Genome-wide pathway analysis highlight the role of Wnt ...seud-congress.com/files/107/COMM_2015/SAMEDI/I/04.-outi-uimari.pdf · Genome-wide pathway analysis highlight the role of

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


Sep 2014




PANTHER 176

pathways

15 pathways

317 pathways

21 pathways

MAGENTA Software



“All” stA stB

PANTHER 176

pathways



All 1320 pathways

All 176 pathways

Panther 176

pathways

MSigC2 1320

canonical pathways


HYPOTHESIS

PATHWAY DATABASES

IDENTIFIED PATHWAYS

ANALYSIS SOFTWARE

DATA

RESULTS




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


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


Sep 2014




PANTHER 176

pathways

15 pathways

317 pathways

21 pathways

MAGENTA Software



“All” stA stB

PANTHER 176

pathways



All 1320 pathways

All 176 pathways

Panther 176

pathways

MSigC2 1320

canonical pathways


HYPOTHESIS

PATHWAY DATABASES

IDENTIFIED PATHWAYS

ANALYSIS SOFTWARE

DATA

RESULTS




RESULTS Hypothesis driven - Candidate gene based

analysis

122 Candidate


Sep 2014




PANTHER 176

pathways

15 pathways

317 pathways

21 pathways

MAGENTA Software



“All” stA stB

PANTHER 176

pathways



All 1320 pathways

All 176 pathways

Panther 176

pathways

MSigC2 1320

canonical pathways


HYPOTHESIS

PATHWAY DATABASES

IDENTIFIED PATHWAYS

ANALYSIS SOFTWARE

DATA

RESULTS




RESULTS Hypothesis driven - Candidate gene based

analysis

DATABASE BIOLOGICAL PATHWAY GENE SET

SIZE

NOMINAL P-VALUE

95% CUTOFF FDR

95% CUTOFF


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


ABOVE 95% CUTOFF


ABOVE 95% CUTOFF

1 PANTHER

INSULIN IGF PATHWAY MAPK CASCADE 30 0.0031 0.0132 2 6


SIZE

NOMINAL P-VALUE

95% CUTOFF FDR

95% CUTOFF


ABOVE 95% CUTOFF


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


Sep 2014




PANTHER 176

pathways

15 pathways

317 pathways

21 pathways

MAGENTA Software



“All” stA stB

PANTHER 176

pathways



All 1320 pathways

All 176 pathways

Panther 176

pathways

MSigC2 1320

canonical pathways


HYPOTHESIS

PATHWAY DATABASES

IDENTIFIED PATHWAYS

ANALYSIS SOFTWARE

DATA

RESULTS




RESULTS Hypothesis driven - Literature review based

analysis


NOMINAL P-VALUE

95% CUTOFF FDR

95% CUTOFF


ABOVE 95% CUTOFF


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


NOMINAL P-VALUE

95% CUTOFF

FDR 95%

CUTOFF


ABOVE 95% CUTOFF


ABOVE 95% CUTOFF

-


NOMINAL P-VALUE

95% CUTOFF FDR

95% CUTOFF


ABOVE 95% CUTOFF


ABOVE 95% CUTOFF

1 PANTHER

ANDROGEN ESTROGEN PROGESTERONE BIOSYNTHESIS 10 0.0131 0.0457 1 3




RESULTS Hypothesis free analysis

122 Candidate


Sep 2014




PANTHER 176

pathways

15 pathways

317 pathways

21 pathways

MAGENTA Software



“All” stA stB

PANTHER 176

pathways



All 1320 pathways

All 176 pathways

Panther 176

pathways

MSigC2 1320

canonical pathways


HYPOTHESIS

PATHWAY DATABASES

IDENTIFIED PATHWAYS

ANALYSIS SOFTWARE

DATA

RESULTS






SIZE

NOMINAL P-VALUE

95% CUTOFF

FDR 95%

CUTOFF


ABOVE 95% CUTOFF


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




SIZE

NOMINAL P-VALUE

95% CUTOFF FDR

95% CUTOFF


ABOVE 95% CUTOFF


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




NOMINAL P-VALUE

95% CUTOFF FDR

95% CUTOFF


ABOVE 95% CUTOFF


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

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Genome-wide pathway analysis highlight the role of Wnt ...seud-congress.com/files/107/COMM_2015/SAMEDI/I/04.-outi-uimari.pdf · Genome-wide pathway analysis highlight the role of