Affymetrix Gene Expression Microarrays
Application to Pulmonary Arterial HypertensionBob 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
Stacher et al., AJRCCM 186, 261 (2012)
Vessel Thickening Muscularization (SMCs) Plexiform Lesion (ECs)
Pulmonary Arterial Hypertension (PAH)
• Elevation in Pulmonary Hemodynamics• Echocardiogram and Right Heart Catherization
Studies• Increased Inflammation
Stacher et al., AJRCCM 186, 261 (2012)
Vessel Thickening vs mPAP (p-value 0.03; r=0.27) Perivascular Inflammation
• 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
Pulmonary Hypertension Breakthrough Initiative
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)
RUL = right upper lobe RML = right middle lobe RLL = right lower lobe H= Hilum.
PHBI Lung Tissue Harvest
Stacher et al., AJRCCM 186, 261 (2012)
• 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
Pulmonary Hypertension Breakthrough Initiative: Sample Summary
Workflow of Gene Expression
Reddy Gali, Ph.D.Microarray Analysis Using
R/Bioconductor
Biological question Experimental design
Tissue / sample preparation
Extraction of Total RNA
Microarray hybridization & processing
Image analysis
Probe amplification & labeling
Data analysisExpression measures - Normalization - Statistical Filtering - Clustering - Pathway analysis
Biological Verification
QC
QC
QC
QC
QC
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
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)
(Yang et al 2008)
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
Stability of ResultsCritical 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”
Affymetrix Gene Expression MicroarraysReport 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 MicroarraysReport Expectations
Annotation Cluster 1 Enrichment Score: 3.980004851748526Category Term Count % PValue Genes List Total Pop Hits Pop Total Fold Enrichment Bonferroni Benjamini FDRSP_PIR_KEYWORDS immunoglobulin c region 5 3.33 1.60E-07 7896697, 8071597, 7981708, 7981601, 8043476118 9 19235 90.56 3.12E-05 3.12E-05 2.00E-04SP_PIR_KEYWORDS immunoglobulin 6 4.00 1.14E-06 7896697, 8043423, 8071597, 7981708, 7981601, 8043476118 31 19235 31.55 2.23E-04 1.12E-04 0.001428INTERPRO IPR007110:Immunoglobulin-like 15 10.00 4.84E-06 8071597, 8043423, 7906613, 8060418, 7981601, 8110237, 8071642, 8125470, 7896697, 8117458, 8117435, 7981708, 8117476, 8043476, 8178833, 8048551110 501 16659 4.53 0.001305368 0.001305368 0.006361INTERPRO IPR003597:Immunoglobulin C1-set 7 4.67 5.38E-06 7896697, 8071597, 8060418, 7981708, 7981601, 8043476, 8178833, 8125470110 68 16659 15.59 0.001451035 7.26E-04 0.007071SP_PIR_KEYWORDS Immunoglobulin domain 14 9.33 5.76E-06 8043423, 8071597, 7906613, 8060418, 7981601, 8110237, 7896697, 8117458, 8117435, 7981708, 8117476, 8043476, 7923917, 8048551118 470 19235 4.86 0.001123285 3.75E-04 0.007194INTERPRO IPR013783:Immunoglobulin-like fold 15 10.00 1.48E-05 8071597, 8043423, 8060418, 7981601, 8110237, 8071642, 8125470, 8117458, 7896697, 8117435, 7981708, 8117476, 8043476, 8178833, 7923917, 8048551110 553 16659 4.11 0.0039884 0.001331238 0.019459SMART SM00407:IGc1 7 4.67 1.75E-05 7896697, 8071597, 8060418, 7981708, 7981601, 8043476, 8178833, 812547075 68 9079 12.46 0.00129556 0.00129556 0.018335GOTERM_MF_FAT GO:0003823~antigen binding 6 4.00 2.80E-05 7896697, 8043423, 8071597, 7981708, 7981601, 804347684 56 12983 16.56 0.006661364 0.006661364 0.036066
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