P ROGRAMS FOR G ENOMIC A PPLICATIONS National Heart, Lung, and Blood Institutes National Institutes of Health

Mission Statement To develop new resources, reagents, and education programs for investigators engaged in NHLBI- related research.

PGA Mission Provide new resources and reagents to link genes to biological function and make these readily available to the NHLBI community. Facilitate workshops, courses, and visiting scientist programs to train investigators in the technologies being applied in the PGAs. Rapidly disseminate data through the world wide web and public databases.

Organizational Structure Coordinating Committee Bioinformatics Subcommittee Proteomics Subcommittee Data Sharing Subcommittee Education Subcommittee Integration Subcommittee Microarray Subcommittee Phenotype Subcommittee

PGA Programs Applied Genomics in CardioPulmonary Disease Johns Hopkins University School of Medicine Genomics of Cardiovascular Development, Adaptation, & Remodeling Harvard Medical School Physiogenomics of Stressors in Derived Consomic Rats Medical College of Wisconsin Genomics of Proteomics of Cell Injury and Inflammation University of Texas S.W. Medical Center Innate Immunity in Heart, Lung, and Blood Diseases The University of Arizona UW-FHCRC Variation Discovery Resource University of Washington Mouse Models of Heart, Lung, and Blood Diseases The Jackson Laboratory Expression Profiling of Rodent Models of Human Disease The Institute for Genomics Research Comparative Genomic Analysis of Cardiovascular Genes Lawrence Berkeley National Laboratory Genomic Analysis of Stress and Inflammation Harvard Medical School NHLBI Bay Area Functional Genomic Consortium The David J. Gladstone Institute

Subcommittee Chairs Bioinformatics Carol Bult, Ph.D., The Jackson Laboratory Data Sharing Isaac Kohane, M.D., Ph.D., Harvard Medical SchoolEducation Scott Weiss, M.D., M.S., Harvard Medical School Genomic Inventory/Integration Edward Rubin, M.D., Ph.D., The Lawrence Berkeley National LaboratoryMicroarray John Quackenbush, Ph.D., The Institute for Genomics ResearchPhenotype Andrew Greene, Ph.D., Medical College of WisconsinProteomics Thomas Kodadek, Ph.D., Univ. Texas S.W. Medical Center

Anticipated PGA Resources/Tools Mouse models of HLBS disorders Rat models of HLBS disorders Microarrays DNA Variations (SNPs - locations, allele frequencies, genotypes and haplotypes) Reagents (clones, antibodies, mice, and rats) Protocols Bioinformatic Resources (software tools and databases)

BayGenomics Apply custom gene-trap vectors to inactivate genes in ES cells and to evaluate the functional importance of these in cardiopulmonary development and disease using computational approaches, expression profiling, in situ hybridization studies, and in select cases in animals. PI: Dr. Stephen G. Young Focus: Cardiopulmonary Development and Disease

CardioGenomics To link genes to function, dysfunction, and structural abnormalities of the cardiovascular system caused by clinically relevant genetic and environmental stimuli. PI: Dr. Seigo Izumo Focus:Cardiovascular Development, Adaptation, and Remodeling Focus:Cardiovascular Development, Adaptation, and Remodeling

HopGenes To identify the genes involved in tissue remodeling using expression profiling to explore the pathology of asthma, chronic obstructive pulmonary disease, cystic fibrosis, lung transplantation, acute lung injury, scleroderma, sarcoidosis, pulmonary hypertension, ischemic cardiomyopathy, and cardiac transplantation. PI: Dr. Joe G.N. Garcia Focus: Tissue Remodeling in Cardiopulmonary Disease

Innate Immunity Explore genetic susceptibility in asthma, chronic obstructive pulmonary disease, myocardial infarction and deep venous thrombosis by evaluating polymorphisms in genes involved in innate immune responses. PI: Dr. Fernando D. Martinez Focus: Genetics of HLB Disorders

JAX PGA Apply a phenotype-driven approach to identify the genetic mechanisms underlying the physiology and pathophysiology of atherosclerosis, hypertension, lung function, blood formation, thrombosis, obesity, inflammation, and sleep function. PI: Dr. Luanne L. Peters Focus: Mouse Models of HLBS Disorders

PARABIOSYS To identify and characterize the gene networks activated by pro-inflammatory, metabolic, and pathogenic stresses affecting cardiovascular and pulmonary systems. PI: Dr. Brian Seed Focus: Genetics of Inflammation and Stress

Berkeley PGA Apply comparative genomics to identify and understand the role of cis-acting regulatory elements that affect the expression of cardiovascular genes. PI: Dr. Edward M. Rubin Focus: Cardiovascular Gene Expression

PhysGen Dissect multigenic common HLBS diseases through the development of panels of chromosomal substitution strains of rats (consomic rat panels). PI: Dr. Howard J. Jacobs Focus: Rat Models of HLBS Disorders

