correct for gene score confounders, STEP4 – estimate gene set enrichment p-value for gene sets of interestb
International EndoGene Consortium, IEC GWA-data: 3,194 cases and 7,060 controls. Cases staged according to rAFS classification system.
“All” stA stB
PANTHER 176
pathways
MSig C2a BioCarta/KEGG/PID
Reactome/SA/SIG/STa 1320 canonical pathways
All 1320 pathways
All 176 pathways
Panther 176
pathways
MSigC2 1320
canonical pathways
HYPOTHESIS DRIVEN ANALYSIS HYPOTHESIS FREE ANALYSIS
HYPOTHESIS
PATHWAY DATABASES
IDENTIFIED PATHWAYS
ANALYSIS SOFTWARE
DATA
RESULTS
a"IPA,"Ingenuity"Pathway"Analysis;"MSig"C2,"Molecular"Signatures"Curated"Gene"sets;"KEGG,"Kyoto"Encyclopedia"of"Genes"and"Genomes;"PID,"Pathway"InteracBon"Database;"SA,"SigmaAldrich;"SIG,"Signaling"Gateway;"ST,"Signal"TransducBon"b"Segre"et"al."PLoSGeneBcs"2010."
Figure I. Study design.
“All” stA stB “All” stA stB
RESULTS Hypothesis driven - Candidate gene based
analysis
122 Candidate
Genes recognised until
Sep 2014
Most Studied Biological Categories
Literature search until Jul 2014 Search terms: endometriosis/
pathophysiology/pathway
PANTHER 176
pathways
15 pathways
317 pathways
21 pathways
MAGENTA Software
MAGENTA algorithm: STEP1 – map SNP’s onto genes, STEP2 – score genes based on regional SNP p-values, STEP3 – correct for gene score confounders, STEP4 – estimate gene set enrichment p-value for gene sets of interestb
International EndoGene Consortium, IEC GWA-data: 3,194 cases and 7,060 controls. Cases staged according to rAFS classification system.
“All” stA stB
PANTHER 176
pathways
MSig C2a BioCarta/KEGG/PID
Reactome/SA/SIG/STa 1320 canonical pathways
All 1320 pathways
All 176 pathways
Panther 176
pathways
MSigC2 1320
canonical pathways
HYPOTHESIS DRIVEN ANALYSIS HYPOTHESIS FREE ANALYSIS
HYPOTHESIS
PATHWAY DATABASES
IDENTIFIED PATHWAYS
ANALYSIS SOFTWARE
DATA
RESULTS
a"IPA,"Ingenuity"Pathway"Analysis;"MSig"C2,"Molecular"Signatures"Curated"Gene"sets;"KEGG,"Kyoto"Encyclopedia"of"Genes"and"Genomes;"PID,"Pathway"InteracBon"Database;"SA,"SigmaAldrich;"SIG,"Signaling"Gateway;"ST,"Signal"TransducBon"b"Segre"et"al."PLoSGeneBcs"2010."
Figure I. Study design.
“All” stA stB “All” stA stB
RESULTS Hypothesis driven - Candidate gene based
analysis
DATABASE BIOLOGICAL PATHWAY GENE SET
SIZE
NOMINAL P-VALUE
95% CUTOFF FDR
95% CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
1 -
DATABASE BIOLOGICAL PATHWAY GENE SET SIZE
NOMINAL P-VALUE
95% CUTOFF
FDR 95%
CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
1 PANTHER
INSULIN IGF PATHWAY MAPK CASCADE 30 0.0031 0.0132 2 6
DATABASE BIOLOGICAL PATHWAY GENE SET
SIZE
NOMINAL P-VALUE
95% CUTOFF FDR
95% CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
1 PANTHER
ANDROGEN ESTROGEN PROGESTERONE BIOSYNTHESIS 10 0.0115 0.0309 1 3
“All” endometriosis
Stage A endometriosis
Stage B endometriosis
RESULTS Hypothesis driven - Literature review based
analysis
122 Candidate
Genes recognised until
Sep 2014
Most Studied Biological Categories
Literature search until Jul 2014 Search terms: endometriosis/
pathophysiology/pathway
PANTHER 176
pathways
15 pathways
317 pathways
21 pathways
MAGENTA Software
MAGENTA algorithm: STEP1 – map SNP’s onto genes, STEP2 – score genes based on regional SNP p-values, STEP3 – correct for gene score confounders, STEP4 – estimate gene set enrichment p-value for gene sets of interestb
International EndoGene Consortium, IEC GWA-data: 3,194 cases and 7,060 controls. Cases staged according to rAFS classification system.
