Overview of the Pharmacogenetics of Asthma Treatment

Overview of the pharmacogenetics of asthma treatment

ST Weiss1, AA Litonjua1, Asthma affects approximately 300 million individuals worldwide. Medica- 2 1 tions comprise a substantial portion of asthma expenditures. Despite the C Lange , R Lazarus , availability of three primary therapeutic classes of medications, there are a 3 4 SB Liggett , ER Bleecker , significant number of nonresponders to therapy. Available data, as well as KG Tantisira1 previous pharmacogenetic studies, suggest that genetics may contribute as much as 60–80% to the interindividual variability in treatment response. In 1Channing Laboratory, Brigham and Women’s this methodologic review, after providing a broad overview of the asthma 2 Hospital, Boston, MA, USA; Harvard School of pharmacogenetics literature to date, we describe the application of a novel Public Health, Boston, MA, USA; 3Department of Medicine, University of Maryland, Baltimore, MD, family-based screening algorithm to the analysis of pharmacogenetic data USA and 4Division of Pulmonary and Critical Care and highlight our approach to identifying and verifying loci influencing Medicine and Center for Human Genomics, asthma treatment response. This approach seeks to address issues related to Wake Forest University School of Medicine, multiple comparisons, statistical power, population stratification, and failure Winston-Salem, NC, USA to replicate from which previous population-based or case–control pharma- Correspondence: cogenetic association studies may suffer. Identification of such replicable loci Dr K Tantisira, Channing Laboratory, Brigham is the next step towards the goal of ‘individualized therapy’ for asthma. and Women’s Hospital, 181 Longwood The Pharmacogenomics Journal (2006) 6, 311–326. doi:10.1038/sj.tpj.6500387; Avenue, Boston MA 02115, USA. E-mail: [email protected] published online 25 April 2006

Keywords: beta-agonist; leukotriene; corticosteroid; FEV1; genetics

Introduction

The goal of this review is to give a comprehensive assessment of asthma pharmacogenetics and highlight the scientific approach being taken by the pharmacogenetics of asthma treatment (PHAT) research group (http:// www.pharmgat.org/) to this research area. Our approach strives to address many of the potential problems inherent in genetic association studies, including those related to multiple comparisons, lack of statistical power, population stratification, and failure to replicate. Moreover, our group of collaborative investigators is well positioned to investigate the molecular biology and functional genomics of identified candidate gene associations. In this review, we will cover some aspects of asthma health care, pathobiology, and review the available pharmacogenetics literature on the important types and pathways of asthma medications: b-agonists, leukotriene (LT) antagonists, methylxanthines, and corticosteroids. We will then present details of our research approach to finding replicated pharmacogenetic associations and suggest what the future may hold for this research area.

Pharmacogenetics – definition and rationale Pharmacogenetics is the study of the role of genetic determinants in the variable, Received 1 December 2005; revised 15 February 2006; accepted 16 February 2006; interindividual response to medications. Numerous examples of heritable published online 25 April 2006 differences in pharmacokinetics (drug distribution and metabolism) in Overview of the pharmacogenetics of asthma treatment ST Weiss et al 312

individuals resulting in varied clinical response to medica- 1 Growth Factors tions have been described. Other mechanisms underlying Endogenous GC Early Environmental Chronic Tissue the genetic response to drugs includes alterations in Factors Inflammation Remodeling Cytokines pharmacodynamics (changes in the drug target), idiosyn- Allergens Endogenous GC Allergens cratic associations (unintended side effects in predisposed Pollution TH2 Pollution Virus Immune Virus individuals), and genetic predisposition to the disease in Cigarettes Deviation Cigarettes which the treatment is to be instituted2,3 Overall, it is Stress Stress Acute estimated that genetics can account for 20–95% of varia- Susceptibility Inflammation Leukotrienes 2 Bronchoconstr Histamine bility in drug disposition and effects. In corticosteroid Genes Endothelins/Chemokines therapy, a number of family and twin studies have demon- ADAM33 Complement FcεRI Endogenous GC strated consistent evidence that endogenous levels of IL4Rα iction glucocorticoids (GC), usually measured as plasma cortisol TGFβ SYMPTOMS 4–8 GPRA Wheezing levels at certain times of the day, are heritable. We have GSTP1 Dyspnea generated similar heritability data for bronchodilator re- Cough 9 sponse in a Chinese population. As the initial sequence of Figure 1 Asthma pathobiology. Asthma is an inflammatory disorder of 10,11 the human genome was published, the concept that the airways influenced by multiple early life and genetic factors. The genetic variation will allow prediction of treatment response inflammatory response, as well as smooth muscle constriction, lead to has been seen as an important and achievable goal.12–14 airway narrowing and symptoms. Further, the food and drug administration has drafted guidelines requiring drug development companies to submit pharmacogenetic information when reporting clinical trial lymphocytes, epithelial cells, and airway smooth muscle results. Ideally, pharmacogenetics will allow for ‘individua- cells play a prominent role.25 Figure 1 shows important lized therapy’ that is tailored to an individual’s genetic pathobiologic features of asthma. Genetics in combination make-up to maximize the potential for therapeutic benefit, with early life environment modulate the development of while minimizing the risk of adverse effects. The potential CD4/lymphocytes towards a Th2 immunophenotype. These for cost savings and for decreasing morbidity and mortality lymphocytes then produce cytokines, such as interleukin is immense because the target therapies are so widely used. (IL)-3, IL-4, IL-5, IL-13, and granulocyte–macrophage col- ony-stimulating factor (GM-CSF) and thereby promote the Asthma – overview and rationale synthesis of IgE, an important allergic effector molecule, resulting in an inflammatory airway milieu. Chemokines, The burden of asthma such as eotaxin, RANTES, and IL-8 produced by epithelial Asthma is a complex disease affecting over 300 million and inflammatory cells, serve to amplify and perpetuate the individuals in the developed world.15 Of all asthma cases, inflammatory events. Several bronchoactive mediators, such 90%, including asthma in adults, has its origins in child- as histamine, LT, and neuropeptides are released into the hood. Of concern are the increases in asthma prevalence16 airways and precipitate an asthma attack by causing airway and hospitalization rates.17 Between 1980 and 1994, the self- smooth muscle constriction, mucus secretion, and edema. reported prevalence of asthma in the USA increased from Over time there is smooth muscle growth and the deposi- 30.7 to 53.8 per 1000, an increase of 75%.16 This increase tion of subepithelial connective tissue, a process referred to has been accompanied by a similar increase in health care as airway remodeling. This basic asthma pathobiology is utilization and mortality over the same time period.16 An important for asthma pharmacogenetics because many estimated 12.6 billion dollars were spent on the diagnosis genes associated with an altered treatment response are also and management of asthma in the US in 1998, of which associated with asthma or its intermediate phenotypes. 58% were direct medical expenditures (DMEs).18 Medication Clinically, asthmatics have difficulty exhaling air because of costs are currently the largest component of DMEs. Despite an increase in airway resistance that is a consequence of the availability of several classes of therapeutic agents for smooth muscle contraction, inflammation and remodeling asthma, it has been estimated that as many as one-half of (Figure 1). In clinical trials, the degree of resistance and asthmatic patients do not respond to treatment with b2- physiological impairment is quantitated most commonly by 3,19,20 agonists, LT antagonists, or inhaled corticosteroids. As the forced vital capacity in 1 s (FEV1) (Figure 2). Forced vital a whole, adverse drug reactions are estimated to cost the US capacity in 1 s is the volume of air a person can ‘blow out’ in $100 billion and over 100 000 deaths a year.21 Asthma one second and is very useful as a measurement of lung therapy has been associated with adverse drug reactions, function because it is easily obtained, reproducible, and 26 including serious and potentially fatal complications.22–24 correlated with asthma severity and therapeutic responses. Another commonly used measurement of the physiological Asthma: definition, clinical features, pathobiology impairment in asthmatics is the peak expiratory flow rate Asthma is a clinical syndrome of unknown etiology (PEFR). Peak expiratory flow rate is defined as the maximal characterized by reversible episodes of airflow obstruction, flow rate achieved during forced exhalation.27 airway hyperresponsiveness, and a chronic inflammatory During an asthma attack, patients experience shortness of process of the airways of which mast cells, eosinophils, T breath, cough, and/or wheezing. Between attacks, patients

