The Pharmacogenomics Journal (2011) 11, 383–392 & 2011 Macmillan Publishers Limited. All rights reserved 1470-269X/11 www.nature.com/tpj FEATURE REVIEW

The pharmacogenetics and pharmacogenomics of therapy

SM Tse, K Tantisira and ST Weiss Despite the availability of several classes of asthma and their overall effectiveness, a significant portion of patients fail to respond to these Channing Laboratory, Brigham and Women’s therapeutic agents. Evidence suggests that genetic factors may partly Hospital and Harvard Medical School, mediate the heterogeneity in asthma treatment response. This review Boston, MA, USA discusses important findings in asthma pharmacogenetic and pharmaco- genomic studies conducted to date, examines limitations of these studies Correspondence: Dr SM Tse, Channing Laboratory, 181 and, finally, proposes future research directions in this field. The focus will be Longwood Avenue, Boston, MA 02115, USA. on the three major classes of asthma medications: b-adrenergic E-mail: [email protected] agonists, inhaled and modifiers. Although many studies are limited by small sample sizes and replication of the findings is needed, several candidate genes have been identified. High-throughput technologies are also allowing for large-scale genetic investigations. Thus, the future is promising for a personalized treatment of asthma, which will improve therapeutic outcomes, minimize side effects and lead to a more cost-effective care. The Pharmacogenomics Journal (2011) 11, 383–392; doi:10.1038/tpj.2011.46; published online 11 October 2011

Keywords: asthma; pharmacogenetics; treatment response

Introduction

Asthma is a chronic inflammatory disease of the airways characterized by variable airflow obstruction and it affects more than 300 million individuals worldwide. In the United States, the prevalence of asthma is 7.7% in adults and 9.6% in children, with similar figures in other developed countries.1 A recent study using data from MEPS (Medical Expenditure Panel Survey) estimated the annual total cost of asthma in the United States, which includes incremental direct costs and productivity costs, to be $56 billion in 2007.2 Prescription medications accounted for the largest direct expenditure for asthma. Despite the wide availability of therapeutic asthma drugs and large studies supporting their efficacy, there is significant interindividual variability in the response to each of the three major classes of asthma medications.3 An efficient method to differentiate nonresponders from responders before initiating treatment would not only avoid unwanted adverse effects, but also significantly decrease the economic burden of this disease. Asthma has long been recognized as a genetic disease through observation of clustering of disease within families and twin studies. Population-based twin studies suggest that heritability estimates for asthma range from 53% up to 92%.4 Received 13 July 2011; revised 16 August 2011; accepted 1 September 2011; Nonetheless, asthma is a complex genetic disease that is likely the result of published online 11 October 2011 multiple gene–gene and gene–environment interactions. The environmental risk The pharmacogenetics and pharmacogenomics of asthma therapy SM Tse et al 384

factors associated with incident asthma include smoke response distribution in exposure, certain viral infections, exposures and ACRN, CARE and CAMP vitamin D deficiency. 30 ACRN The following sections highlight a selection of important CAMP as well as recent studies in the pharmacogenetics and 25 CARE pharmacogenomics of asthma. Each of the three major therapeutic drug classes will be discussed, which includes 20 b2-adrenergic agonists, corticosteroids, and leukotriene modifiers. 15

Harmonization of asthma treatment response 10 phenotypes Percent of subjects (%)

