1. The Genetics of Pulmonary Fibrosis Mark P. Steele, MD Associate Professor of Medicine Pulmonary, Allergy, and Critical Care Duke University Medical Center

2. Idiopathic Pulmonary Fibrosis (IPF) Onset between 50 and 70 years of age Progressive shortness of breath and hypoxemia Median survival is 3-5 years and untreatable Environmental lung disease - cigarette smoke, asbestos, silica, beryllium, tungsten carbide, avian antigens, and molds

3. LIP: Lymphocytic Interstitial Pneumonia DIP: Desquamative Interstitial Pneumonia RB-ILD: Respiratory Bronchiolitis Interstitial Lung Disease COP: Cryptogenic Organizing Pneumonia AIP: Acute Interstitial Pneumonia IPF: Idiopathic Pulmonary Fibrosis (IPF) NSIP: Nonspecific Interstitial Pneumonia Idiopathic Interstitial Pneumonia (IIP)

4. Familial pulmonary fibrosis appears to be inherited as an autosomal dominant trait with variable penetrance Genetic Basis of Pulmonary Fibrosis Marshall. Thorax 2000; 55:143 Raghu. Sem Respir Med 1993; 14:323 5060 5853515249 Pulmonary fibrosis is associated with specific complex genetic disorders and autoimmune diseases Variable susceptibility to fibrogenic agents is evident among workers and laboratory animals

5. Search for Genes in Pulmonary Fibrosis Gene expression studies Linkage studies

6. Search for Genes in Pulmonary Fibrosis Gene expression studies – total genomic response to identify pathogenic genes IN LUNG. Linkage studies

7. Microarray  Whole human genome array (44k probes, 41k genes/ESTs)  25 genes, 114 EST clusters, and 370 singleton ESTs in the “FPF critical region” on Chromosome 10 Samples of Lung Tissue  surgical lung biopsy, transplant, or autopsy  9 normal controls  16 sporadic IIP (14 UIP and 2 NSIP)  10 FPF (6 UIP and 4 NSIP) Gene Expression Profiling of FPF Specimens Yang. A83 Mini-Symposium Mark Steele Jordan Savov Ivana Yang

8. 135 transcripts 5% FDR (SAM) > 1.8 fold change Disease vs. Normal Study Group Normal (n=9) Sporadic IIP (n=16) FPF (n=10) Histology Normal (n=9) UIP (n=20) NSIP (n=6)

9. FPF Candidate Genes (n=62) [Genes with FPF/sporadic IIP > 1.8] Cell adhesion Chemokines ECM Cell prolif/death Growth factors CXCR4 is the receptor for CXCL12

10. The Search for Genes in Pulmonary Fibrosis Gene expression studies Linkage studies – genomic screen to identify regions of DNA (loci) that contain a gene or genes that predispose individuals to develop pulmonary fibrosis

11. Linkage Analysis: Segregation Analysis 1 2 2 2 12 2 2 22 12 12 22 22 22

12. Linkage Analysis: Strategies for Segregation Analysis More and Larger Pedigrees More chromosome markers –N=1000 microsatellite –N= 300,000 SNPs

13. Linkage Analysis Identified > 350 families over the past 5 years 114 Familes phenotyped 83 Families phenotyped and genotyped

14. Diagnosis of Pulmonary Fibrosis [IPF/UIP, NSIP, COP, RB-ILD, DIP, and AIP] Possible IIP – suggestive CXR with no additional confirmatory tests Probable IIP – HRCT scan with reticular or ground glass opacities with either class 2 dyspnea or DLCO < 80% Definite IIP – surgical lung biopsy or autopsy ATS/ERS. Am J Respir Crit Care Med 2000;161:646 ATS/ERS. Am J Respir Crit Care Med 2002;165:277

15. Phenotype of 114 Families 714 Family Members 418 Unaffected296 AffectedSelf-Report 356 Unaffected 34 Possible Consensus Diagnosis 28 Probable/ Definite 35634 28

16. Phenotype of 114 Families 359 Unaffected 59 Possible Consensus Diagnosis 296 Probable/ Definite 35634 28 25268 714 Family Members 418 Unaffected296 AffectedSelf-Report Accuracy of Self Report87% 3

