The Pharmacogenetics of Asthma: a Candidate Gene Approach

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to more effectively treat this syndrome The pharmacogenetics of asthma: a would be of great benefit. While much is known about the candidate gene approach pathobiology of asthma and the inflammatory mediators and effector ES Silverman1, SB Liggett2, EW Gelfand3, LJ Rosenwasser3, cells that produce the clinical pheno- RM Baron1, S Bolk4, ST Weiss1 and JM Drazen1 type, the genetic and environmental causes of the disease have remained 5 1Department of Medicine, Channing Laboratory, Brigham and Women’s Hospital and obscure (reviewed in Kay, and 6 Harvard Medical School, Boston, MA; 2Department of Medicine, University of Cincinnati Holgate ). Although a small percent- College of Medicine, Cincinnati, OH; 3Departments of Medicine and Pediatrics, National age of asthma cases may result from a Jewish Medical and Research Center, University of Colorado Health Sciences Center, single environmental or genetic cause, Denver, CO; 4Whitehead Institute for Genome Research, Massachusetts Institute of most cases result from the interaction Technology, Boston, MA, USA of multiple genetic and environmental factors. Each of these factors acting alone would be expected to contribute INTRODUCTION describe a candidate gene approach a small amount to the development of Asthma is a common respiratory syn- that is being used to uncover asthma. However, when combined drome characterized by partially additional polymorphisms with phar- with other factors, the ultimate reversible airflow obstruction, airway macogenetic potential. phenotype or a particular character- hyperresponsiveness, and airway istic of an individual’s phenotype, inflammation. The syndrome is caused such as atopy, severity, or response to by a complex interaction between gen- ASTHMA AND ITS TREATMENT a drug is established. Moreover, since etic and environmental factors that Asthma is a clinical syndrome with asthma is a syndrome reflecting many results in a spectrum of biologic and three defining characteristics: (1) pathophysiologic pathways, it is likely clinical features. Among the spectrum recurrent episodes of airway obstruc- that different sets of genes and of features is a highly variable response tion that resolve either spontaneously environmental factors lead to asthma to asthma therapeutics. There are cur- or as a result of therapy; (2) increased or a particular phenotype in different rently three major classes of asthma airway hyperresponsiveness, which is patients or populations. Thus, therapeutics available: (1) beta2-agon- an increased bronchoconstrictor although several studies based on link- ists (␤-agonist); (2) glucocorticoids response to stimuli that would have age analysis have defined genomic (GC); and (3) inhibitors of the little or no effect in nonasthmatic sub- regions of interest, it should come as cysteinyl-leukotriene pathway (cLI). jects; and (3) chronic airway inflam- no surprise that a single ‘asthma gene’ Asthmatic patients vary greatly in mation, often with abundant eosino- 7,8 their response to all three classes of phils.1–3 Asthma severity is usually has not been identified. Neverthe- drugs and it is estimated that up to 60– measured by the presence or absence less, these genome-wide searches for 80% of this variability may have a of symptoms, the frequency of exacer- linkage have consistently identified pharmacogenetic basis. Over the past bations, and the degree of airflow important chromosomal regions that 10 years, several polymorphic loci resistance as measured by the forced have highlighted known genes as have been discovered in genes that expiratory volume in one second being good ‘asthma candidates’ with the potential to contribute to the play a role in the action of these drugs. (FEV1). As a clinical problem, asthma Data suggest that these polymor- is both prevalent and associated with asthma phenotype if a particular allele phisms directly or indirectly alter an substantial morbidity. There are is present in an individual. asthmatic’s response to therapy and approximately 17 million asthmatic At the cellular level, eosinophils, can be used to predict the response to patients in the United States (6% of mast cells, alveolar macrophages, lym- certain asthma drugs, thereby maxim- the adult population), and asthma is phocytes and neutrophils recruited to izing efficacy and avoiding adverse the number one cause of hospitaliza- the airways of asthmatics produce a effects. It is likely that many, as yet tions and emergency room visits for variety of inflammatory mediators, undiscovered, polymorphisms exist children. The health care costs such as histamine, kinins, neuropep- given the large number of gene pro- approximate 8 billion dollars per year tides, and leukotrienes, which lead to ducts involved in the pharmacodyn- of which 35–40% are due to medi- bronchial smooth muscle constriction amic and pharmacokinetic pathways cation costs. Moreover, the problem and obstruction of airflow, and the of all three classes of asthma drugs. In has been getting worse with a world- perpetuation of airway inflam- this review we discuss the established wide increase in prevalence of 30% mation.9,10 An understanding of the asthma pharmacogenetic loci and over the two past decades.4 Strategies inflammatory processes and the mol- Asthma pharmacogenetics ES Silverman et al 28