“All” stA stB
PANTHER 176
pathways
MSig C2a BioCarta/KEGG/PID
Reactome/SA/SIG/STa 1320 canonical pathways
All 1320 pathways
All 176 pathways
Panther 176
pathways
MSigC2 1320
canonical pathways
HYPOTHESIS DRIVEN ANALYSIS HYPOTHESIS FREE ANALYSIS
HYPOTHESIS
PATHWAY DATABASES
IDENTIFIED PATHWAYS
ANALYSIS SOFTWARE
DATA
RESULTS
a"IPA,"Ingenuity"Pathway"Analysis;"MSig"C2,"Molecular"Signatures"Curated"Gene"sets;"KEGG,"Kyoto"Encyclopedia"of"Genes"and"Genomes;"PID,"Pathway"InteracBon"Database;"SA,"SigmaAldrich;"SIG,"Signaling"Gateway;"ST,"Signal"TransducBon"b"Segre"et"al."PLoSGeneBcs"2010."
Figure I. Study design.
“All” stA stB “All” stA stB
RESULTS Hypothesis driven - Literature review based
analysis
DATABASE BIOLOGICAL PATHWAY GENE SET SIZE
NOMINAL P-VALUE
95% CUTOFF FDR
95% CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
1 REACTOM
E
REGULATION OF HYPOXIA INDUCIBLE FACTOR HIF BY OXYGEN 22 0.0005 0.0275 1 6
2 REACTOM
E DEFENSINS 19 0.0016 0.0366 1 5
DATABASE BIOLOGICAL PATHWAY GENE SET SIZE
NOMINAL P-VALUE
95% CUTOFF
FDR 95%
CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
-
DATABASE BIOLOGICAL PATHWAY GENE SET SIZE
NOMINAL P-VALUE
95% CUTOFF FDR
95% CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
1 PANTHER
ANDROGEN ESTROGEN PROGESTERONE BIOSYNTHESIS 10 0.0131 0.0457 1 3
Stage B endometriosis
Stage A endometriosis
“All” endometriosis
RESULTS Hypothesis free analysis
122 Candidate
Genes recognised until
Sep 2014
Most Studied Biological Categories
Literature search until Jul 2014 Search terms: endometriosis/
pathophysiology/pathway
PANTHER 176
pathways
15 pathways
317 pathways
21 pathways
MAGENTA Software
MAGENTA algorithm: STEP1 – map SNP’s onto genes, STEP2 – score genes based on regional SNP p-values, STEP3 – correct for gene score confounders, STEP4 – estimate gene set enrichment p-value for gene sets of interestb
International EndoGene Consortium, IEC GWA-data: 3,194 cases and 7,060 controls. Cases staged according to rAFS classification system.
“All” stA stB
PANTHER 176
pathways
MSig C2a BioCarta/KEGG/PID
Reactome/SA/SIG/STa 1320 canonical pathways
All 1320 pathways
All 176 pathways
Panther 176
pathways
MSigC2 1320
canonical pathways
HYPOTHESIS DRIVEN ANALYSIS HYPOTHESIS FREE ANALYSIS
HYPOTHESIS
PATHWAY DATABASES
IDENTIFIED PATHWAYS
ANALYSIS SOFTWARE
DATA
RESULTS
a"IPA,"Ingenuity"Pathway"Analysis;"MSig"C2,"Molecular"Signatures"Curated"Gene"sets;"KEGG,"Kyoto"Encyclopedia"of"Genes"and"Genomes;"PID,"Pathway"InteracBon"Database;"SA,"SigmaAldrich;"SIG,"Signaling"Gateway;"ST,"Signal"TransducBon"b"Segre"et"al."PLoSGeneBcs"2010."
