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 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)

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

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 GenderLow 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)

(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 ReportingAlain Dupuy , Richard M . Simon (JNCI 2007)

“Simon Says Top 40”

Nature Biotechnology 28, 827 (2010)

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