In pharmacogenetic studies, response to asthma therapy has 5 been frequently assessed using quantitative methods, such as change in FEV1 (forced expiratory volume in 1 s), PC20 0 (provocative concentration of methacholine causing a 20% 0 to 5 drop in FEV1, which reflects the degree of airway hyper- -5 to 0 5 to 10 10 to 15 15 to 20 20 to 25 25 to 30 30 to 35 35 to 40 responsiveness), and bronchodilator response (change in -10 to -5 b FEV1 shortly after administration of a short-acting 2- less than -10 Bronchodilator response (%) agonist (SABA)). These measures of lung function are greater than 40 reproducible and their quantitative nature allows for Figure 1 Distribution of the bronchodilator response across the three increased statistical power in genetic analyses. Peak expira- major asthma networks in SHARP. Note that despite highly varying tory flow rate (PEFR) has also been used as a response enrollment criteria, the overall distribution of response is remarkably phenotype, although it correlates poorly with symptoms similar between the clinical trial networks. This supports the ability and may lack reproducibility.5,6 Although qualitative mea- to harmonize asthma pharmacogenetic phenotypes and the likely sures, such as asthma exacerbations or presence/absence of presence of common genetic determinants of response. Abbreviations: ACRN, Asthma Clinical Research Network; CAMP, Children Asthma symptoms, do not fully correlate with measures of lung 7–9 Management Program; CARE, Childhood Asthma Research and function, they may nonetheless be useful pharmaco- Education. genetic outcome measures. In general, the change in lung function associated with asthma treatment administration follows a near-normal distribution, demonstrating substan- networks contributing to SHARP, heterogeneity of response tial interindividual variability,10,11 with a significant propor- within each group of network subjects as well as similarity of tion of both nonresponders and high responders to therapy. response distribution between groups are noted (Figure 1). This wide variability in interindividual response, combined Together, the data from both the inhaled and with high intraindividual repeatability, suggests a genetic bronchodilator response distributions indicate that there are basis to the heterogeneity in asthma treatment response.3 factors, such as genetics, independent of the drug itself that Harmonization of the treatment outcome phenotypes is determine relative degree of response to asthma medications essential for comparison between pharmacogenetic studies and that these factors are common across a wide range of for several reasons, including the assurance that a given subjects. These findings suggest that there is a consistent pharmacogenetic locus, if valid, will be generalizable. pharmacogenetic effect across populations independent of Moreover, different study populations are often combined asthma severity. In the context of genome-wide association to increase the statistical power to detect associations. We analyses of asthma treatment response, this supports the have previously demonstrated that, despite widely variant combination of different clinical trials for more powerful entrant criteria, the lung function response to inhaled analyses, as well as the ability to successfully independently corticosteroids follows both a relatively normal distribution replicate pharmacogenetic loci. and is remarkably similar across multiple clinical trial populations.12 Although the acute bronchodilator response distribution is slightly skewed, it too demonstrates remark- The b2-adrenergic receptor agonist pathway able reproducibility across populations. For instance, the SNP Health Association Resource (SHARe) Asthma Resource The b2-adrenergic receptor agonists are the most commonly project (SHARP) (www.ncbi.nlm.nih.gov/projects/gap/cgi- prescribed medications in the treatment of asthma. SABAs bin/study.cgi?study_id ¼ phs000166.v1.p1) is conducting are the mainstay of treatment for acute symptoms of genome-wide analyses of adults and children who have , whereas long-acting b2-agonists (LABAs) participated in National Heart, Lung, and Blood Institute’s are used as adjuncts to inhaled corticosteroid therapy to clinical research trials on asthma. In the evaluation of acute provide long-term asthma control.13 Both SABAs and LABAs bronchodilator response across the three major asthma exert their therapeutic effects via their binding to the active

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site of b2-adrenergic receptors (b2-ARs), which are densely association was found between the number of Arg16 alleles located on the cells of the lower respiratory and responsiveness to a single dose of albuterol among tract. When stimulated, the b2-AR couples to adenylate asthmatic Puerto Ricans with a baseline FEV1 o80% cyclase through a trimeric G-protein, leading to increased predicted.22 This relationship was not present in Mexican production of cyclic adenosine monophosphate and protein asthmatics. Along with the finding of different linkage kinase A. The protein kinase A phosphorylates key regula- disequilibrium patterns between ADRB2 SNPs in Puerto tory proteins involved in the control of muscle tone whereas Ricans and Mexicans, this study underscores ethnic-specific cyclic adenosine monophosphate results in sequestration of pharmacogenetic differences in the effect of Arg16Gly intracellular Ca þ , both causing relaxation of the airway genotype on drug response. smooth muscle.14 The relationship between the Arg16Gly genotype and long-term use of SABAs has also been examined. Israel Short-acting b2-agonists et al.23 genotyped the Arg16Gly and Glu27Gln SNPs in 190 A substantial fraction of the interindividual variability in subjects who were randomized to regular plus as-needed response to SABA treatment is attributed to genetic factors.3 albuterol or as-needed albuterol. When stratified by Many studies have examined the pharmacogenomics and genotype, regular b2-agonist use was associated with a pharmacogenetics of b2-agonists. However, as a whole, they decline in morning and evening PEFRs in patients who have proven to be inconclusive because of the wide were homozygous for Arg16, but not other genotypes. variability in study design and their definition of therapeu- Although this genotype-specific effect was noted during tic end points.15 the 16-week study period, the effect was greatest during the The literature on b2-agonist treatment heterogeneity in run-out period, during which all patients returned to using asthma is dominated by studies on the ADRB2 gene, which as-needed albuterol. The authors postulated that Arg/Arg encodes the b2-AR. This gene is a small, intron-less gene patients could be more susceptible to the tachyphylactic located on chromosome 5q31,16 and has been resequenced effect of regular b2-agonist use as previous work has shown in several populations.17,18 Most studies have focused on that b2-ARs are more downregulated at baseline in Gly/Gly Arg16Gly and Glu27Gln, two common nonsynonymous patients compared with Arg/Arg patients. The higher single-nucleotide polymorphisms (SNPs) at positions þ 46 concentration of b2-ARs at baseline in Arg/Arg patients and þ 79 on ADRB2, respectively19 (Figure 2). The reported would also be consistent with the findings above that minor allele frequency for Arg16Gly (Gly16 being the minor Arg/Arg patients have a greater bronchodilator response to allele) is B40% for whites and African Americans.17,20 single-dose albuterol. These results led to the first genotype- Martinez et al.21 genotyped 269 children of Hispanic or stratified, prospective placebo-controlled trial of asthma Caucasian descent, 78 (29.2%) of whom reported episodes of therapy.24 The BARGE trial (Beta-Adrenergic Response by wheezing during the previous year. They found that Genotype) randomized 78 subjects to either regular albu- children who were homozygous for Arg16 were 5.3 times terol or placebo for 16 weeks, followed by a crossover in more likely to respond to a single dose of albuterol than the treatment for another 16 weeks. Arg/Arg subjects had lower children who were homozygous for Gly16. There was a PEFRs during treatment with regular albuterol compared nonsignificant trend for association between Glu27Gln and with placebo (10 l min–1, range 19 to 2). Although this bronchodilator responsiveness. Similarly, in a family-based is statistically significant, the small difference may not study of Latino Americans with asthma, a highly significant be clinically significant. As was used as rescue throughout the study, it was hypothe- sized that even as-needed use of SABAs may be unsafe for Arg16Gly Arg/Arg patients. NH2 Long-acting b2-agonists Gln27Glu The concern for safety of b2-agonists was also extended to LABAs. Wechsler et al.25 examined multiple SNPs on ADRB2 in a sample of subjects previously enrolled in clinical trials involving the LABA . They found that Arg/Arg patients had a diminished therapeutic response to the regular use of salmeterol compared with those with Gly/ Gly, as evidenced by a decrease in PEFR. However, this study’s impact was limited by its small sample size. Bleecker et al.26 performed a similar analysis but found contradictory results, with no evidence of association between the Arg16Gly genotype and response to salmeterol in combina- COOH tion with inhaled corticosteroid (ICS). This led to two Figure 2 Polymorphisms in ADRB2. Arg16Gly and Gln27Glu are larger, genotype-stratified, randomized controlled trials. In located at positions þ 47 and þ 79, respectively, and are shown in the LARGE study (Long-Acting Beta Agonist Response the transmembrane b2-adrenergic receptor. by Genotype), a total of 87 patients were randomized to

