Genetics of Asthma

1. Genetics of Asthma Paul E. Moore, M.D. Vanderbilt University School of Medicine May 9, 2013

2. Definition of asthma • Chronic inflammation • Airway reactivity to specific triggers • Reversible airway obstruction • Manifest as symptoms that can include cough, wheeze, and dyspnea

3. Weiss, Pharmacogenomics, 2006

4. Phenotype • Phenotype: observable properties of an organism that are produced by the interactions of the genotype and the environment • Asthma: collection of different phenotypes, rather than a single disease • Asthma is a heterogeneous phenotype resulting from interaction between multiple genetic and environmental influences Wenzel, Nature Medicine, 2012

5. Genetics of Asthma • Twin studies and family studies suggest a heritable pattern. • Some genes influence asthma development (susceptibility) or progression (severity) • Other genes modify response to therapy (pharmacogenetics). • Gene-environment interactions add to the level of complexity.

6. Issues in Studying Asthma Genetics • Polygenic disease • No "gold standard" to define asthma • Inconsistent application of clinical parameters • Gene-environment interactions

7. Measurable Asthma Phenotypes • Presence of asthma • Asthma-related traits • Bronchial hyperresponsiveness • IgE levels, Skin test reactivity • Response to asthma medication • Physiologic improvement • Quality of life measurements

8. Genetic Approaches • Candidate-gene association studies • Genome-wide linkage studies in families • Genome-wide association studies (GWAS)

9. Association Studies • Hypothesis-driven • Determine the relationship between specific polymorphisms in candidate genes and certain disease characteristics. • To date, close to 200 candidate genes in over 500 published studies have been linked to different asthma phenotypes.

10. Categories of asthma susceptibility genes • Innate immunity and immunoregulation • TH2-cell differentiation and effector functions • Epithelial biology and mucosal immunity • Lung function, airway remodeling, and disease severity Vercelli D, Nat Rev Immunol, 2008

11. Susceptibility genes associated with asthma phenotypes in 20+ association studies IL13 5q31 TH2 effector function IL4 5q31.1 TH2 differentiation CD14 5q31.1 Innate immunity ADRB2 5q31-32 ASM relaxation TNF 6p21.3 Inflammation HLA-DRB1 6p21 Antigen presentation FCERIB 11q13 Fc receptor for IgE IL4R 16p12.1 a-chain of IL4R,13R

12. Limitations of association studies • Selection of genes is based on available knowledge. • Each variant makes only a modest contribution to overall heritability in a polygenic disease.

13. Linkage studies • Allows identification of new genes and pathways • Technique: statistical approach using DNA markers, followed by positional cloning • To date, ~20 linkage scans have been reported in different asthma study populations.

14. ADAM33 • Original study (2002): 460 Caucasian families found linkage to chromosome 20p13 • Gene: A disintegrin and metallopeptidase domain 33, expressed in lung fibroblasts and smooth muscle cells • Series of replications studies inconclusive • Single gene with modest contribution to overall population risk Van Eerdewegh P, Nature, 2002

15. Genes identified by linkage scans • DPP10 (dipeptidyl-peptidase X) • PHF11 (plant homeodomain finger protein) • GPRA (G-protein coupled receptor) • HLA-G • Cytoplasmic fragile X mental retardation protein (FMR) interacting protein 2 (CYFIP2) • IRAKM (IL-1 receptor associated kinase) • COL29A1 (Collagen XXIX) • PCDH1 (Protocadherin 1)

16. Limitations of linkage scans • Difficult to identify candidate gene • Challenge to link specific locus or gene to asthma or asthma-related traits

17. Genome-wide association studies • International HapMap resource • Availability of dense genotyping chips • Collection of sets of large and well characterized human samples

18. First GWAS: 17q21 • 317,000 SNPs in 994 patients with childhood-onset asthma and 1,243 non-asthmatics • Multiple markers on chr 17q21 strongly associated with childhood asthma • Independent replication studies of the 17q21 locus showed strong association with diagnosis of childhood asthma: • 2,320 subjects in a cohort of German children • 3,301 subjects in the British 1958 Birth Cohort Moffatt MF, Nature, 2007