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Asthma: a Outcome Measures 16 beclomethasone nonasthmatic 14 12 10 montelukast asthmatic 8

from Baseline, % 6 1 Volume (L) 4 placebo 2 0 132 • Airway Inflammation Time (sec) -2 036 9 12 • Airway Remodeling Change in FEV -4 Weeks • Airway Hypersensitivity Forced Expiratory FEV1 = • Reversible Airflow Obstruction Volume at 1 Second b 30 Figure 2 Forced vital capacity in 1 s is decreased in asthma. In the Montelukast patient with asthma, inflammation and bronchoconstriction lead to 25 Beclomethasone decreased ability to exhale rapidly and forcefully. This is measured 20 clinically by the FEV1. 15

Patients, % 10 may be asymptomatic or they may have chronic symptoms of breathlessness with mild-to-moderate exertion or epi- 5 sodes of nocturnal awakening due to airway narrowing at 0 night. In clinical drug trials, these asthma symptoms are <-30 -30- -20- -10- 0- 10- 20- 30- 40- >50 commonly graded and recorded by the patient to give an <-20 <-10 <0 <10 <20 <30 <40 <50 Change in FEV from Baseline, % index of the symptomatic response to asthma treatment.28 1 Other potentially useful indices of the effects of asthma Figure 3 Interindividual variability in response to asthma therapy. The treatment are measures of healthcare utilization including average lung function response to therapy with leukotriene inhibitors doctor visits, urgent care emergency room visits, and and inhaled corticosteroids in asthma is significant when compared to placebo (a). However, when evaluated at the individual level, there are a hospitalizations. Changes in FEV1, PEFR, symptom scores, and healthcare utilization that occur during the course of significant number of individuals who do not respond to therapy and actually may have lung function decrements with a given treatment (b). therapy are important outcome indicators that we will refer (Adapted from Malmstrom et al.31, with permission) to throughout this review.

Asthma therapy although available data on LT and theophylline will be Standard guidelines for the diagnosis and management of mentioned. asthma have been published.29 The two main types of There is large interindividual variation in the treatment asthma drugs are the so-called reliever drugs that target the response to asthma medications.19,30 For example, in a study acute bronchconstriction and the so-called controller drugs by Malmstrom et al.31 comparing the efficacy of the inhaled that are used to reduce the severity of airway inflammation steroid beclomethasone (200 mg bid) with the LT antagonist and the severity and frequency of obstruction.29 The main montelukast (10 mg/qd), it is clear that both drugs are reliever drugs are rapid-acting b2-agonists (e.g., albuterol, effective over a 12 week course of treatment with a mean metaproterenol, pirbuterol, and levalbuterol), which relax increase in FEV1 of 13.1% for the beclomethasone group and the bronchial smooth muscle by activating b2-adrenergic 7.4% for the montelukast group (Figure 3a). However, when receptors (b2AR). This is the treatment of choice for very these same data are viewed from a different perspective, mild asthma. For moderate and severe persistent asthma, focusing on the number of individuals as a function of the reliever treatment is usually combined with controller percent change in FEV1 from baseline, it is clear that many treatment. The two commonly used classes of controller patients had little response (Figure 3b). In fact, 22% of agents are the inhaled GC and the leukotriene receptor patients appear to have had an adverse response to antagonists (LTRA). Inhaled GC (e.g., budesonide, beclo- treatment with a decline in FEV1 at 12 weeks compared methasone, flunisolide, and fluticasone) and LTRAs (e.g. with baseline. The mean therapeutic improvement in FEV1 montelukast and zafirlukast) modify the inflammatory for all patients is driven by a dramatic increase in FEV1 in a microenvironment of the airway to reduce airway obstruc- minority of trial subjects. In a similar study, 38% of patients tion and hyperresponsiveness. randomized to inhaled budesonide or fluticasone demon-

This review will focus on b2-agonists and GC because they strated improvements in FEV1 of under 5% over the course are the most effective and commonly used asthma drugs and of 24 weeks.30 A unique subset of up to 25% of the we have outstanding clinical trials data for these pathways nonresponders can be classified as having GC-resistant