17. Type of Idiopathic Interstitial Pneumonia [IPF/UIP, NSIP, COP, RB-ILD, DIP, and AIP] 68 (60%) families had uniform IPF/UIP 46 (40%) families had more than one type of IIP 82% Idiopathic Pulmonary Fibrosis (UIP) 6% Non-specific Interstitial Pneuomonia 2% Cryptogenic Organizing Pneumonia 1% Respiratory Bronchiolitis-ILD 9% Unclassifiable ILD

18. Risk Factors for FPF [IPF, NSIP, COP, RB-ILD, DIP, and AIP] Steele. Am J Resp Crit Care Med 2005; 172:1146 Age at Onset Number of Subjects Age of onset highly correlated among sibs Older age (68 years vs 53 years; P<0.001) Male gender (56% vs 37%; P=0.004) Cigarette smoking (68% vs 34%; OR adj = 3.6 [1.3-9.8]; P<0. 01)

19. Epidemiology of FPF [IPF, NSIP, COP, RB-ILD, DIP, and AIP] Autosomal dominant pattern - vertical transmission with male to male inheritance Cigarette smoking enhances the risk of transmission Phenotypic heterogeneity of IIP suggests that tobacco smoke (or other exposures) may initiate common pathogenic mechanisms that place individuals at risk for IIPs Steele. Am J Resp Crit Care Med 2005; 172:1146

20. Histopathology of FIP The most common diagnosis is an unclassifiable chronic fibrosing interstitial pneumonia with septal fibrosis, fibroblast and smooth muscle proliferation and honeycombing that may be diffuse or sub-pleural. ATS 2006: A242

21. Genomic Screen on 83 Families with FPF [1100 markers  5 cM; 242 affected individuals] Mark Steele Marcy Speer Nell Burch Anastasia Wise Jim Loyd Kevin Brown Marvin Schwarz Hakon Hakonarson cM (recombinant distance) LOD Score

22. cM (recombinant distance) on Chr 11 LOD Score Chr11 Fine Map in 83 Families with FPF Multipoint LOD = 3.5

23. Chr11 SNP Map for FPF and IPF [150 FPF, 167 IPF, and 237 Controls]

24. Genomic Screen on 83 Families with FPF [1100 markers  5 cM] cM (recombinant distance) LOD Score LOD = 3.5 LOD = 2.1 Mark Steele Marcy Speer Nell Burch Anastasia Wise Jim Loyd Kevin Brown Marvin Schwarz Hakon Hakonarson

25. Chr10Chr11 25 Families 26 Families Younger Smokers Influence of Loci on Chr10 and Chr11 Nonsmokers 17 Both

26. cM (recombinant distance) LOD Score Influence of Smoking on Chr10 LOD LOD = 3.0 36 Families – 100% affected smoke

27. Determine whether SNPs in Chr11 candidate gene map with disease in families Identify Chr11 gene variants in founder population and other IIPs Genotype and sequence all candidates in Chr10 region Focus on unique phenotypes (rapid progressors and early disease) 3 million base pairs Future Research Approaches

28. Gene expression studies novel genes potentially involved in pathogenesis (CXCR4, CXCL12, tenacin C, osteopontin, MMPs, cathepsin G, etc) FPF is quite different than sporadic IIP UIP and NSIP appear somewhat similar Linkage studies Chr 11 and Chr10 looks promising Evidence for gene by environment interaction Phenotypic heterogeneity suggests common pathogenic mechanisms place individuals at risk for IIPs Search for Genes in Pulmonary Fibrosis: Summary

29. Nell Burch Tony Church Hadley Hartwell Zareen Kapadia Jason Thacker Kevin Brown Marvin Schwarz Jim Loyd Gunner Gudmunson Hakon Hakonarson Genetic Determinants of Familial Pulmonary Fibrosis Jordan Savov Ivana Yang Marcy Speer Aretha Herron Susan Slifer Frank Zhao David David Schwartz Mark Steele

30. Genetic Determinants of Familial Pulmonary Fibrosis A Special Thank You To all the family members Without whom This would not be possible.