19. First GWAS: 17q21 • Independent replications in North Americans of European ancestry, Puerto Rican, Mexican, Japanese, and Chinese populations • No associations yet described with African-American populations • In Caucasians, even stronger association with early tobacco smoke exposure and early-onset asthma Bouzigon E, New Engl J Med, 2008

20. First GWAS: 17q21 • Transcript levels of 19 genes in EBV-transformed lymphoblastoid cell lines • Rationale: Variation in gene transcription may mediate disease susceptibility, and transcript abundance may be directly modified by polymorphisms in regulatory elements. • ORMDL3: member of a gene family that encodes transmembrane proteins anchored in the endoplasmic reticulum Moffatt MF, Nature, 2007

21. GWAS and Asthma Susceptibility • 2large meta-analyses of asthma susceptibility identified 4 regions that were associated with asthma in individuals of different ethnic backgrounds: • loci in the ORMDL3 region of 17q21 • IL1RL/IL18R on chromosome 2q • TSLP on 5q22 • IL33 on chromosome 9p24 Slager Clin Chest Med 2012

22. GWAS and Asthma Severity • Genes that are associated with asthma subphenotypes, such as lung function, biomarkers levels, and asthma therapeutic responses, can provide insight into mechanisms of asthma severity progression. • A joint model of risk variants in lung function genes identified in the general population were highly associated with lower lung function and increased severity in asthma populations. Slager Clin Chest Med 2012

23. GWAS and Pharmacogenetics • A pharmacogenetic genome-wide screen identified 2 correlated genetic variants in the GLCCI1 gene related to response to inhaled glucocorticoids. Slager Clin Chest Med 2012

24. Benefits of GWAS • Identification of novel genes not previously linked to asthma: ORMDL3, DENND1B, HHIP • Suggestion of link between lung development, affecting COPD and asthma

25. Limitations of GWAS • Some genes identified by GWAS do not appear biologically plausible • Challenges in finding the specific mutation or gene within a region of interest • Single genes unlikely to be sufficient for disease causation • Relatively small sample size precludes results of statistical significance • Replication limited in non-European populations • Heterogeneity in environmental exposures • Challenges in bioinformatics

26. Gene-Environment Interactions • Concordance rates among monozygotic twins: 50-60% • Allergens • Respiratory infections • Tobacco smoke • Air pollution • Diet

27. β2-Adrenergic Signaling Pathway

28. Questions about deleterious effects of b-agonists • Concern: whether b-agonist use is associated with poor asthma control • In Beta Agonist in Mild Asthma (BAGS), AM PEF no different with regular albuterol use • Meta-analysis: Just 1 to 6 weeks of regular b-agonist use significantly reduced the bronchodilator response Sears MR, Lancet, 1990; Drazen JM, NEJM, 1996; Salpeter SR, Ann Int Med, 2004

29. Questions about deleterious effects of long-acting b-agonists • Concern: whether long-acting b-agonist use is associated with poor asthma control • In Salmeterol Multi-Center Asthma Research Trial (SMART): higher number of asthma-related deaths or life-threatening experiences with Serevent • Meta-analysis: Long-acting b-agonists increased exacerbations requiring hospitalization and life-threatening exacerbations. Nelson HS, Chest, 2006; Salpeter SR, Ann Int Med, 2006

30. b2-Adrenergic Receptor Liggett, AJRCCM, 1997

31. No association of ADRB2 polymorphisms and the diagnosis of asthma Amino acid Genotype Frequency (%) Position Normal Asthmatic 16 Homozygous Arg 13.5 14.6 Heterozygous 29.2 32.2 Homozygous Gly 57.3 53.1 27 Homozygous Gln 28.0 26.0 Heterozygous 44.0 50.0 Homozygous Glu 28.0 24.0 adapted from Liggett et al., AJRCCM, 1997