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asthma.32 These patients are nonresponsive to even high to prospectively determine response to drugs in asthmatics doses of oral steroids. Furthermore, a careful analysis of based upon genetic testing.43 Although the PHAT research these studies indicates that individuals responsive to one focus is on b2-agonists and GC, we will review examples of class of asthma drug may not necessarily be the individuals asthma pharmacogenetics for each of four therapeutic responsive to a different class of asthma drug. Despite these pathways for completeness. studies, there is no universally accepted definition of a steroid (or b2-agonist) nonresponder. These types of data b2-adrenergic receptor polymorphisms and response to b-agonists illustrating variable drug efficacy are not limited to asthma b2-Adrenergic receptor agonists (b2-agonists) are the most drug trials but can be found for almost all classes of drugs. commonly used class of medication in the treatment of 36 The degree to which this interindividual variability is asthma worldwide. A b2-agonist inhaler is discharged genetic remains uncertain and probably differs with each around the world 19 times a second! b2-agonists produce class of drug. Moreover, interindividual variability may bronchodilitation (increase FEV1) in asthmatics by binding depend on the particular preparation within a class of to and directly simulating b2ARs on airway smooth muscle asthma drug. Calculations of the repeatability (r) of the cells. b2ARs are cell surface receptors (GPCR) that signal to treatment response for all three classes of asthma drugs, the interior of the cell by multiple mechanisms. In brief, defined as the fraction of the total population variance that agonists binding to the receptor result in the activation of results from within individual differences, show values for r adenylyl cyclase via stimulatory G proteins (Gs). This leads between 60 and 80% indicating the upper limit of the to an increase in cAMP and activation of protein kinase A genetic component and suggesting that a substantial (PKA). Protein kinase A phosphorylates several target fraction of the variance of the treatment response could be proteins, decreases intracellular Ca2 þ , and leads to smooth 19 genetic. In support of these data, several genetic variants muscle relaxation. Once b2ARs are activated by b2-agonists and haplotypes have recently been discovered in genes that a complex process of tachyphylaxis or ‘desensitization’ play a role in the action or metabolism of asthma drugs and begins. Desensitization functions to attenuate the effects are associated with altered therapeutic responses. These of b2-agonists and is mediated by both short-term (e.g. examples are discussed below. It is likely that many, as yet uncoupling of receptor) and long-term mechanisms undiscovered, polymorphisms exist, given the many gene (e.g., downregulation of receptor expression). The intricacies products involved in the pharmacodynamic and pharmaco- of this pathway as it related to this proposal are discussed in kinetic pathways of asthma drugs. The ultimate goal of detail below. A diagram of this pharmacological pathway is asthma pharmacogenetics is to identify this information shown in (Figure 4) and may be viewed on the PharmGKB and use it to tailor an individual’s therapeutic regimen in website (http://www.pharmgkb.org/index.jsp). This diagram order to maximize efficacy and minimize side effects. accentuates the fact that multiple genes in addition to b2AR likely interact in the response to this class of medications.

The b2AR is highly polymorphic with over 55 single- Asthma pharmacogenetics to date nucleotide polymorphisms (SNPs) (B1 every 300 bp) recorded in public databases (http://snpper.chip.org). Two To date, genetic variants in multiple genes have been common nonsynonymous SNPs, rs1042713, and rs1042714, associated with altered therapeutic response to four classes have been the most extensively studied. rs1042713 (A/G, of asthma drugs. Examples of asthma pharmacogenetics that 0.44 avg. het.) results in Arg vs Gly at position 16 and will be discussed include the b2AR, 5-lipoxygenase (ALOX5), rs1042714 (C/G, 0.49 avg. het.) results in Gln vs Glu at leukotriene C4 synthase (LTC4S), corticotropin releasing position 27. Over the last decade nonsynonymous coding 37 factor receptor type 1 (CRHR1) and cytochrome p450 1A2 SNPs of the human b2AR were identified and characterized (CYP1A2) genes. Our laboratories discovered three of these in cells by us.38–41 Arg or Gly can be found at amino-acid 16, examples; each has been replicated in at least one other whereas Gln or Glu can be found at position 27. A rare clinical drug trial. In the case of the ALOX5 polymorphisms, polymorphism Thr164-Ile (5% heterozygous frequency) these genetic variants have been discovered to be important has also been detected. In transfected cells, Arg16, Gly16, 33–35 in the context of atherosclerosis as well as asthma. Gln27, and Glu27 b2AR (in all possible haplotypic combina- Despite progress in the discovery of asthma pharmacoge- tions) appeared to have the same agonist binding affinities netic loci, to date no single variant interrogated has been and coupling to adenylyl cyclase39 However, agonist- found to explain more than a small percentage (o10%) of promoted downregulation of receptor number was greatest the total phenotypic variance of treatment response that is for Gly16/Gln27 and Gly16/Glu27, compared to Arg16/ characteristic of any asthma drug.19 Part of the reason is that Gln27.39 Interestingly, Arg16/Glu27 failed to downregulate, the exact location of the functional variants remains unclear but this diplotype is very rare in the human population. for each of these associations. The molecular mechanisms of Subsequent studies in human airway smooth muscle cells their action are also unclear and appear to be complex and expressing the various 16/27 SNPs gave similar results.41 multifaceted.36 It is also highly likely that sequence variants However, when other end points were utilized the pheno- in other genes contribute significantly to the heterogeneity types were not always as would have been predicted from 42,43 of therapeutic response. Nevertheless, progress has been the transfected cell data. The rare Ile164 b2AR has made and studies are underway to investigate the potential decreased agonist affinity, depressed coupling to adenylyl

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Figure 4 The b-agonist pathway. The b2-adrenergic receptor is a G protein (Gs) coupled receptor with genetic influences and interactions at multiple levels. (From PharmGKB – http://www.pharmgkb.org/index.jsp.) cyclase, and a decreased duration of action (by B50%) in uled albuterol had a slow decline in AM PEFR, which 38,40 response to the long-acting b2-agonist salmeterol. This continued even during the washout phase. Neither Gly functional work on the b2AR needs to be expanded to homozygous individuals or Arg homozygotes on as needed include the promotor and 3’ UTR variants newly identified. (PRN) albuterol showed no such decline. In another study of Clinical studies have not revealed an association between similar design, except that prospective enrollment was by these SNPs and asthma per se, but a myriad of subpheno- homozygous position 16 genotype (BARGE), we noted types have been reported to be associated with one or both analogous changes in PEFR, although there were some of these variants. In terms of pharmacogenetics, several fundamental difference.45 In this study, Arg/Arg individuals studies have reached similar conclusions regarding associa- showed no significant decline in lung function while on tions between position 16 SNPs and the response to chronic regular albuterol, but did display a decrease during washout.

(regularly scheduled) b2-agonist albuterol. These association Gly/Gly patients on regular albuterol showed improved studies represent the first successful efforts in asthma function and no decrement during washout.44,45 These two pharmacogenetics to take data from a retrospective associa- studies both point to homozygosity at Arg16 being asso- tion study and apply it prospectively. Such prospective ciated with an unsatisfactory response to albuterol. How- studies will comprise an important component of future ever, the clinical relevance of these findings remains research in this field. Israel et al.44 reported on the changes uncertain, since the genotype-attributable treatment differ- in PEFR during albuterol therapy in patients with mild ence (the effect of treatment in Arg/Arg patients minus that asthma stratified by b2AR genotype. Patients with Arg/Arg observed in Gly/Gly patients) was only À24 l/min, which is (i.e., homozygous for Arg16) treated with regularly sched- quite modest. Moreover, since chronic administration of

