P ROGRAMS FOR G ENOMIC A PPLICATIONS

1. PROGRAMS FORGENOMICAPPLICATIONS National Heart, Lung, and Blood Institutes National Institutes of Health

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

3. 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.

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

5. 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

6. Bioinformatics • Carol Bult, Ph.D.,The Jackson Laboratory Data Sharing • Isaac Kohane, M.D., Ph.D.,Harvard Medical School Education • Scott Weiss, M.D., M.S.,Harvard Medical School Genomic Inventory/Integration • Edward Rubin, M.D., Ph.D.,The Lawrence Berkeley National Laboratory Microarray • John Quackenbush, Ph.D.,The Institute for Genomics Research Phenotype • Andrew Greene, Ph.D.,Medical College of Wisconsin Proteomics • Thomas Kodadek, Ph.D.,Univ. Texas S.W. Medical Center SubcommitteeChairs

7. 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)

8. BayGenomics http://baygenomics.ucsf.edu Focus: Cardiopulmonary Development and Disease • 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

9. CardioGenomics http://www.cardiogenomics.org Focus: Cardiovascular Development, Adaptation, and Remodeling • 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

10. HopGenes http://www.hopkins-genomics.org Focus: Tissue Remodeling in Cardiopulmonary Disease • 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

11. Innate Immunity http://innateimmunity.net Focus: Genetics of HLB Disorders • 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

12. JAX PGA http://pga.jax.org Focus: Mouse Models of HLBS Disorders • 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

13. PARABIOSYS http://genetics.mgh.harvard.edu/Parabiosys/ Focus: Genetics of Inflammation and Stress • To identify and characterize the gene networks activated by pro-inflammatory, metabolic, and pathogenic stresses affecting cardiovascular and pulmonary systems. PI: Dr. Brian Seed

14. Berkeley PGA http://pga.lbl.gov Focus: Cardiovascular Gene Expression • 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

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

16. Seattle SNPs http://pga.mbt.washington.edu Focus: Inflammation and Genetic Variability • 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

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

18. TREX http://pga.tigr.org Focus: Gene Expression in HLBS Disorders • 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

19. NHLBI PGA Research Network • SeattleSNPs - Seattle . . • PhysGen - Milwaukee • JAX PGA - Bar Harbor . • CardioGenomics - Boston . • ParaBioSys - Boston . . • HopGenes - Baltimore • BayGenomics • - San Francisco . . • TREX - Rockville • Berkeley PGA • - Berkeley . . • InnateImmunity • - Tucson • Southwestern - Dallas

20. NHLBI PGA Web Sites PROGRAMSFORGENOMICAPPLICATIONS http://www.nhlbi.nih.gov/resources/pga/index.htm

21. PGA Web Sites (cont.) BayGenomics - http://baygenomics.ucsf.edu CardioGenomics - http://www.cardiogenomics.org HopGenes - http://www.hopkins-genomics.org InnateImmunity - http://innateimmunity.net JAX PGA - http://pga.jax.org ParaBioSys - http://genetics.mgh.harvard.edu/Parabiosys/

22. PGA Web Sites (cont.) Berkeley PGA - http://pga.lbl.gov PhysGen - http://pga.mcw.edu SeattleSNPs - http://pga.mbt.washington.edu Southwestern - http://pga.swmed.edu TREX - http://pga.tigr.org

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

24. 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,

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

27. 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.

28. 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,

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

30. Strategy: BAC Skims BAC skims to cover 90% of genome Originally 20,000 150 kb `de-randomized` BACs @ 1.0 x coverage, (384 reads per BAC) = 1 x clone coverage = 67% - 80% coverage ~ 5,000 gaps for walks, Now 15,000 200 kb BACs @768 reads per BAC and 1.7 X coverage

31. Strategy: BAC Skims (cont`d) Reads in BACs: 2kb paired end (dye term) +++ 10kb paired end (dye term) -- M13 (with primer chemistry) ?

32. Strategy: WGS Reads 2kb paired end (dye term) +++ 10kb paired end (dye term) +++ 5kb and 50kb discussed, M13 (with primer chemistry) ?

33. Challenges: - Usual - Assembly - Data `purity`

34. Atlas: A whole genome assembler

35. high salt & salt depletion normoxia hypoxia Systemic Vascular Cardiac Studies & Renal Studies Genomics Component Genotyping & Animal Production (Transponders and transfer) 2,640 15 strains/yr Phenotyping Component Conditioning Protocol 330 330 330 330 330 330 330 330 high salt low salt hypoxia normoxia Respiratory Lung Studies Isolated Vessel Studies Blood Studies Genomics Component Expression Research Services Component Profiling Bioinformatics Component TIGR Consomic Expression Data Consomic Rat Functional Strains Baseline & Stressed Baseline & Stressed DNA & Genotyping Mapping Physiological Data Expression Data Data Rat distribution

36. Phenotyping Team 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

37. Phenotyping Component Component Director -Allen W. Cowley, Jr, Ph.D. • 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

39. Frequency of Non-Augmented Breaths in Room Air (Eupnea) 140 120 100 Frequency (Breaths/minute) 80 60 40 20 0 BN SS FHH Frequency of Augmented Breath in Room Air (Eupnea) 0.7 P < 0.05 0.6 0.5 Augmented Breaths/Minute 0.4 # 0.3 0.2 0.1 0 BN SS FHH P = 0.223

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

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

50. Cardiac Conditioning and Phenotyping Protocol 8 week old rats