ecular pathways of these mediators has led to the development and wide- spread use of several pharmacologic agents that mitigate airway inflammation and bronchoconstriction. Three major classes of asthma therapeutics are currently in use: (1) ␤-agonist (eg albuterol, salmeterol, fenoterol); (2) GC (eg, beclomethasone, triam- cinolone, prednisone); and (3) cLI (eg, montelukast, zafirlukast, zileuton). In the early 1990s, a classification scheme was developed for defining asthma severity and choosing appropriate regi- mens from among these classes of drugs.2 Reliever drugs (inhaled, short acting ␤-agonist) are usually adequate for mild intermittent asthma and pro- vide rapid ‘rescue’ for acute airway obstruction when it infrequently occurs. In contrast, Controller drugs (inhaled GC, long acting ␤-agonist, Ϯ Figure 1 Mean percentage change from baseline ( SE) in FEV1 over the 12-week treat- and cLI) are necessary for mild, moder- ment period. Black squares represent patients receiving inhaled beclomethasone, 200 ␮g ate or severe persistent asthma in twice daily; white triangles represent patients receiving montelukast, 10 mg once daily; addition to reliever drugs, because and black circles represent patients receiving placebo. Beclomethasone increased mean they modify the airway environment, FEV1 approximately 13.1% at 12 weeks, whereas montelukast increased mean FEV1 and lessen the frequency of acute air- approximately 7.4%. Modified from Malmstrom et al.11 way narrowing in the more sympto- matic patients. When asthmatics are treated according to these established the montelukast group11 (Figure 1). lations based on the Repeatability (r) guidelines, the vast majority of However, when these same data are of the treatment response for all three patients can be effectively managed viewed from a different perspective, classes of asthma drugs, defined as the with minimal morbidity, whether it is focusing on the number of individuals fraction of the total population vari- directly related to asthma or indirectly as a function of percent change in ance which results from among indi- caused by drug toxicity. However, FEV1 from baseline, it is clear that vidual differences, shows values for r there is an important caveat that must many patients had little response. In between 60–80% indicating the upper be considered with this statement— fact, several patients appear to have limit of the genetic component and not all asthmatics are the same and had an adverse response to treatment suggests that a substantial fraction of there is tremendous heterogeneity in with a decline in FEV1 at 12 weeks the variance of the treatment response therapeutic response to each asthma compared with baseline (Figure 2). The could be genetic in nature.12 The ulti- drug. increase in mean FEV1 for all patients mate goal of asthma pharmacogenet- is eschewed by a dramatic increase in a ics is to identify this genetic infor- HETEROGENEITY OF few individuals. Furthermore, a careful mation and use it to tailor an THERAPEUTIC RESPONSE analysis of these studies indicates that individual’s therapeutic regimen in Although all three classes of asthma individuals responsive to one class of order to maximize efficacy and minim- drugs are effective when examined in asthma drug may not necessarily be ize side effects. large clinical drug trials, many studies the individuals responsive to a differ- indicate that there is wide latitude ent class of asthma drug. These types Asthma Pharmacogenetics in inter-individual response. For of data illustrating variable drug effi- Obviously, sequence variants that lead example, in a study by Malmstrom et cacy are not limited to asthma drug to the identification of novel meta- al comparing the efficacy of the leuko- trials but can be found for almost all bolic pathways or novel mechanisms triene antagonist montelukast (10 mg classes of drugs. The degree to which for inflammation would have great a day) with the inhaled steroid beclo- this inter-individual variability is gen- potential for the development of new methasone (200 ␮g twice a day), it is etic remains uncertain and probably drugs. However, such fortuitous dis- clear that both drugs are effective over differs with each class of drug. More- coveries are unlikely to occur very a 12-week course of treatment with a over, inter-individual variability may often, and will not represent the mean increase in FEV1 of 13.1% for the depend on the particular preparation majority of pharmacogenetic para- beclomethasone group and 7.4% for within a class of asthma drug. Calcu- digms. There are three general para-

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stimulates adenylyl cyclase (AC) to cat- alyze the conversion of ATP to cAMP. The ␤␥ subunits, particularly from Gi, also carry out signal transduction in some cells, including alteration of the activities of AC and phospholipase C (PLC) and activation of the mitogen- activated protein kinase (MAP-kinase). ␤ The bronchodilating action of 2AR is