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b-agonists on a regularly scheduled basis is not part of of asthmatics that are potent bronchoconstrictors. Of the recommended treatment guidelines, the clinical applicabil- many enzymes involved in the formation of the LT, ity of these findings is also uncertain. In another study of polymorphisms in two, ALOX5 and LTC4S have been mild–moderate asthmatics,46 exacerbations were signifi- associated with altered response to LT inhibitors. 5-lipox- cantly greater for Arg/Arg16 individuals compared to ygenase catalyzes the conversion of arachidonic acid to heterozygotes or Gly/Gly patients, and indeed were even LTA4, which is a rate limiting step involved in the synthesis greater than Arg/Arg patients on placebo. The PEFR trended of all LT.49 In 221 patients with asthma who received either towards showing a decrease over time for Arg/Arg patients, high-dose ABT-761, an ALOX5 inhibitor similar to zileuton, but this did not reach statistical significance. (n ¼ 114) or placebo (n ¼ 107) treatment, we found that The acute bronchodilatory response to albuterol has not approximately 6% of asthmatic patients had no wild-type been associated with position 16 in any of the above studies. allele at the ALOX5 promoter locus and had a diminished 50 However, certain extended b2AR haplotypes appear to response to ABT-761 treatment. These findings were confer a differential response. In one study, we identified consistent with the hypothesis that repeats of the – 13 SNPs (from the promoter forward through the coding GGGCGG– sequence other than the wild type are associated region but not including the 30 UTR) organized into 12 with decreased gene transcription and ALOX5 product 47 haplotypes. The change in percent predicted FEV1 was production. This has recently been confirmed in cells 51 related to b2AR haplotype pair (P ¼ 0.007 by ANCOVA), but obtained from patients with asthma. not any of the individual SNPs. Of the homozygous Another enzyme of the LT pathway, LTC4S, is responsible haplotype pairs, patients with haplotype 4/4 had the poorest for the adduction of glutathione at the C-6 position of the response compared with those with haplotype 2/2. In arachidonic-acid backbone to form LTC4, a potent bronch- transfected cells, haplotype 4 had lower mRNA and protein oconstrictor. There is a known SNP in the LTC4S promoter, expression compared with haplotype 2.47 A-444C, with a C allele frequency of 0.19 in normal subjects 52 A more recent study by our group utilized restricted b2AR and of 0.27 in patients with severe asthma. The À444C genotyping of patients and their parents in the Childhood allele creates an activator protein-2 binding sequence that Asthma Management Program (CAMP). Here, the most appears to be functional,52 suggesting that the À444C intriguing finding was an association between a synon- variant is associated with enhanced cysteinyl LT production. ymous SNP at nucleotide position þ 523 in the 30 end of the Sampson et al.53 found that, among asthmatic subjects coding region and bronchodilator response. These findings treated with zafirlukast (20 mg bid), those homozygous for 0 suggest that additional variants, likely in the 3 UTR, are in the A allele (n ¼ 10) at the À444 locus had a lower FEV1 linkage-disequilibrium with þ 523 and may represent the response than those with the C/C or C/A (n ¼ 13) genotype. causal polymorphisms for bronchodilator response. As a Given the previous associations, we have recently colla- result of these findings, deep resequencing of the b2AR gene borated to perform genotyping on 28 SNPs in five LT has occurred. Indeed, several novel variants, both 50 and 30 pathway genes in subjects participating in a clinical trail of the gene have now been described.48 Of interest, a repeat comparing the efficacy of montelukast vs low-dose theo- region consisting of a variable number9–14 of ‘C’ alleles at phylline vs placebo as add on therapy in mild–moderate position 1269 has now been identified. Moreover, this poly- asthma.54 DNA was obtained in 248 participants and the C track also contains additional SNP variation, adding to the primary analysis was performed on 61 Caucasians taking complexity of the region. By imputing haplotypic substruc- montelukast. Outcomes assessed included change in FEV1 ture, it is clear that the poly-C region subdivides the and presence of exacerbations over 1, 3, and 6 months of aforementioned haplotypes further. Moreover, preliminary time. At 6 months, two SNPs were associated with change in association studies of these subdivided haplotypes suggests FEV1, one in the MRP1 gene (rs119774) and the second in that this region may influence both lung function and ALOX5 (rs2115819). Of potentially greater interest, variants bronchodilator response. Confirmatory studies are under- from three genes were associated with risk of exacerbations. way. Variation in one LTA4H SNP (rs2660845) was associated with

Taken together, some aspects of b2-agonist pharmacoge- a fourfold increase in the risk of at least one exacerbation netics have been ascertained, but predictability remains low. over the 6 month follow-up, whereas variation in the This lack of predictability is due to an incomplete under- previously described ALOX5 promoter repeat and the the standing of the haplotypic structure of the gene and the LTC4S A-444C SNP was associated with a 73% and a 76% need to identify and characterize additional functional reduction in exacerbations, respectively. Although these variants. Even with this information explained phenotypic findings require replication, they provide evidence of variation is low. Therefore, genetic variants in other genes in multiple LT pathway pharmacogenetic loci in modulating the pathway are likely to be important determinants of b2- the therapeutic response to these agents. agonist response and need to be characterized (Figure 4). CYP1A2 polymorphisms and theophylline metabolism

5-lipoxygenase and leukotriene C4 synthase polymorphisms and Theophylline, and related methylxanthine derivatives, have response to leukotriene antagonist weak bronchodilator and anti-inflammatory effects and are The LT are a family of lipoxygenated eicosatetraenoic acids considered second line drugs for asthma.29 Dose-related derived from arachidonic acid and produced in the airways toxicities, such as tachycardia, headache, nausea, and

