Affymetrix Gene Expression Microarrays
Application to Pulmonary Arterial Hypertension
Bob Stearman
02/24/2014
Pulmonary Arterial Hypertension (PAH)
• Rare Disease: USA ~1000 new cases/yr
• More Common in Females (2:1) with Earlier
Presentation (30s-40s vs 40s-50s)
• BMPR2 Mutations Found in HPAH with
Incomplete Penetrance
• Combination Drug Therapy Common
•
•
PDE5 and Endothelin Inhibitors plus Prostacyclin
Slows Progression But No Cure (5-yr 66%)
• Elevations of Pulmonary Hemodynamics
Pulmonary Arterial Hypertension (PAH)
• Hyperproliferation of PAECs and SMCs
•
Structural Changes in Lung Vasculature
Vessel Thickening
Muscularization (SMCs)
Plexiform Lesion (ECs)
Stacher et al., AJRCCM 186, 261 (2012)
Pulmonary Arterial Hypertension (PAH)
• Elevation in Pulmonary Hemodynamics
•
Echocardiogram and Right Heart Catherization
Studies
• Increased Inflammation
Vessel Thickening vs mPAP
(p-value 0.03; r=0.27)
Perivascular Inflammation
Stacher et al., AJRCCM 186, 261 (2012)
Pulmonary Hypertension Breakthrough
Initiative
• Multi-Center Lung Tissue Procurement
• 9 Geographically Spread Procurement Centers
•
•
“Excessively” Complex Tissue Dissection
3+ Hours To Carryout
• 1 Central Tissue Repository
• Genomics Core & Secondary Tissue Repository
(Frozen and RNALater)
• Rare Disease = Slow Recruitment
PHBI Clinical Information
Stacher et al., AJRCCM 186, 261 (2012)
PHBI Pathological Information
• Vessel Thickness (measured and fractional)
•
Total, Intima, Media, Internal Diameter
• Plexiform Lesions
•
Number and Density
• Inflammation
• Thrombi
• Pulmonary Vein Remodeling
Stacher et al., AJRCCM 186, 261 (2012)
PHBI Lung Tissue Harvest
RUL = right upper lobe RML = right middle lobe RLL = right lower lobe H= Hilum.
Stacher et al., AJRCCM 186, 261 (2012)
Pulmonary Hypertension Breakthrough
Initiative: Sample Summary
• PAH Patients: End-stage disease getting transplant
•
4 General Types of PAH: HPAH, IPAH, APAH, Other
• Failed Donors (FDs; controls): No match for lungs
but available for research
• Slow Recruitment: MAs run in 3 “annual” batches
• PAH: Female Bias (2:1 F:M) and older
• FDs: Male Bias (1:2 F:M) and younger compared to
PAH
Workflow of Gene Expression
Biological question
Experimental design
QC
Tissue / sample preparation
Extraction of Total RNA
QC
Probe amplification & labeling
QC
Microarray hybridization & processing
Image analysis
Data analysis
Biological Verification
QC
QC
Expression measures - Normalization Reddy Gali, Ph.D.
Statistical Filtering - Clustering Microarray Analysis Using
Pathway analysis
R/Bioconductor
PHBI: Experimental Design
• PAH Patients vs. FD
•
•
•
Disease-specific gene expression patterns?
4 General Types of PAH: HPAH, IPAH, APAH, Other
Will Gene Expression Patterns Detect Clinical Subclasses?