due primarily to G␣s activation of adenylyl cyclase in bronchial smooth muscle, with resultant increases in intracellular cAMP.14 cAMP activates protein kinase A (PKA), which acts to phosphorylate myosin light-chain kinase, cell surface potassium channels, a sodium potassium ATPase, phospho- lamban, and one or more pumps that lead the sarcoplasmic reticulum to calcium uptake and inhibition of inositol phosphates. The net effect is a decrease in intracellular calcium and phosphorylation of contractile proteins, which leads to smooth muscle relax- 15 ␤ ation. 2AR function is dampened by ␤ Figure 2 Distribution of treatment responses for FEV . Response distributions (% of its phosphorylation by the AR kinase 1 ␤ patients) are shown as histograms for predefined intervals of % change in FEV1. Striped and the binding of arrestin. PKA ␤ bars represent patients treated with montelukast and black bars represent patients treated phosphorylation of 2AR also with beclomethasone. Note wide variation in therapeutic response among patients. uncouples the receptor and promoters promote coupling to Gi. ␤ digms by which sequence variants in an unintended consequence of a 2AR, as well as other cell surface may lead to heterogeneity in thera- drug outside of its therapeutic target G protein-coupled receptors, can peutic response and impact asthma (idiosyncratic). A third possible para- natively toggle in the absence of agon- treatment (Table 1). The most obvious digm consists of sequence variants in ist between the activated (RA, also and best-described paradigm in phar- the drug target or the drug target path- called R*) and inactivated (RI, also 16 macogenetics is sequence variants that way that leads to altered drug efficacy called R) state. In the absence of alter the metabolism, distribution or (pharmacodynamic) or a difference in agonist, the majority of receptors are excretion of a drug resulting in the phenotypic expression of asthma. in the RI state. In the presence of agon- enhanced or inhibited drug effects With regard to asthma pharmaco- ist, RA is stabilized and accumulates (pharmacokinetic). A second paradigm genetics, only examples of the pharm- such that this state predominates. involves sequence variants that result acodynamic paradigm have been Inverse agonists stabilize the RI state conclusively demonstrated. These thereby lowering basal AC activity. examples will be discussed for each Partial agonists stabilize the RA confor- mation but not as efficiently as full Table 1 Pharmacogenetic paradigms category of asthma drug after a brief agonists, such that maximal partial with implications for asthma treat- review of the relevant pharmacodyn- ment amic pathway. agonist-stimulated AC activity is less than that found with full agonists. ␤ (1) Genetic variants associated with altered PHARMACOGENETICS OF - Neutral antagonists stabilize both RA uptake, distribution, or metabolism of AGONISTS IN ASTHMA and RI. Agonist binding also initiates a drug (Pharmacokinetic). ␤-agonists, such as albuterol, act via series of events leading to a loss of ␤ ␤ binding to 2-adrenergic receptors 2AR function over time. This waning (2) Genetic variants resulting in an ␤ ( 2AR), a cell surface G protein- of the response despite continuous unintended action of a drug outside of ␤ coupled receptor (Figure 3). 2AR stimulation is termed desensitiz- its therapeutic indication (Idiosyncratic). 17 ␤ primarily couple to the stimulatory ation. 2AR and many other G pro- (3) Genetic variation in the drug target guanine nucleotide-binding protein, tein-coupled receptors can undergo 13 leading to altered drug efficacy Gs. Gs is a heterotrimeric G protein, agonist-specific desensitization (Pharmacodynamic). which upon activation by receptor dis- (termed homologous) or heterologous ␣ ␤␥ sociates into and subunits. G␣s desensitization due to actions of

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block polymorphisms with minor allele frequencies of 39% and 43%, respectively. In contrast, the Ile 164 and the Met34 polymorphisms are rare. No differences in the frequency of any polymorphism between the asthmatic and normal cohorts24–26 have been found except for one report in pediatric asthma.27 It is thus unlikely that any of these polymorphisms or combination of polymorphisms is a major risk factor for asthma. The effects of these sequence variants on gene expression and receptor function at a molecular level have been the subjects of intense research efforts. For example, direct compari- sons of the wild-type and mutant ␤ forms of the 2AR in vitro indicate that the agonist and antagonist binding affinities and coupling efficiencies to ␤ AC of the Gly16 and Glu27 receptors Figure 3 Simplified view of the 2-adrenergic pathway. Only the major signal transduc- ␤ are similar to those of the so-called tion components are shown but there are many candidate proteins in this pathway. 