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seizures are major problems that have limited their use. is unknown. Multiple theories exist and focus on abnorm- Serum levels must be monitored closely because of a narrow alities in cellular immune response, GC signal transduction, therapeutic window and a plethora of drug interactions29 receptor gene conformation, and receptor isoform b-expres- may have anti-inflammatory effects, however, that may not sion68–70 CRHR1 was chosen as a candidate gene because it is necessitate monitoring.55 Obase et al.56 have recently a major regulator of GC synthesis, it has been implicated in described a common promoter polymorphism in the the pathogenesis of inflammatory diseases, and it is located CYP1A2 gene that is associated with altered clearance of on chromosome 17q21–22, a region linked to asthma in theophylline. Specifically, the A allele of a G/A SNP at some genomewide screens.71 It is also located in a genomic position À2964 was associated with decreased clearance region that is orthologous with a region of the mouse (increased serum levels) in a group of 75 Japanese asth- genome on chromosome 11 that has been linked to AHR.72 matics. Although these findings need to be replicated, they Corticotropin releasing factor receptor type 1 plays a major are biologically plausible because CYP1A2 is known to role in the stress response via its regulation of endogenous degrade theophylline to 1,3-dimethyl uric acid before GC and catecholamine production. Our studies of CRH, the excretion in the urine and poor metabolizers of theophyl- major ligand of CRHR1, indicate that in the absence of CRH line are known to exist. If true, this association is the first endogenous GC production decreases and allergen-induced example of an asthma pharmacogenetic response that is airway inflammation and lung mechanical dysfunction caused by a polymorphism in the pharmacokinetic pathway increase dramatically.73 By extrapolation, genetic or ac- of a drug. quired defects in CRHR1 could cause a similar increase in allergen-induced airway inflammation because of insufficient GC production. We postulate that this would result in Corticotropin releasing factor receptor type 1 polymorphisms and an increased response to exogenous GC, and that this is the response to inhaled glucocorticoids mechanism responsible for the association between CRHR1 Glucocorticoids are the most potent and commonly pre- polymorphisms and asthmatic GC response. In this regard, scribed anti-inflammatory agents for the treatment of our laboratories have recently identified polymorphisms in asthma.57–59 Glucocorticoids bind to the glucocorticoid the CRHR1 that are associated with therapeutic response to receptor (GR) and function as transcription factors that GCs.74 Corticotropin releasing factor receptor type 1 poly- may activate or repress the expression of many genes. In morphisms are associated with GC response in asthmatics, general, GCs activate anti-inflammatory genes and repress we genotyped 12 of these SNPs in adult asthmatics (Adult the expression of proinflammatory genes.60 Treatment of Study), 311 childhood asthmatics (CAMP), and 336 adult asthmatics with GCs attenuates airway inflammation and asthmatics (Asthma Clinical Research Network (ACRN)). hyperresponsiveness by inhibiting inflammatory cell re- The primary outcome measure of the association analyses cruitment and the production of proinflammatory cyto- was our continuous FEV phenotype, %DFEV over time in kines. The majority of asthmatics respond to inhaled GC 1 1 response to inhaled corticosteroids, from baseline to 8 weeks therapy with a reduction in the numbers of activated for the Adult Study and CAMP, and baseline to 6 weeks in eosinophils, mast cells, and T lymphocytes and a decrease ACRN. We found individual SNPs or haplotypes that were in the concentration of cytokines/chemokines in the air- associated with a significant enhanced response to GC in all ways (Table 1) lists some of the many effects of GCs on the three populations. Specifically, SNP rs242941 (minor allele airways of asthmatics. frequency B30%) was associated with positive treatment Although inhaled corticosteroids are very effective in response in both the Adult Study and CAMP (P ¼ 0.025 and those asthmatics who respond, many fail to respond (Figure 0.006, respectively). The mean %DFEV for homozygotes for 3b) and the potential for side effects in those receiving 1 the minor allele vs homozygotes for the major allele was higher doses can be significant.61 These side effects can 13.2873.11 vs 5.4971.40 in the Adult Study and include growth retardation in children,62 adrenal suppres- 17.8076.77 vs 7.5771.50 for CAMP. Haplotypic analysis sion63,64 bone demineralization,65 skin changes,66 and revealed one common haplotype (frequency 27%), termed cataract formation.67 The precise reason for GC resistance GAT (rs1876828, rs242939, and rs242941), associated with a significantly enhanced response to inhaled corticosteroids Table 1 Effects of glucocorticoids in asthma in both the Adult Study and CAMP (P ¼ 0.02 and 0.01, respectively). The estimated short-term improvement in m Eosinophil apoptosis FEV1 for those subjects imputed to have the homozygous m Epithelial apoptosis k Mucus GAT/GAT haplotype was over twice that for those homo- k Goblet cell hyperplasia zygous for non-GAT haplotypes in the Adult Study k Inflammatory cell infiltrate (13.7373.80 vs 5.5471.29%), and nearly three times that k AHR in CAMP (21.8378.07 vs 7.3571.41%). These findings k IgE in vivo represent the first genetic association involving GC ther- m IgE in vitro apeutic response that has been successfully replicated. It is k Airway remodeling? unknown if any of these variants is functional.74)A k Airway smooth muscle growth systematic analysis of the sequence variation and haplotype k Subepithelial fibrosis structure of the CRHR1 gene will be required to identify

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these variants along with mouse models of airway responsiveness.

Interactions between the glucocorticoid and the b2-agonist pathways Dynamic interactions exist between endogenous GC and catecholamines in vivo. In animals, adrenalectomy results in a generalized loss of responsiveness to catecholamines75,76 Molecular interactions between the pathways have also gained increasing recognition77–79 For instance, both classes of drugs can activate C/EBP-a and the GR.80,81 Moreover, both classes of medications combined, even at lower doses, can synchronously activate both transcription factors, resulting in an enhanced antiproliferative effect.75,79–82 b-2-agonists (particularly long-acting preparations) may Figure 5 Interaction of glucocorticoids, adenylate cyclase 9 (AC9), and affect GR nuclear localization through modulation of GR b-agonist response in a transfected cell model. N ¼ 3 experiments. phosphorylation and, further may prime GR functions within the nucleus by modifying GR or GR-associated protein phosporylation.83 Glucocorticoids may in turn regulate b2AR function by increasing expression and sone for 12 h, and cAMP accumulation ascertained at inhibiting b2AR downregulation, thereby directly modulat- baseline and in response to 10 min exposure to 10 mM ing contraction of airway smooth muscle.81,84 Pharmacolo- isoproterenol (Figure 5). We found that dexamethasone gical interactions between GC preparations and b2-agonists had little effect on b2AR signaling in the absence of AC9 have been observed, and these interactions are reviewed expression. However, isoproterenol stimulated cAMP levels elsewhere.79,85 In clinical trials, the concomitant adminis- were significantly greater in AC9 expressing cells treated tration of inhaled corticosteroids and long-acting b2-ago- with dexamethasone compared to untreated. Thus, these nists have shown improvements in control of asthma results indicate an interaction between corticosteroids and 86,87 symptoms. Thus, investigating whether interactions the function of AC9 and b2AR signaling, which is consistent between genetic variants in these pathways are able to with the clinical findings.88 Owingto this potential interac- better predict asthma treatment response is an important tion, we now screen all of the genes in both the b-agonist goal of this proposal. Practically, all except patients with and the steroid pathways for potential associations with mild asthma are on both medications (i.e. an inhaled both b-agonist related bronchodilator response and lung corticosteroid and at least a short acting b-agonist), includ- function changes related to inhaled corticosteroids. ing most of our trial subjects. Moreover, given the extent of the crosstalk between these therapeutic pathways, it is distinctly possible that candidate genes for the GC pathway Overview of pharmacogenetics of asthma treatment may be significantly associated with b2-agonist phenotypic study design outcomes, and vice versa. Indeed, we have found just such an effect with adenylate cyclase 9 (AC9). We genotyped a Introduction and rationale nonsynonymous AC9 SNP in CAMP and the minor allele Although we have had reasonable success in our candidate frequency for this SNP was 28%. We noted a significantly gene approach to the identification of asthma pharmacoge- enhanced mean bronchodilator response (9.2 vs 7.9%, netic genes to date, we have recently altered our approach to overall effects by ANOVA, P ¼ 0.006; interaction of response that of a more comprehensive screening approach. The by genotype, P ¼ 0.0015) in association with the presence of advantages of such an approach are outlined in the any variant allele, but only in those children randomized to paragraphs that follow and in (Table 2). These changes were the inhaled corticosteroid treatment group. The mechanism predicated by the increasing availability of high-throughput of the apparent interaction of AC9 genotype, long-term b2- genotyping and novel statistical methodology. Our global agonist response, and corticosteroids is not clear. To begin to approach is outlined in (Figures 6, 7 and 8). Utilizing explore this, we sought a model cell system whereby b2AR is biologic, pathway, and expression array analysis to identify endogenously expressed but AC9 is not, and overnight candidate genes, we are selecting variants in these genes to incubation with corticosteroids induces classic changes in genotype and test for association with therapeutic response multiple gene expression. The cell line we found that fits in the CAMP population. After high-throughput genotyp- these criteria is the A431 line, derived from a lung ing, the screening algorithm developed by Lange et al.89,90 adenocarcinoma. Using these as the host cells, the wild- will be applied using the available pharmacogenetic phenotype AC9 was stably transfected using the AC9 cDNA types and genetic data in the CAMP population. The without its endogenous promoter, and a line expressing screening algorithm will identify the most promising AC9 at levels comparable to endogenous AC5 was chosen for hypotheses to explore with formal hypothesis testing with- study. Cells were exposed to vehicle or 10 mg/ml dexametha- out affecting the significance level of the subsequent