Figure I. Study design.
“All” stA stB “All” stA stB
RESULTS Hypothesis free analysis
DATABASE BIOLOGICAL PATHWAY GENE SET
SIZE
NOMINAL P-VALUE
95% CUTOFF
FDR 95%
CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
1 REACTOM
E
GRB2 SOS PROVIDES LINKAGE TO MAPK SIGNALING FOR INTEGRINS 14 0.0001 0.0055 1 6
2 SA WNT SIGNALING 85 0.0259 0.0259 4 9
3 REACTOM
E P130CAS LINKAGE TO MAPK SIGNALING FOR INTEGRINS 14 0.0003 0.0305 1 5
4 REACTOM
E
REGULATION OF HYPOXIA INDUCIBLE FACTOR HIF BY OXYGEN 22 0.0002 0.0465 1 6
“All” endometriosis
RESULTS Hypothesis free analysis
DATABASE BIOLOGICAL PATHWAY GENE SET
SIZE
NOMINAL P-VALUE
95% CUTOFF FDR
95% CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
1 ST ERK1 ERK2 MAPK PATHWAY 32 0.0015 0.0015 2 7 2 SA TRKA RECEPTOR 17 0.0016 0.0031 1 5
3 SIG
PIP3 SIGNALING IN CARDIAC MYOCYTES 67 0.0013 0.0036 3 10
4 ST
PHOSPHOINOSITIDE 3 KINASE PATHWAY 36 0.0083 0.0083 2 6
5 ST G ALPHA S PATHWAY 15 0.0049 0.0093 1 4
6 ST
DIFFERENTIATION PATHWAY IN PC12 CELLS 45 0.0073 0.021 2 7
7 SA
B CELL RECEPTOR COMPLEXES 24 0.0269 0.0269 1 4
8 SIG
INSULIN RECEPTOR PATHWAY IN CARDIAC MYOCYTES 49 0.0313 0.0313 2 6
9 SA PTEN PATHWAY 17 0.0488 0.0408 1 3
Stage A endometriosis
Stage B endometriosis
DATABASE BIOLOGICAL PATHWAY GENE SET SIZE
NOMINAL P-VALUE
95% CUTOFF FDR
95% CUTOFF
EXPECTED NO OF GENES
ABOVE 95% CUTOFF
OBSERVED NO OF GENES
ABOVE 95% CUTOFF
1 -
DISCUSSION
MAPK and ENDOMETRIOSIS MAPK/Erk1/2 pathway activation by estrogen – promote proliferation, adhesion and invasion on ectopic endometrial stromal cells. (Li et al. Hum Reprod 2013) MAPK signaling pathway abnormal activation – higher proliferation and migration rates on eutopic endometrial cells. (Yotova et al. Hum Reprod 2011) P38 MAPK down-regulation – anti-inflammatory, anti-proliferative and anti-invasive effects on ectopic endometrial tissue. (Wu et al. Br J Pharmacol 2014)
DISCUSSION
Wnt signaling and endometriosis Estrogen’s role for Wnt signaling - factor for endometrial hyperplasia and cancer. (Wang et al. 2010) Targeting Wnt signaling inhibits endometriotic lesion migration and invasion. (Matsuzaki and Darcha 2013) Wnt signaling pathway down-regulation – impaired during implantation. (Matsuzaki et al. 2010) Wnt signaling - a mediating role in fibrogenesis formation in endometriosis. (Matsuzaki and Darcha 2013)
estrogen
progesterone
inflammation
immunology
angiogenesis
CONCLUSION
Wnt signaling
MAPK
Acknowledgements
All women who participated in the studies Clinicians who recruited women to the OXYGENE and GBE studies Wellcome Trust Centre for Human Genetics, University of Oxford, UK Nilufer Rahmioglu, Andrew Morris, Krina Zondervan Nuffield Department of Obstetrics and Gynaecology, University of Oxford, UK Christian Becker, Stephen Kennedy, Krina Zondervan Queensland Institute of Medical Research, Australia Dale Nyholt, Grant Montgomery Consortia International Endogene Consortium Funded by