2AR, ␤ ␤ ␤ 28–30 2-adrenergic receptor; ARK, AR kinase; CysLT1, cysteinyl leukotriene receptor 1; Gs, wild-type receptor; however, there heterotrimeric guanine nucleotide-binding protein with ␣, ␤ and ␥ subunits; AC, adenylyl are clear differences in agonist traf- cyclase; PKA, protein kinase A; PLC, phospholipase C; MAP-kinase, mitogen-activated pro- ficking of these receptors. The Gly16 tein kinase; SR, sarcoplasmic reticulum. The opposing effects of one receptor are shown receptor undergoes enhanced downre- to illustrate how crosstalk occurs between pathways. gulation (loss of receptor number) compared with the wild-type Arg16 receptor. In contrast, the Glu27 recep- ligands at other receptors and their interaction with ␤-agonist and its cen- tor undergoes no downregulation resultant intracellular signaling. Clini- tral role in the ␤-agonist pathway; compared with the wild-type receptor. cally, desensitization is typically however, sequence variants in many Additional studies showed that these termed tachyphylaxis, which refers to other candidate genes are possible alterations in downregulation occur at a loss of clinical effectiveness of a given the large number of interacting a step distal to the internalization pro- therapeutic agent upon repetitive proteins comprising the ␤-agonist cess, at the point where degradation or administration. In asthma, chronic ␤- pathway. Early studies revealed four recycling to the cell surface occurs. agonist administration has been asso- coding regions and one promoter The polymorphism at position Ϫ47, ciated with tachyphylaxis in some18,19 polymorphism by directly sequencing localized within a small open reading but clearly not all20,21 studies. In PCR-derived fragments in cohorts of frame encoding a peptide that modu- ␤ ␤ addition, excessive -agonist use has normal and asthmatic subjects lates translation of 2AR, has been ␤ 31 been associated with increased asthma (Table 2). At amino acid position 16 shown to affect 2AR expression. mortality22 and morbidity.23 These Arg or Gly can be present, and at pos- These in vitro observations are con- associations with excess ␤-agonist use ition 27 Gln or Glu can be present. sistent with many clinical studies ␤ are not universally found, though, and These represent the common coding examining potential roles of 2AR it is not known whether tachyphylaxis per se is the basis of such observations. ␤ To explain inter-individual differ- Table 2 Common sequence variants in 2-adrenergic receptor ences in response to ␤-agonist and sporadic reports of tachyphylaxis, Lig- Nucleic acid Nucleic acid Amino acid ‘Wild type’ Polymorphic Minor allele gett and colleagues hypothesized that position position amino acid amino acid frequency sequence variants exist in the genes of 46 A or G 16 Gly Arg Arg (0.39) the ␤-agonist pathway and alter pro- 79 C or G 27 Gln Glu Glu (0.43) tein function or gene expression. 100 G or A 34 Val Met Met (Ͻ0.001) ␤ Efforts focused on the 2AR gene, an 491 C or T 164 Thr Ile Ile (0.05) intronless gene localized to chromo- Ϫ47 C or T 19 Cys Arg Arg (0.47) some 5q31–32, because of its direct

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polymorphisms in asthma. Some of ness to methacholine compared with thesized that tachyphylaxis to albut- ␤ these clinical studies have examined their responsiveness when they did erol was associated with a 2AR geno- potential disease modifying effects, not receive regular treatment. This type. To test this hypothesis, 32 33,34 ␤ such as severity, phenotype, and finding is consistent with patients who genotyping at the 2AR 16 and 27 pos- physiologic parameters.35 A few stud- are homozygous for the Arg 16 form of itions was carried out with genomic ␤ 38 ies have examined pharmacogenetic the 2AR being predisposed to an DNA obtained from 190 patients. relationships between these polymor- adverse reaction to the regularly Individuals with the Arg/Arg genotype phisms and the response to ␤-agonists. scheduled use of this ␤-agonist. receiving regularly scheduled albuterol For example, Martinez et al36 exam- An Asthma Clinical Research Net- experienced a significant decline in ined the bronchodilating response to work (ACRN) study was performed to AM peak expiratory flow rate (PEFR) the administration of a single dose of assess whether chronic regular-use over the course of the study (P = 0.012) albuterol in children. The study group albuterol was associated with deleteri- (Figure 4). This tachyphylaxis was not consisted of 191 normal children and ous clinical or physiologic sequella observed when treatment was given 78 children with a history of wheez- compared with as-needed dosing in on an as-needed basis to patients with ing. Of this latter group 37 had a diag- mild stable asthmatics.20 The study the same genotype. In contrast, those nosis of asthma. In the asthmatic was a 16-week, placebo-controlled, with the Gly/Gly genotype, even when group, 60% of those with homozygous double-blind design with 255 asth- receiving regularly scheduled albut- Arg16 had a positive (Ͼ15.3% increase matics. For the entire population the erol, showed no tachyphylaxis. A simi- in FEV1) response to albuterol, com- results showed that, on average, regular pattern was observed with PM pared with 13.3% who were homo- larly scheduled (two puffs, four times PEFR. The findings were not influ- zygous for Gly16. While borderline a day) albuterol had no clinical or enced by the position 27 genotype, statistically significant (P = 0.05 for physiological deleterious effects com- race, sex, or initial lung function. We trend), it should be noted that the pared with an as-needed schedule. conclude from these data that poly- ␤ homozygous Arg16 group consisted of Although, tachyphylaxis was not morphisms of