1. Inherit the Wheeze: The Genetics of Asthma Benjamin Raby, MD.CM, MPH, F.R.C.P.C. Respiratory Environmental and Genetic Epidemiology Channing Laboratory Brigham and Women’s Hospital Boston, MA BRIGHAM AND WOMEN’S HOSPITAL HARVARD MEDICAL SCHOOL

2. The Post-Genomic Era: February 13th, 2001

3. Growth of Genomic Information: 1992-2002 Venter et al. 2003 Nature Genetics 33:219

4. (Some) Notable Events in Pulmonary Genetics 1960s: Alpha-1 Antitrypsin genetics 1985-1990: PCR for diagnosis of M.Tb 1989: CFTR identified as cause of Cystic Fibrosis 1994: INH-resistance gene 1994: GM-CSF and Pulmonary Alveolar Proteinosis 2000: BMPR2 mutations identified in Primary Pulmonary Hypertension 2001: Surfactant C mutations in Interstitial Fibrosis 2002: ADAM33 identified as an asthma gene

5. Overview of Complex Trait Genes Demonstrate Familial Aggregation of Complex Trait Positional Cloning Candidate Gene Approach Localize Susceptibility Gene(s) Identify Disease Susceptibility Gene and Functional Variants Diagnostics Determine Fraction of Explained Variation Treatment

6. Describe Asthma and Complex Trait Genetics Heritability Genome-wide linkage analysis Example: ADAM33 Fine-mapping efforts and genetic association Example: Chromosome 12q Outline

7. Describe Asthma and Complex Trait Genetics Heritability Genome-wide linkage analysis Example: ADAM33 Fine-mapping efforts and genetic association Example: Chromosome 12q Outline

8. Recessive Inheritance Dominant Inheritance Asthma ?

9. Complex Genetic Disease Airway anatomy Mucin Production Lymphocyte signaling Epithelium Integrity Smooth Muscle Properties Cytokine metabolism Genetic Heterogeneity Eosinophil behavior

10. Complex Genetic Disease Environmental Phenocopy RSV infection RADS Smoker Bronchitis COPD

11. Complex Genetic Disease Reduced Penetrance

12. Complex Genetic Disease Genes confer increased risk of disease, not causative alone! Simple Disease Complex Disease

13. The Genetics of Asthma

14. Describe Asthma and Complex Trait Genetics Heritability Genome-wide linkage analysis Example: ADAM33 Fine-mapping efforts and genetic association Example: Chromosome 12q Outline

15. Risk to relatives Risk to relatives: disease more common among individuals with family history compared to those with out.

16. Twin studies Twin concordance studies: phenotype more similar among monozygotic vs. dizygotic twins even though environment shared

17. Heritability estimates Heritability estimates: variability in trait explained by familial resemblance

18. Asthma and intermediate phenotypes are highly heritable. Risks to relatives 2-4 times for asthma »Sibbald et al. Thorax 1980 Heritability of Airways responsiveness 30-66% »Hopp et al. JACI 1984 »Palmer et al. AJRCCM 2000 Atopy-related phenotypes highly heritable –Total and specific serum IgE levels: 56-79% »Hopp et al. JACI 1984 »Hanson et al. AJHG 1991 »Palmer et al. AJRCCM 2000 –Skin test reactivity heritable »Hanson et al. AJHG 1991

19. Australian Twin Studies : 3,808 Pairs 0.64 1 1 Hay fever Atopy Asthma 0.8 0.6 0.4 0.2 0 0 0.12 0.86 0.21 0.76 0.19 MZ DZ MZ DZ Duffy et al. AJRCCM 1990

20. Describe Asthma and Complex Trait Genetics Heritability Genome-wide linkage analysis Example: ADAM33 Fine-mapping efforts and genetic association Example: Chromosome 12q Outline

21. Linkage Analysis Analyze the distribution of DNA markers within families to determine if a region of the genome contains a gene related to the phenotype Intermarker Distance Trait Locus (alleles unobserved) Marker Loci (alleles unobserved) Isolated Population Outbred Population sib pairs pedigrees