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Table 2 Advantages of our current asthma pharmacogenetic study design

Technique Advantage How implemented

Screening algorithm Helps address multiple comparisons issues Screen using noninformative families before performing FBAT analysis Family-based association test Population stratification is not an issue FBAT testing methodology using probands and parents Principal components analysis Increases statistical power Each longitudinal outcome measure contributes to the observations. Replication population testing Helps to assure generalizability and address Two population-based replication studies for each false positives gene identified via screening, before confirmation Functional studies Extends association studies into the Use of gene expression, in vitro, and animal models functional domain to test specific candidate genes and variants

Candidate Genes Resequenced Replicated Candidates from Pathways, Expression, from Specific Aim 1 Literature, & Biology

Functional Negative, Screen and and Discard Test in CAMP Molecular Biological Evaluation

Negative, Replicate in Adult Studies Discard

Replicated Candidates Gene In vitro Animal Expression Models Models Resequence Functional Markers and Mechanisms of Action Specific Aim 3 Specific Aim 2 Specific Aim 3

Figure 6 Flow chart for our first specific aim. We screen candidate Figure 8 Flow chart for our second specific aim. Our replicated genes single-nucleotide polymorphisms (SNPs) within candidate genes using a from specific aim 1 are explored for functional basis of genetic effects, family-based screening approach, which helps to address issues of using gene expression studies, cellular models, and animal models. multiple comparisons and allows ranking based upon power for replication of a pharmacogenetic effect. After family-based screening, 90 we genotype genes with the most power for replication in our testing. The statistical techniques to test the hypotheses population-based replication cohorts. Replicated genes are then generated in the screening stage will include family-based resequenced and evaluated functionally. association methods as well as population-based regression strategies at the single SNP and haplotype levels. These techniques will be applied to the CAMP population and CAMP genes that indicate association with pharmacogenetic replication 1 replication 1 phenotypes will be sequentially explored in a second and third clinical trial population Significant association results in CAMP that are replicated in the other trial populations β ADULT STUDY: Steroid ADULT STUDY: 2Agonist will be retested utilizing the method of genomic control to exclude the potential effects of population stratification. replication 2 replication 2 Genes with confirmed replications are completely resequenced and functionally evaluated to characterize the etiologic basis underlying the association. Upon completion ACRNβ Agonist Trials ACRN Steroid Trials 2 of the association testing and functional work, the findings Figure 7 Flow of specific aim 1 emphasizing the available study are being utilized to develop a predictive model of treatment populations for the pharmacogenetic investigation of the corticosteroid response, which will be tested on multiple population and b-agonist pathways in asthma. samples to assess its predictive capabilities.

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Pharmacogenetics of asthma treatment asthma response of inhaled budesonide vs nedocromil vs placebo (prn b populations agonist). Trial design and methodology have been The PHAT research group utilizes phenotypic information previously published.91 The mean duration of follow-up and DNA from multiple clinical trial populations. Although was 4 years.92 Childhood Asthma Management Program varying in demographic characteristics, and duration of enrolled 1041 children ages 5–12 years with mild-to- follow-up, all of the subjects included in our analyses met moderate chronic asthma over 23 months. 311 were on on strict definitions for asthma diagnosis and were enrolled in b22-agonist (albuterol). Entry criteria included asthma protocol-defined clinical trials. All patients or their legal symptoms and/or medication use for 6 or more months in guardians provided both consent for the study protocol and the previous year. Among the exclusion criteria were: for ancillary genetic testing. Our primary population prednisone bursts more than five times or more than one consists of a 4-year clinical trial of childhood asthmatics hospitalization for asthma in the preceding year, prior enrolled in CAMP. The two inhaled corticosteroid popula- intubation for asthma, FEV1 o65% of normal, and tions currently available for replication include an 8-week other active pulmonary disease. All study participants clinical trial of adult asthmatics on flunisolide (Adult Steroid had asthmatic range airway responsiveness with a Study), and the 6-week run-in period of a group of adult provocative concentration of methacholine causing a 20% asthmatics on triamcinolone enrolled through the Asthma reduction in FEV1 (PC20) p12.5 mg/ml. Follow-up visits Clinical Reserch Network of NHLBI (ACRN). There are also occurred at 2 months then every 4 months and spirometry two populations available for b2-agonist replication ana- was performed twice yearly. We have chosen to use the lyses, including a study comparing the efficacy of regular vs CAMP study as our primary population for the following levalbuterol sponsored by a pharmaceutical company reasons: the available phenotypic data are extremely referred to as the adult b2-agonist Study, and a compilation rich and well-documented, the sample size is large enough of studies assessing short-term b2-agonist response as part of to explore both GC and b2-agonist pathways, and the ACRN. Basic information from these trials is shown in the availability of familial genetic data enables the (Table 3). application of powerful family-based association screening and testing strategies The family-based association screening Primary analysis population. Childhood Asthma Manage- and testing strategies will generate and test for genetic ment Program is a multicenter, longitudinal, randomized, associations with pharmacogenetic phenotypes without double-blinded clinical trial testing the safety and efficacy incurring substantial penalty for multiple comparisons or

Table 3 Asthma trials summary

Trial Background RX RX Albuterol response Archive DNA+

ACRN BAGS None b-agonist 173 173 SOCS/SLIC Inhaled steroids ICStaper/LABA 168 327 DICE None ICS 143 143 MICE None ICS 21 21 BARGE None b-agonist 72 270 IMPACT None ICSorLTA or PLA 233 343 SMOG None ICS/LTA 89 125 SLIMSIT Inhaled steroids/LTA LABA/ICS or LTA 153 191 PRICE None ICS 23 23 1616

CAMP None ICS 1041 981 Adult study (S) Inhaled steroids ICS 470 470 Adult study (b) None b-Agonist 631 631 2082 DNA totals ACRN Other Total

Total on inhaled steroids 654 781 1435 Phenotypes available: lung Total on LTA 208 — function, symptom score, and Total on b2-agonists 754 1030 1784 airway responsiveness PC20, IgE (alone)

Abbreviations: ACRN, Asthma Clinical Reserch Network; CAMP, Childhood Asthma Management Program; ICS, inhaled corticosteroid; LTA, leukotriene antagonist;

LABA, long acting b2-agonist; SOCS/SLIC, salmeterol or corticosteroids/salmeterol7inhaled corticosteroids; + ¼ archive at Channing Lab, Brigham and Women’s Hospital.