the 2AR at locus 16 are only five patients. Children with a his- observed in the entire group, some a significant genetic predictor of tory of wheezing failed to have a stat- individuals did display tachyphylaxis, tachyphylaxis to the ␤-agonist albut- istically significant difference, while particularly in the group allocated to erol. Our results are consistent with normal children again showed a regular albuterol use. Thus we hypo- the notion that the Gly16 receptor is greater percentage of positive responses in the homozygous Arg16 group (P = 0.04). When the groups were analyzed together, the odds ratio for having a positive bronchodilator response to albuterol (adjusted for asthma and wheezing) was 5.3 for homozygous Arg16 children (P = 0.007). This study suggests that the initial response to albuterol in chil- ␤ dren is affected by 2AR genotype at position 16, although the data in asthmatics suffer from a small sample size. The results suggest that the Gly16 receptor is chronically downregulated (presumably by endogenous catecholamines), thereby resulting in a depressed response to the initial dose of albuterol. Recently, Hancox and colleagues37 reported the genotypes of a group of 61 asthmatics treated with regular vs as-needed fenoterol.23 An earlier report showed deterioration in asthma control with regularly scheduled fenoterol Figure 4 Time course of the change in morning peak expiratory flow (AM PEF) among ␤ ␤ ␤ treatment compared with as-needed different 2AR position 16 genotypes in response to 2A treatment. 16-Arg/Arg ␤ treatment. During treatment with (homozygous for Arg allele) patients who received regularly scheduled 2-agonist treat- -regularly scheduled fenoterol, subjects ment (regular) experienced a 30.5 Ϯ 12 l min؊1 decline in AM PEF compared with regu who were homozygous for Arg16 had larly scheduled ␤16-Gly/Gly patients or as needed ␤16-Arg/Arg patients. Modified from 38 an increase in bronchial responsive- Israel et al.

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downregulated maximally by protein (hsp) 90, and one molecule target proteins regulated by GC could endogenous catecholamines and thus each of hsp 70, hsp 56, hsp 26, and have pharmacogenetic significance. cannot display additional downregul- other proteins.41 The binding of GC to There is only one GR gene located ation by albuterol. On the other hand, the GR results in dissociation of the on chromosome 5q31. However, clon- the Arg16 receptor is less downregul- hsp subunits and exposure of the DNA ing of the human GR cDNA and gene ated by endogenous catecholamines, binding site on the GR. The activated has revealed that alternative splicing but is downregulated with chronic GR nuclear complex regulates tran- of the GR pre-mRNA generates two administration of albuterol. This con- scription by binding to specific palin- homologous protein isoforms, termed cept of dynamic regulation13 is con- dromic sequences of DNA, ie, GRE, or GR␣ and GR␤.42 These two protein iso- sistent with the results of Martinez et by direct interactions with other tran- forms have the same first 727 NH2-ter- al,36 which showed a decreased initial scription factors. Usually, two GRE minal amino acids in common and response to albuterol in patients half sites are arranged as inverted pal- thus both contain the DNA binding homozygous for Gly16. Additional indromes, allowing the GR to bind the domain. GR␣ is the ligand-activated studies are underway to determine if DNA as a homodimer. This interaction transcription factor which, in the hor- genotypic data can be used prospec- changes the rate of transcription and mone-bound state, modulates the tively to prevent tachyphylaxis. results in either induction or expression of GC-responsive genes. We have also recently shown that repression of gene expression. Admin- GR␤ differs from GR␣ only in its the 5Ј upstream promoter region of istration of GC to asthmatics, either COOH-terminus, with replacement of ␤ the 2AR gene is highly polymorphic. parenterally or directly to the airways the last 50 amino acids of the latter Indeed, there are at least 13 SNPs in by metered dose inhalers, has pro- with a unique 15-amino acid the promoter and coding blocks of this found anti-inflammatory effects due to sequence. These differences render gene. Of the 213 = 8192 possible com- their ability to alter the expression of GR␤ unable to bind GCs and reduce its binations of these SNPs, there are only many pro-inflammatory genes binding affinity for DNA recognition 12 haplotypes that we could identify (Figure 5). Sequence variants in the sites and its ability to transactivate in an index reporting consisting of genes encoding for the GR, nuclear GC-sensitive genes.43–46 The impor- four ethnic groups.39 In a recombinant proteins that interact with the GR, or tance of the GR in determining clinical expression system, certain haplotypes appeared to differentially affect receptor function. For patients bearing the low expression haplotype, the bronchoconstrictor response to albuterol was found to be depressed as compared to those with other haplotypes. These data suggest that in highly polymorphic genes it will be important to con- sider SNPs within the context. That is, as they appear with other SNPs rather than in isolation.