32. Clinical Correlation Importance of early in vitro studies No consensus on which polymorphism influences asthma severity. 4 studies from the Asthma Clinical Research Network: Arg16

33. BAGS and BARGE: Patients homozygous for the Arg16 allele showed worsening in AM PEF while on regularly scheduled albuterol. Israel E, AJRCCM, 2000; Israel E, Lancet, 2004

34. SOCS and SLIC: Arg16 subjects who received salmeterol alone had worsening AM PEF. Wechsler, AJRCCM, 2006

35. Effect of ADRB2 polymorphisms on response to LABA The Gly16 genotype had no effect on the percentage of participants with severe exacerbations across all treatment groups (primary endpoint). Bleecker et al., Lancet, 2008

36. The inability to reproduce genotype-phenotype associations is related to a number of factors: • Measurements of many different parameters relative to asthma • Coding block haplotypes may be markers for more functionally significant haplotypes in the promoter region.

37. Promoter/coding block haplotypes • Resequencing of a 5.3-kb region in 429 whites and 240 African Americans revealed 31 SNPs with minor allele frequency >3%. • 4 extended haplotypes account for >90% of the persons genotyped from 4 ethnic backgrounds. Drysdale et al, PNAS, 2000 Hawkins et al, AJRCCM, 2006

38. ADRB2 haplotype influences spirometric response • Genotype: ADRB2 SNPs in 99 adults admitted for asthma exacerbation • Primary endpoint: Change in FEV1 24 h after admission • Results: Complex promoter/Arg16 coding block haplotype present only in African Americans associated with decreased FEV1 response Moore et al, Clin Trans Sci, 2008

39. SPARE RECEPTORS NO SPARE RECEPTORS C. 25,000 receptors A. 50,000 receptors ASM ASM Desensitization: reduced b2AR number =spare receptor ASM ASM B. 30,000 receptors: No reduction in maximal responsiveness; thus, no change in functional desensitization D. 5,000 receptors: Reduced maximal effectiveness; thus, increased functional desensitization Drury et al., Brit J Pharm, 1997 Nishikawa et al, AJRCMB, 1994 Rousseau et al, Eur J Pharm, 1997

40. Other regulatory elements of the ADRB2 • Rare genetic variations in the promoter region • Poly-C repeat in the 3’-untranslated region: length associated with FEV1 in African American. • In vitro studies suggest that length of the poly-C repeat alters mRNA stability and ADRB2 expression. Hawkins et al. , AJRCCM, 2006. Panebra et al, AJP: Lung, 2008.

41. Gene-gene interactions • Adenylyl Cyclase IX • GRK5 • Regulation of ADRB2 signaling by S-nitrosylation of GRK2 may provide a link between NO and ADRB2 function. Whalen EJ, Cell, 2007. Tantisira KG, Hum Mol Gen, 2005 Zhang G, Eur Respir J, 2007.

42. Gene-environment interactions • ADRB2 genotype effects may be manifest only after specific environmental exposures, including cigarette smoke and viral infection. • Children homozygous for the Arg16 allele exposed to maternal smoking in utero: 3-fold increased risk for lifetime wheezing Moore et al, AJRCMB, 2006 Wang et al, AJRCCM, 2001 Wang et al., Pediatrics, 2008

43. Summary • Asthma is a heterogeneous phenotype resulting from interaction between multiple genetic and environmental influences. • Genes can influence asthma susceptibility, severity, or response to therapy. • Genome-wide association studies have helped to identify genes not previously linked with asthma.

46. Resources • Vercelli D. Discovering susceptibility genes for asthma and allergy. Nat Rev Immunol 2008; 8:169–182. • Sleiman P and Hakonarson H. Recent advances in the genetics and genomics of asthma and related traits. Curr Opin Ped 2010; 22:307-312. • Slager RE et al. Genetics of Asthma Susceptibility and Severity. Clin Chest Med 2011;33:431-43. • Wenzel S. Asthma phenotypes: the evolution from clinical to molecular approaches. Nat Med 2012;18:716-25.