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producing false positive associations due to population populations, including a primary pediatric test population stratification. and separate replicate adult corticosteroid and b2-agonist populations, to evaluate our hypotheses. Populations for follow-up corticosteroid analysis. The Adult Steroid Study was a multicenter 8 week clinical trial used to compare the effect of once daily high-dose inhaled Analysis phenotypes flunisolide vs ‘standard inhaled corticosteroid therapy’. We plan to assess three phenotypes: one continuous and Patients were randomized in a 4:1 flunisolide vs other two categorical (Table 4). We have explored values other therapy ratio and the study subjects ranged in age from 16 to than 20% (conventional dosage of inhaled corticosteroids). 75 years, with a slight (58%) female preponderance. We have also replicated results using these phenotypes. Inclusion criteria included a history of asthma and documentation within the previous year of at least 12% Association analysis strategy and statistical methodology improvement in FEV1 with administration of the short A major criticism of genetic association studies has been the acting b2-agonist, albuterol. Additionally, all subjects were failure to provide demonstrable replication of findings.95–97 required to have been using inhaled steroids at specified Failure to replicate findings in genetic association studies doses. Exclusion criteria included a history of significant has been attributed to variations in study design, including pulmonary disease other than asthma, smoking in excess of differing study populations and definitions of pheno- 10 pack-years, and recent upper respiratory infection or types.96,97 Conversely, the replication of associations in asthma exacerbations. Subjects were followed by phone other, distinct, populations with differing clinical character- contact on a weekly basis, and had follow-up spirometry at 4 istics, such as each of our clinical trial populations, supports and 8 weeks. In total, 470 moderate to severe adult both the validity and generalizability of these findings. asthmatics (mean FEV1 72% at enrollment) had an average Using recent advances in statistical genetics, we have improvement in prebronchodilator FEV1 of 7.0% at the end implemented a three-stage statistical analysis strategy to of the trial period. As the change in the FEV1 in both identify genetic variants in the candidate genes that treatment groups was the same (P ¼ 0.30), we will combine demonstrate robust and replicated associations with the the treatment groups for our analyses. treatment response phenotypes. The first and second stage Two of the completed trials conducted by the ACRN, the consist of a screen and family-based association test (FBAT) 7 salmeterol or corticosteroids (SOCS) and salmeterol in- strategy using data from the CAMP population. Genes that haled corticosteroids (SLIC) trials, had a common initial 6 are identified as positively associated with treatment week run-in period utilizing four puffs twice daily of response phenotypes in the CAMP population will be triamcinolone before separate randomization to one of genotyped and tested in additional clinical trial populations these two trials and have already been utilized (Table 3). in the third stage. This multistage strategy optimizes our Details regarding the entry criteria, run-in period, and chances of finding robust and replicated genetic associations randomization have been published with the primary trial with the treatment response phenotypes and minimizes the 93,94 results. These individuals had an average improvement probability that the significant associations are due to in their FEV1 of 6.8% during the run-in period. The other problems arising from multiple testing or population ACRN steroid trial subjects in Table 3 are currently being stratification. There are many pitfalls to robust genetic plated for use in genotyping. association studies including how to pick the candidate genes and their variants, how to reduce genotyping error, Populations for follow-up b2-agonist analyses. Initial how to avoid population stratification, and multiple replication for any positive b2-agonist association in CAMP comparisons, how to have adequate power. A comprehen- will be performed in an adult study of b2-agonist. Subjects sive assessment of all of these issues is beyond the scope of were over the age of 18 years and had the full range of this review. However, we believe our approach to association asthma severity. Subjects were randomized through three is sound and will achieve the desired goal to ultimately different treatment arms and a placebo PRN albuterol arm. achieve robust replicable and generalizable results. The three treatment arms included 0.63 mg of levalbuterol, 1.25 mg of levalbuterol, and 2.25 mg of racemic albuterol and all treatments were delivered by nebulizer three times Table 4 Phenotypes for glucocorticoids and b2-agonist daily. Subjects had pulmonary function determined during response the run-in period at baseline at randomization and at 2, 4, 6, and 8 weeks of treatment. Dose–effect curves to an acute GC b2-Agonist dose of nebulized b2-agonist were determined at each time point and pre- and postbronchodilator FEV1 and AM peak Continuous phenotype (CP) + + flows are available at each time point. Our primary analytic Categorical phenotype nonresponder (CatPNR) + + end point is the bronchodilator response at randomization. Categorical phenotype responder (CatPR) + + However, subsequent analyses using repeated measures Definitions: CP FEV% ¼ (Post-FEV1 – Pre-FEV1/Pre-FEV1) Â 100; CatPNR ¼ bottom could add to the richness of the analyses. We have 20% of CP distribution vs others; CatPR ¼ top 20% of CP distribution vs others. multiple, richly characterized, asthma clinical trial Abbreviations: GC, glucocorticoids; FEV1, forced vital capacity in 1 s.