22. Recombination A B C D A B C D A B C D Duplication A B C D A B C D Cross-over A B C D A B C D Recombination

23. Linkage Analysis A2A5A2A5 A1A6A1A6 A1A2A1A2 A3A4A3A4 A1A3A1A3 A2A3A2A3 A1A4A1A4 A1A4A1A4 A2A4A2A4 A2A3A2A3 NR R

24. Linkage Analysis: Non-Parametric Models Affected Sib-Pair Allele Sharing A1A2A1A2 A3A4A3A4 A1A2A1A2 A3A4A3A4 A1A3A1A3 A 1 A 3 = 35% A 1 A 4 = 35% A 2 A 3 = 15% A 2 A 4 = 15% A1A3A1A3 A1A3A1A4A2A3A2A4A1A3A1A4A2A3A2A4 = 50% = 25%

25. Positional Cloning of ADAM33 460 Caucasian families Affected sib-pairs Asthma diagnosis and medication use Genome-wide linkage analysis Chromosome 20p13 2.5 Megabase region Van Eerdewegh et al. Nature 2002 2-allele sharing (%) LOD score Asthma 31%2.94 Asthma + BHR 35%3.93

26. Describe Asthma and Complex Trait Genetics Heritability Genome-wide linkage analysis Example: ADAM33 Fine-mapping efforts and genetic association Example: Chromosome 12q Outline

27. 0.04 Asthma Linkage to 12q: Genome-wide and Regional Screens 0.0295.56 -101.1AsthmaGerman 0.01154 -159AsthmaDanish 0.00003133.3EosinophilsFrench 0.03130AsthmaU.S Hispanic 0.001111.9 -125.3AsthmaJapanese <0.001109.5 -125.3AsthmaNetherlands 0.03111.9AHRFrench 0.00493.65AsthmaU.S Caucasian 83.19 AsthmaFrench 0.00880.52AsthmaHutterites 0.0162.1- 75.2AHRGerman P-value Chromosome position (in cM from pter) PhenotypePopulation 0.002 0.0009 0.000887.9AsthmaAfro-Caribbean 0.0283.19AtopyFrench Canadian 0.000467.63AsthmaItalian 95.56IgEGerman 133.33AsthmaU.S. Caucasian

28. Asthma Linkage to Chromosome 12q in 55 Sib-pairs Hum Mol Gen 2003 12:1973

29. Fine-Mapping of Chromosome 12q Asthma Locus Goal :Perform association mapping of 28.5 Mb centromeric locus on chromosome 12q using high-resolution SNP map Study Design :Family-based association study using 400 Caucasian parent-offspring trios ascertained through an asthmatic child participating in the Childhood Asthma Management Program (CAMP) Linkage disequilibrium mapping using LD-tagging SNPs identified from the HapMap project.

30. Association Studies Family-Based Case Control

31. Transmission Disequilibrium Test (TDT) AAAG AG:AA 50:50 AAAG AA AG AA AG AAAG AA AGAA AGAG AGAG AGAG AGAG AGAG AGAG AGAG AGAG AGAG AGAG AGAG AG:AA 66:33

32. Linkage Disequilibrium Recombination Ancestral chromosomes a b c d Contemporary chromosomes a b c d

33. SNP Selection From HapMap Data reduction of 2525 SNP across region to 1611 with high residual LD

34. SNP Associations With Asthma in CAMP

35. Haplotype Block Associations FrequencyT:U ratiop-value 0.58180:1580.23 0.1878:1240.001 0.1492:820.45 0.0744:430.92 0.0323:120.06

36. Summary and Future Directions This initial fine mapping effort has identified 19 regions totaling ~2Mb with evidence of association with asthma. These regions contain 46 expressed genes, few of which have been previously implicated in asthma pathobiology. We intend to further characterize the genetic patterns across these regions using higher resolution maps and to attempt to replicate our associations in other asthmatic populations. Replicated regions will then be resequenced to identify disease-susceptibility variants for functional analysis.

37. Acknowledgements Channing Laboratory Scott Weiss Edwin Silverman Ross Lazarus Many others… Harvard School of Public Health Kristel Van Steen Christoph Lange McGill University Thomas Hudson Alexandre Montpetite Jean-Francois Olivier Funding National Institutes of Health National Heart Lung Blood Institute Canadian Institutes of Health Research We thank all the CAMP children and their parents for their ongoing participation in this study