• PAH Patient Gender Bias
• PAH vs FD Gender and Age (?) Bias
• Microarrays run in 3 “annual” batches
•
•
Different lots of arrays and reagents
Changes in labeling methodology
PHBI Lung RNA Samples (in RNALater) Are
Variable
PHBI: Normalization
Uncorrected PHBI Data
PHBI: Normalization
Corrected PHBI Data By Batch and Gender
PHBI: Normalization
Corrected PHBI Data By Batch and Gender
Low Variance Genes Removed (33K->6K)
PHBI: Clustering
PHBI: Expression Correlation With Clinical
Parameters
PHBI: Expression Correlation With Clinical
Parameters
Sources of Reproducibility (Experimental
Design- Randomization)
C4 Hybridized Sample B & A1 (Day 1); A6 & A15 (Day 2) [while same day samples had
very similar #DE genes as C1, C2, and C3]
C5 Hybridized Samples B & A1 (Day 1); A6 (Day 2); A15 (Day 3)
C2 and C3 Stored Male Arrays ON, 4oC while Female Arrays were Wash/Stain
(Yang et al 2008)
Sources of Reproducibility (Experimental
Design- Randomization)
Each dataset is self-consistent
Training on Center 3 generally
groups M and F by sample type
Training on Center 4 produces
poor correlation between M and
F by sample type
(Yang et al 2008)
Stability of Results
Critical Review of Published Microarray Studies for Cancer
Outcome and Guidelines on Statistical Analysis and Reporting
Alain Dupuy , Richard M . Simon (JNCI 2007)
“Simon Says Top 40”
Nature Biotechnology 28, 827 (2010)
Affymetrix Gene Expression Microarrays
Report Expectations
•
•
•
•
Experimental Design and Questions Summary
Signal Intensities Before and After Normalization
PCA plots
Significant Genes Listing including FC, p-value, FDR
analysis
• Cluster Diagram (+/- Signal Intensities)
• Over-represented Gene Ontology Categories
•
• RDAVID??
Experimental Conclusions and Discussion
Affymetrix Gene Expression Microarrays
Report Expectations
Annotation Cluster 1
Category
SP_PIR_KEYWORDS
SP_PIR_KEYWORDS
INTERPRO
INTERPRO
SP_PIR_KEYWORDS
INTERPRO
SMART
GOTERM_MF_FAT
Enrichment Score: 3.980004851748526
Term
immunoglobulin c region
immunoglobulin
IPR007110:Immunoglobulin-like
IPR003597:Immunoglobulin C1-set
Immunoglobulin domain
IPR013783:Immunoglobulin-like fold
SM00407:IGc1
GO:0003823~antigen binding
Count
%
5
6
15
7
14
15
7
6
PValue
3.33
4.00
10.00
4.67
9.33
10.00
4.67
4.00
Genes
List Total Pop Hits Pop Total Fold Enrichment Bonferroni
Benjamini
FDR
1.60E-07 7896697, 8071597, 7981708,118
7981601, 8043476
9
19235
90.56
3.12E-05
3.12E-05 2.00E-04
1.14E-06 7896697, 8043423, 8071597,118
7981708, 7981601,
31
19235
8043476
31.55
2.23E-04
1.12E-04 0.001428
4.84E-06 8071597, 8043423, 7906613,110
8060418, 501
7981601,16659
8110237, 8071642, 8125470,
4.53 7896697,
0.001305368
8117458,
0.001305368
8117435, 7981708,
0.0063618117476, 80434
5.38E-06 7896697, 8071597, 8060418,110
7981708, 7981601,
68
16659
8043476, 8178833, 8125470
15.59
0.001451035
7.26E-04 0.007071
5.76E-06 8043423, 8071597, 7906613,118
8060418, 470
7981601,19235
8110237, 7896697, 8117458,
4.86 8117435,
0.001123285
7981708, 8117476,
3.75E-04 8043476,
0.0071947923917, 80485
1.48E-05 8071597, 8043423, 8060418,110
7981601, 553
8110237,16659
8071642, 8125470, 8117458,
4.11 7896697,
0.0039884
8117435,
0.001331238
7981708, 8117476,
0.0194598043476, 81788
1.75E-05 7896697, 8071597, 8060418, 75
7981708, 7981601,
68
8043476,
9079
8178833, 8125470
12.46
0.00129556 0.00129556 0.018335
2.80E-05 7896697, 8043423, 8071597, 84
7981708, 7981601,
56
12983
8043476
16.56
0.006661364 0.006661364 0.036066