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Robust pharmacogenetic associations will be explored at Mutation location the molecular, cellular, and integrative level to establish functional variants and pharmacogenetic mechanisms. Promoter Protein coding Splicing site Intron/Other Functional methods will involve bioinformatics tools, mRNA profiling, and cellular and animal models of asthma AA change ? RNA stability Alternative Reporter assays therapeutic responsiveness (Figure 8). Perhaps no issue in transcript genetics is as complex as getting the proper functional No Altered Yes No variants out of a gene that has replicated in several transcription ? YeYes No association studies. Linkage disequilibrium can make it very Ligand binding Stop Stop difficult to definitively pinpoint a specific variant that Stop G protein coupling Analysis of DNA-protein might be causal. The mouse ova model is commonly used interactions (EMSA, Footprint) to assess physiology in the whole animal. Less frequently Receptor stability used is an animal model for asthma drug treatment Desensitzation Cotransfection response, that is, an asthma mouse model that can be assessed for b-agonist or steroid treatment response. Simi- Figure 9 Algorithm for establishing the effect of corticotropin releasing factor receptor (CRHR) mutations – mutation location will be the major larly, mouse models with knock in/knock outs of the gene of determinant of our approach for designing assays to test functional interest are available and frequently used for asthma. More significance. challenging is to create mice with alternative genetic constructs that contain the specific genetic variant or haplotype of interest but these are also achievable. Sequence variants in the gene promoter region. Given the high sequence variability found in 50 flanking sequences of genes, In vitro assays to test the impact of select variants on gene function we anticipate that the CRHR1 and Corticotropin releasing or expression factor receptor type 2 (CRHR2) promoters will contain For variants that are associated with asthma treatment several common polymorphisms. We have previously response and located in conserved regions or regions of described polymorphisms in promoter regions with potential functional interest, we propose to establish their clinically significant effects on gene expression. For biological significance by demonstrating, on the basis of ALOX5 and TGFB1, we have defined transcriptional experimental data, how a given variant could affect protein mechanisms that may be responsible for genetic function or production and thus how an individual associations and plan to use a similar approach.98,99 We possessing such a sequence variant could have altered have had extensive experience and success with these assays; asthma therapeutic response. The approach for a given to save space, we have not included experimental protocols, mutation will depend largely on the location of the but refer the reviewer to our recent publications on mutation (i.e., protein coding, splicing region, promoter, polymorphisms in promoter regions with clinically 50 or 30 untranslated region, or intron). The first step is to significant effects on gene expression. place the mutation in the context of what is already known about the CRHR1 and CRHR2 genes and their proteins. This Sequence variants in other regions. Sequence variants in is possible because the general structure and function of intron, intron–exon junctions, or 50-, 30-untranslated both genes have been well defined. According to the regions may affect RNA slicing or stability and result in location of the mutation, it should be possible to make an alternative or truncated protein or altered steady-state levels educated guess as to how the mutation alters receptor of RNA. These events can be detected using RNA stability production (i.e., promoter, splicing region, or untranslated assays or real-time RT-PCR with primers that flank the region mutation) or function (ligand binding, Gs coupling). intron.100 Differences in the intensity or length of amplified A general algorithm describing our approach is shown in bands may indicate abnormalities caused by the (Figure 9). There are four major types of sequence variants to polymorphism. These variants may be particularly consider in CRHR1 and CRHR2: protein-coding variants important in asthma pharmacogenetics and in complex affecting protein function; splicing-site variants affecting trait genetics generally. This is because regulatory variation RNA splicing; intron/UTR/Other variants affecting RNA tends to be common, evolutionarily conserved and hence stability, splicing, or processing; and promoter variants easily detectable with moderate sample sizes and with affecting transcription. hapmap identified SNP’s.

Animal models of corticosteroid response – the CRH pathway Sequence variants in the protein-encoding region. Non- The CRH pathway consists of a family of ligands and synonymous protein coding variants are the most obvious receptors and is more complex than was first appreciated. and fairly straightforward to test. In addition to their effects Corticotropin releasing factor receptor type 1 and the on protein synthesis and structure, variants modifying homologous receptor, CRHR2, are the two GPCRs that protein-encoding sequence may also alter ligand binding, mediate the biological actions of the CRH pathway. The Gs coupling, and receptor processing. receptors differ significantly in their tissue distributions,

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ligand affinities, and physiological actions.101 The binding with CRHR1, these splice variants may affect ligand binding, of agonist ligands to these receptors causes a conformational and their relative expression depends on tissue type. change that transduces signals through activated hetero- Corticotropin releasing factor receptor type 1 is found trimeric Gs. Corticotropin releasing factor receptor type 1 mainly in the anterior pituitary and other regions of the and CRHR2 signal mainly through Gs, increasing adenylyl brain where it regulates the hypothalamus–pituitary–adre- cyclase activity and cAMP levels; however, these receptors nal (HPA) axis and endogenous GC. Corticotropin releasing may also couple to Go, Gq, and Gi and signal through other factor receptor type 1 is a GPCR that is the major receptor for pathways that may involve phospholipase C, protein kinase corticotropin-releasing factor (CRH). A diagram of the first B/Akt, MAPKs, NOS/guanylyl cyclase, and intracellular portion of the GC pathway is shown in (Figure 10), the full Ca2 þ .102 The two CRHR receptors are encoded by different pathway may be viewed on the PharmGKB website (http:// genes located on different chromosomes. They are 70% www.pharmgkb.org/index.jsp). Corticotropin-releasing fac- homologous at the amino-acid level and have the char- tor is best known as the hypothalamic regulator of pituitary acteristic structure of GPCRs, which includes a membrane- adrenocorticotropin (ACTH) secretion, which stimulates spanning region consisting of seven hydrophobic p-helices, adrenal GC synthesis. Corticotropin-releasing factor is a an external N-terminal region for ligand binding, and an 41-amino-acid peptide produced by the paraventricular internal C-terminal region for signaling. Corticotropin nuclei of the hypothalamus and secreted into the hypophy- releasing factor receptor type 1 has seven known mRNA sial portal system in response to inflammatory, physiologi- splice variants and their relative expression varies with tissue cal, and behavioral forms of stress.103 Corticotropin- type. Some of these splice variants have significant effects releasing factor binds CRHR1 on corticotrophs of the on ligand binding and signaling, and it is possible that some anterior pituitary and stimulates the release of ACTH, which of the polymorphisms associated with asthma may affect is an essential regulator of GC and catecholamine secretion CRHR1 function by altering RNA splicing. Corticotropin by the adrenal glands. Mice deficient in CRH or CRHR1 releasing factor receptor type 2 has at least 3 mRNA splice show adrenal gland atrophy and an inability to produce variants that modify exon 1 and the N-terminal region. As increased GCs in response to stress.104 Glucocorticoids

Figure 10 The CRF pathway. Within the central nervous system, endogenous corticosteroid production begins with the release of corticotropin releasing factor from the hypothalamus. (From PharmGKB – http://www.pharmgkb.org/index.jsp.)

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production, as modulated by CRH and the HPA axis, may Receptors in Asthma. We thank all families for their enthusiastic represent one mechanism by which excessive activation of participation in the Camp Genetics Ancillary Study, supported by the immune system is restrained.105 The mechanisms by the National Heart, Lung, and Blood Institute, NO1-HR-16049. which CRHR1 variants affect GC response are unknown. The OVA-sensitization and -challenge mouse model will play a References prominent role in the experimental tasks of our ongoing research efforts. It is fortunate that the genes we propose to 1 Weinshilboum R. Inheritance and drug response. N Engl J Med 2003; study have been successfully targeted and that KO mice are 348: 529–537. 2 Evans WE, McLeod HL. Pharmacogenomics – drug disposition, drug available for our studies. We initially hypothesize that CRH- targets, and side effects. 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