PHARMACOGENETICS OF GLUCOCORTICOIDS IN ASTHMA GC exert their biological effects by passively penetrating the plasma membrane of target cells and binding to a specific cytoplasmic glucocorticoid receptor (GR). The GR has three major domains: (a) the hormone- or ligand-binding domain at the COOH- terminus; (b) the DNA-binding domain with zinc finger structures that confer high affinity for glucocorticoid response elements (GRE); and (c) an amino-terminal transcription regu- Figure 5 Simplified view of the glucocorticoid pathway. Glucocorticoids (GC) bind to 40 latory domain. The unliganded cytoplasmic receptors (GR) and translocate to the nucleus where they interact with other receptor is thought to be a heterohex- transcription factors to regulate the expression of many inflammatory genes. Many candi- amer containing the receptor with a date proteins need to be considered. NF-kB, NFAT and STAT6 are only a few of the single GC-binding and DNA-binding important signal transduction components and transcription factors that interact with GC. 79 subunit, two molecules of heat shock Modified from Barnes.

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responsiveness to GC is supported by processes associated with the atopic arachidonic acid and exhibit a wide the discovery of mutations in the GR, state including IgE isotype switching, range of pharmacological and physio- as is best illustrated in primary cortisol mast cell proliferation, and eosinophil logical actions.60,61 In biological sys- resistance.47 These patients present survival and activation. Several invest- tems, their actions are limited by their with elevated total plasma cortisol igative groups studying families with relative rates of synthesis and degra- concentrations, but no Cushingoid diverse phenotypes, all of which are dation.62,63 Of the three enzymes features. The molecular basis for GC relevant to asthma (IgE levels, skin-test exclusively involved in the formation resistance in the various reported kin- reactivity, airway responsiveness, or a of the leukotrienes, namely ALOX5, dreds includes reduced GR numbers, diagnosis of asthma itself), have ident- LTC4 synthase, and LTA4 epoxide decreased binding afﬁnity for GC, or iﬁed genetic linkage of the presence of hydrolase, ALOX5 is the enzyme poor DNA binding of their GR to these phenotypes to a region on required for the production of both 48,49 GRE. human chromosome 5q31–33 in the cysteinyl leukotrienes (LTC4, LTD4,

Although most asthmatics respond which the genes encoding many T cell and LTE4 and LTB4 (Figure 6). Over the to GC therapy, a subset of asthmatics cytokines, including those encoding past 10 years, it has been shown that have poorly controlled asthma even for ILs- 3, 4, 5, 9, 12␤, and 13 and GM- pharmacological inhibition of the when treated with systemic GC. These CSF, are clustered.53–56 Although gen- action of ALOX5 or antagonism of the patients have been referred to as etic loci directly related to GC-resistant action of the cysteinyl leukotrienes at steroid-resistant or steroid-insensitive asthma have yet to been determined, their receptor is associated with an asthmatics. In a recent workshop on sequence variants associated with amelioration of asthma.64 GC insensitivity in asthma, it was pro- asthma, asthma severity and atopy Although a variety of biological posed that GC-resistant asthma could have been detected in the IL-4 pro- actions have been ascribed to LTB4,it be defined by the failure to improve moter (Ϫ590, C to T), TNF-␣ promoter is predominantly a chemotactic moi- baseline morning prebronchodilator (Ϫ308, A to G), and Fc⑀RI␤ (576, Ala ety for eosinophils and neutro- Ͼ 57–59 65,66 FEV1 15% following at least 7–14 to Arg) in some but not all studies. phils; these actions occur via signal 67 days of 20 mg twice-daily oral predni- transduction at the LTB4 receptors. sone or its equivalent. It should also be PHARMACOGENETICS OF The cysteinyl leukotrienes were ident- pointed out that in most patients, ster- LEUKOTRIENES IN ASTHMA ified as a result of the search for the oid resistance is not an absolute state The leukotrienes are a family of chemical structure of the materials but a significant shift in the dose- polyunsaturated lipoxygenated