Seattle SNPs To identify variable sites in human genes to expand the resources available to explore the role of inter- individual variation and its relationship to disease risk, outcome and treatments for common human disorders. PI: Dr. Deborah A. Nickerson Focus: Inflammation and Genetic Variability

Southwestern Elucidate the basic mechanisms underlying cell injury and inflammation through a combination of genomic and proteomic approaches. PI: Dr. Stephen A. Johnston Focus: Cell Injury and Inflammation

TREX Explore gene-environment interactions using rodent models of human disease and cDNA microarray assays to elucidate patterns of gene expression in heart, lung, blood, and sleep disorders. PI: Dr. John Quackenbush Focus: Gene Expression in HLBS Disorders

NHLBI PGA Research Network HopGenes - BaltimoreHopGenes - Baltimore CardioGenomics - BostonCardioGenomics - Boston PhysGen - MilwaukeePhysGen - Milwaukee Southwestern - DallasSouthwestern - Dallas InnateImmunityInnateImmunity - Tucson SeattleSNPs - SeattleSeattleSNPs - Seattle JAX PGA - Bar HarborJAX PGA - Bar Harbor TREX - RockvilleTREX - Rockville Berkeley PGABerkeley PGA - Berkeley ParaBioSys - BostonParaBioSys - Boston BayGenomicsBayGenomics - San Francisco

NHLBI PGA Web Sites P ROGRAMS FOR G ENOMIC A PPLICATIONS

PGA Web Sites (cont.) BayGenomics - BayGenomics - CardioGenomics - CardioGenomics - ParaBioSys - ParaBioSys - JAX PGA - JAX PGA - HopGenes - HopGenes - InnateImmunity - InnateImmunity -

PGA Web Sites (cont.) Berkeley PGA - Berkeley PGA - TREX - TREX - PhysGen - PhysGen - Southwestern - Southwestern - SeattleSNPs - SeattleSNPs -

Baylor College of Medicine NHGRI/NHLBI Howard Jacob MCW Marco Marra UBC Shaying Zhao TIGR Peter Tonellato Rat GDB CHO Pieter de Jong Univ Utah Robert Weiss RAT GENOME SEQUENCING PROJECT

Goals Produce a `draft sequence` of the genome, Assembly and first-pass annotation, Cross-species comparison, A public assembler, Foundation for a finishing effort, Interface with community,

Fold-Coverage Timelines (Year One) (Cumulative) All in MOU (with data release policy)

Strategy: Strain Selection: Howard Jacob’s BN/SsNHsd This Brown Norway strain has only seven heterozygous markers in the more than 4,000 tested i.e. it is very `isogenic` as a representative strain.

New BAC Library Made by Pieter de Jong from BN/SsNHsd female, One enzyme so far ~200 kb inserts 10 X coverage Second enzyme to come? Doug Smith pursuing chr Y library,

Fingerprints: UBC, ~40,000 for `de-randomization` ~200,000 for f`print assembly, BES: TIGR, ~200,000 paired ends

Strategy: BAC Skims BAC skims to cover 90% of genome Originally 20, kb `de-randomized` 1.0 x coverage, (384 reads per BAC) = 1 x clone coverage = 67% - 80% coverage ~ 5,000 gaps for walks, Now 15, kb reads per BAC and 1.7 X coverage

Strategy: BAC Skims (cont`d) Reads in BACs: 2kb paired end (dye term) kb paired end (dye term) -- M13 (with primer chemistry) ?

Strategy: WGS Reads 2kb paired end (dye term) kb paired end (dye term) +++ 5kb and 50kb discussed, M13 (with primer chemistry) ?

- Usual - Assembly - Data `purity` Challenges:

Atlas: A whole genome assembler

Lung Studies hypoxia normoxia Cardiac Studies hypoxia normoxia Systemic Vascular & Renal Studies high salt & salt depletion Isolated Vessel Research Services Component Phenotyping Component Conditioning Protocol Genomics Component Profiling Respiratory Studies Bioinformatics Component Consomic Rat Strains Functional Mapping DNA & Genotyping Data Baseline & Stressed Expression Data Rat distribution Baseline & Stressed Physiological Data Genomics Component Genotyping & Animal Production (Transponders and transfer) TIGRConsomic Expression Data Expression low salt high salt Blood Studies 2, strains/yr 330

Mary Pat Kunert, RN, Ph.D., Project Supervisor Mindy Dwinell, Ph.D. Project Supervisor Meredith Skelton, M.S., Sr. Research Associate Julie Messinger, M.S., Program Coordinator Cardiac Studies:Jessica Laessig and William Hutchins Renal Studies:Larry Clowry and Mike Bregantini Biochemistry:Candace Jones Lung Studies:Jess Powlas and Bernadette Cabigas Isolated Vessel Studies:Janelle Yarina Respiratory Studies:Jenny Hogan and Andrea Trevett Animal Delivery and Care: Alison Kriegel Laboratory Support Services:Jennifer Labecki Phenotyping Team