eicosa- that constituted the contractile response curve such that these tetraenoic acids that are derived from activity known as slow-reacting sub- patients require much higher quan- tities of GC for optimal response to therapy than patients with steroid- sensitive asthma.50 Studies carried out in our laboratory and others have demonstrated that patients with GC- resistant asthma have decreased cellular immune responses to GC.51,52 These abnormalities involving their T cells, monocytes, and eosinophils make GC treatment less effective in suppressing the activation of cells from GC-resistant asthmatics than from GC-sensitive asthmatics. To elucidate the genetic basis of GC- resistant asthmatics, screening of many pro-inflammatory genes regulated by GC for sequence variants with pharmacogenetic effects has begun. Much of this effort has focused on the many cytokines regulating T-lympho- cytes phenotype, a characteristic central to the development of asthma and other allergic diseases5 and profoundly altered by GC. These cytokines, which include ILs- 3, 4, 5, 9, and 13 and gra- Figure 6 Simplified view of the leukotriene pathway. Multiple enzymes and cofactors nulocyte-macrophage colony-stimul- interact to form leukotrienes. cPLA2, cytoplasmic phopholipase A2; FLAP; 5-lipoxygenase ating factor (GM-CSF), regulate several activating protein; ALOX5, 5-lipoxygenase; LTR, leukotriene receptors.

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stance of anaphylaxis (SRS-A)60,61 and are among the most potent bronchoconstrictor substances ever identified;68,69 their actions result from signal transduction at the CysLT1 receptor. In addition, a myriad of other, mostly proinflammatory biological activities have been attributed to the cysteinyl leukotrienes, from mitogenic effects to the modification of vascular integrity (see reviews by Badr,70 Bend & Karmazyn,71 and Denzlinger72). Among the human dis- Figure 7 Location of ALOX5 polymorphisms. WT allele has five Spl/Egr-1 transcription ease conditions thought to result from factor consensus binding sites. Alleles M(3), M(4), and M(5) have three, four, and five excess availability of the cysteinyl leu- binding sites, respectively. Allele frequencies are noted. kotrienes is asthma.64,73 Inhibition of ALOX5 activity or blockade of the

CysLT1 receptor by cLI is associated with clinically significant improve- ments in asthma outcome.74 The regulation of ALOX5 is multifa- ceted; known mechanisms include regulation of action of the mature protein and regulation of ALOX5 gene transcription and translation. The ALOX5 gene promoter contains numerous consensus binding sites for many known transcription factors, including Sp1, Sp3, Egr-1, Egr-2, NF- kB, GATA, Myb, and AP family mem- bers. Of particular importance, Hoshiko et al75 have identified a GϩC- rich sequence, between Ϫ212 and Ϫ88 bp relative to the translational start site, as being necessary for promoter-reporter construct activity in HeLa and HL-60 cells; these observations, which we have confirmed, led us to examine this region of the ALOX5 promoter for mutations that might alter gene transcription. We Figure 8 ALOX5 genotype predicts anti-leukotriene response. Improvement in FEV1 at 84 identified a family of naturally occur- days of treatment was significantly greater for WT subjects treated with ABT-761 (300 mg ring mutations that alter Sp1 and Egr- day؊1) compared with subjects possessing any ALOX5 mutant allele. ABT-761 is an ALOX5 1 consensus binding sites by the inhibitor similar to zileuton. Modified from Drazen et al.78 addition of one or the deletion of one or two -GGGCGG- sequences (Figure 7).76 Sp1 and Egr-1 were cap- more than a simple tandem nucleotide ABT-761 resulted in a significant able of binding this region of the pro- repeat; rather, they were positioned so improvement in asthma as measured moter, and the mutations altered the as to modify gene transcription and by FEV1 compared with placebo con- pattern of binding as detected by elec- therefore to potentially modify trol. When the 221 individuals who trophoretic mobility shift assays ALOX5 product formation. received either high-dose ABT-761 (EMSAs) with nuclear extracts and The significance of these promoter (n = 114) or