Co-investigators: Cardiac Studies:John Baker, M.D. Lung Studies:Christopher Dawson, Ph.D. Biochemistry Studies:Kirkwood Pritchard, Ph.D. Respiratory Studies:Hubert Forster, Ph.D. Isolated Vessel Studies:Julian Lombard, Ph.D. Vascular and Renal Studies:David Mattson, Ph.D. Richard Roman, Ph.D Component Director - Allen W. Cowley, Jr, Ph.D. Phenotyping Component

Frequency of Non-Augmented Breaths in Room Air (Eupnea) Frequency (Breaths/minute) BNSS FHH P = Frequency of Augmented Breath in Room Air (Eupnea) BNSSFHH Augmented Breaths/Minute P < 0.05 #

Treadmill Protocol (MAP) Rest Walk Run 5 minutes 400  l Blood (3rd min.) 400  l Blood (3rd min.) 400  l Blood (3rd min.) Rectal Temp Rectal Temp Continuous measurement of MAP & HR: Analyze Data from 30sec. before blood draws.

Mean arterial pressure in SS and BN parental rats and consomic chr. 13 and 16 on high salt (4.0%)

Cardiac Conditioning and Phenotyping Protocol 8 week old rats

Post ischemic infarct size (%)

Recovery developed ventricular pressure (%)

blood draw from carotid catheter in anesthetized rats following chronic exposure to hypoxia or normoxia ANP-15 (Marshfield Clinic)  Small Animal Profile (ANP-1) Glucose, AST, ALT, Alkaline Phosphatase, Total Bilirubin, Total Cholesterol, Total Protein, Albumin, Urea Nitrogen, Creatinine, Phosphorus, Calcium, Sodium, Potassium, Chloride, Bicarbonate, Anion Gap  Complete Blood Count With Differential (CBC) RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, RDW, Platelet, WBC and Differential Biochemical Measurements

Lung Studies hypoxia normoxia Cardiac Studies hypoxia normoxia Systemic Vascular & Renal Studies high salt & salt depletion Isolated Vessel Research Services Component Phenotyping Component Conditioning Protocol Genomics Component Profiling Respiratory Studies Bioinformatics Component Consomic Rat Strains Functional Mapping DNA & Genotyping Data Baseline & Stressed Expression Data Rat distribution Baseline & Stressed Physiological Data Genomics Component Genotyping & Animal Production (Transponders and transfer) TIGRConsomic Expression Data Expression low salt high salt Blood Studies 2, strains/yr 330

Airway Resistance at 100 ml/min/gm

PGA Respiratory Protocol Timeline Measuring changes in Pulmonary Ventilation (V E ), Tidal Volume (V T ) and frequency (f) during Hypoxia (12% O 2 ) and Hypercapnia (7% CO 2 ). Treadmill Studies Hyper- capnia Study. Treadmill Adaptation & Data Analysis Hypoxia Study Treadmill & Box Adaptation with N 2 Treadmill & Box Adaptation with CO 2 Treadmill & Box Adaptation Surgery Catheter Implantation in Femoral Artery Treadmill Adaptation Treadmill Adaptation - 11 week old rats Monday Tuesday Wednesday Thursday Friday Week 1 Pre-surgical Conditioning Week 2 Surgery & Adaptation Week 3 Studies Measuring Mean Arterial Pressure and Blood Gas Responses to Exercise Treadmill Studies

11 week old rats Vascular Conditioning and Contraction Protocol

Vascular Conditioning and Relaxation Protocol 11 week old rats 12:00 pm

Renal Cardiovascular Phenotyping Protocol 13 Week old Rats Week 1 Week 2

Carotid catheter & Endotrachael Tube Insertion Methacholine Challenge Heart & Lung Extraction P/Q Curve, MB+ & FAPGG Experiments 10 Week old rats Lung Prep Timeline Marshfield Blood Samples via catheter ~25 minutes Collect Data: Resistance Compression Tidal Volume Breathing Frequency Minute Ventilation Extract Lungs for Perfusion Extract Heart for RV & LV Weights Liver Specimen for DNA Processing Collect Data:   Change in Pressure w/ flow   FAPGG Absorbance   MB+ Absorbance   Lung Wet Weight ~35 minutes Anesthetize Rat Place Rat in Plethysmograph & Insert Esophageal Balloon Remove Rat to Surgery Attach Trachea & Pulmonary Artery to Perfusion Apparatus Anesthetize Next Rat

Hypoxia & Hypercapnia Protocol (Ventilation and MAP) Put rat in box 20 Min. Control Inject gas N 2 or CO 2 Equil. Stop Recording 1 st Rectal Temp 5” 1” min. 2 & 3 min. 4,5 & 6 min. 7,8 & 9 2 nd Rectal Temp Blood draw 400  l (Min. 3) Blood draw 400  l (Min 8) Calibration (30 Sec.) Analyze Data (H1) Analyze Data (H2)