placebo (n = 107) treat- recombinant protein. Moreover, these polymorphisms has been examined ment for 84 days were stratified by mutations had significant functional from a pharmacogenetic perspective as genotype at the ALOX5 core promoter consequences when studied in the part of a clinical drug trial examining locus, dramatic differences in response context of CAT reporter constructs.77 the effects of ABT-761, a ALOX5 were detected (Figure 8). Patients with These data indicated that the various inhibitor similar to zileuton, in the wild-type allele had an approxi- 78 polymorphisms we identified were patients with asthma. As expected, mate 19% improvement in FEV1, com-

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pared with Ϫ1% in FEV1 among indi- in the context of other sequence vari- 8 Bleeker E, Postma D, Meyers D. Evidence for viduals with mutant alleles ants in the same gene and sequence multiple genetic susceptibility loci for asthma. Am J Resp Crit Care Med 1997; 156: = ϫ Ϫ5 (P 2.5 10 ). These data are con- variants in other genes involved in the S113–116. sistent with the idea that individuals pharmacologic pathway.39 Despite 9 Holgate ST. Asthma: a dynamic disease of harboring any of the mutant forms of these challenges, significant progress inflammation and repair. Rising Trends in the ALOX5 core promoter have has been made and it is now possible Asthma 1997; 2: 6–34. 10 Hogg JC. The pathology of asthma. APMIS decreased ALOX5 gene transcription to genotype asthmatics at a few loci 1997; 105: 735–745. and thus downregulated ALOX5 and use this information to make 11 Malmstrom K, Rodriguez-Gomez G, Guerra expression. If ALOX5 products were therapeutic decisions that improve J, Villaran C, Pineiro A, Wei L et al. Oral among the several mediators contribu- drug efficacy and mitigate compli- montelukast, inhaled beclomethasone, and placebo for chronic asthma: a randomized, ting to the airway obstruction in cations. With completion of the contolled trial. Ann Intern Med 1999; 130: asthma, and if patients harboring the human genome sequence, and the 487–495. mutant genotypes were equivalent to availability of high-density single 12 Drazen JM, Silverman EK, Lee TH. Hetero- patients who were receiving ABT-761 nucleotide polymorphism maps and geneity of therapeutic responses in asthma. Br Med Bull (in press). treatment that inhibited ALOX5, then high-throughput genotyping tech- 13 Liggett SB, Green SA. Molecular biology of the former patients would have airway nology, the pace of newly discovered the b2-adrenergic receptor: focus on inter- obstruction mediated by mechanisms loci with pharmacogenetic relevance actions of agonist with receptor. In: other than the leukotrienes. As a to asthma is likely to increase expo- Pauwels R, Lofdahl CE, O’Byrne P (eds). Beta2-Agonists in Asthma Treatment. Marcel consequence of this natural inhi- nentially over the next few years. Dekker: New York, 1996, pp 19–34. bition, patients harboring the mutant 14 Liggett SB. Molecular basis of G-protein genotypes have a diminished response ACKNOWLEDGEMENTS coupled receptor signaling. In: Crystal R, to exogenous ALOX5 inhibition. It Supported by UO1 HL65899 from the West JB, Weibel ER, Barnes PJ (eds). The Lung: Scientific Foundations. Raven Press: may be possible to genotype asth- National Heart, Lung, and Blood Institutes of the National Institutes of Health. New York, 1997, pp 19–36. matics at the ALOX-5 locus and pre- 15 Hakonarson H, Grunstein M. Regulation of dict which patients will improve with second messengers associated with airway DUALITY OF INTEREST leukotriene inhibitors; asthmatics with smooth muscle contraction and relaxation. None declared. Am J Respir Crit Care Med 1998; 158: mutant alleles at the ALOX-5 should S115–S122. probably avoid cLI because they are Correspondence should be sent to 16 Samama P, Cotecchia S, Costa T, Lefkowitz unlikely to derive clinical benefit. Scott T Weiss, MD, MS, Director, Respiratory, RJ. A mutation-induced activated state of Environmental, and Genetic Epidemiology, the b2-adrenergic receptor. J Biol Chem 1993; 268: 4625–4636. Channing Laboratory, Brigham and Women’s CONCLUSION 17 Liggett SB, Lefkowitz RJ. 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