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18 weeks of treatment with salmeterol or placebo with a In contrast, there was no association between individual subsequent crossover.27 Open-label use of beclomethasone SNPs and bronchodilator response, suggesting that haplo- and ipratropium bromide was allowed throughout the trial. type analysis is more powerful in detecting associations than Both Arg/Arg and Gly/Gly patients showed improved PEFRs single SNP analysis. More recently, Hawkins et al.17 compre- after treatment with salmeterol. Interestingly, Gly/Gly hensively sequenced a 5.3-kb region of ADRB2 in 429 patients had enhanced bronchoprotection after treatment white and 240 African-American subjects. They identified with salmeterol and inhaled corticosteroids, as evidenced 20 previously unreported SNPs found in regulatory regions by a higher PC20, whereas there was no such difference in that contribute to haplotypic structure and to genetic Arg/Arg patients. Recently, Bleecker et al.28 published a large association with asthma-related phenotypes. These studies genotype-stratified clinical trial where 544 asthmatic sub- support the need for comprehensive analysis of the ADRB2 jects with Arg/Arg, Gly/Gly or Arg/Gly at codon 16 were gene, including haplotype analysis, in future pharmacoge- randomized to salmeterol alone or in combination with netic studies. Animal studies have noted the importance of inhaled propionate for 16 weeks. Both Arg/Arg the 30 untranslated region to ADRB2 gene function, and and Gly/Gly subjects showed improvement in morning studies of regulatory variation in the gene would also be of PEFR with salmeterol alone and salmeterol in combination interest. with , although the differences in In addition to ADRB2, a number of other genes from the PEFR were not statistically significant. Furthermore, Arg16- b2-agonist pathway have been examined for pharmacoge- Gly genotypes did not modify other secondary treatment netic effects. These include S-nitrosoglutathione reductase responses, including evening PEFR, FEV1, as-needed ipra- (GSNOR), which metabolizes the endogenous bronchodila- tropium bromide use and percentage of symptom-free days. tor S-nitrosothiol. Increased GSNOR expression and The authors conclude that there was no evidence of a decreased S-nitrosothiol levels have been demonstrated in pharmacogenetic effect of ADRB2 gene polymorphisms on the bronchoalveolar fluid of adults with mild asthma.32 salmeterol response, in particular at the Arg16Gly position. Furthermore, GSNOR expression in bronchoalveolar lavage Although the number of black subjects was small and cell lysates was positively correlated with airway hyperreac- unbalanced by genotype (n ¼ 130, with more Arg/Arg tivity. Choudry et al.33 recently sequenced the GSNOR gene compared with Arg/Gly and Gly/Gly), post hoc analyses in in a subset of a Mexican and Puerto Rican asthmatics cohort this subgroup showed that there was also no differential and identified 13 SNPs with an allele frequency of 45%. response to salmeterol based on the Arg16Gly genotype of Association studies between five GSNOR SNPs (selected the subjects. These results corroborate with previous pub- based on location and linkage disequilibrium pattern) lications showing no correlation between Arg16Gly and identified gene–gene interactions between GSNOR and adverse outcomes associated with LABA use in the black ADRB2 in Mexicans and Puerto Ricans combined, although population.26,29 Such findings are particularly relevant in these SNPs and their haplotypes were not associated the context of the previous SMART trial (Salmeterol Multi- with bronchodilator response after Bonferroni correction center Asthma Research Trial), a randomized controlled trial for multiple testing. Specifically, asthmatic patients with the comparing the safety of salmeterol or placebo added to usual GSNOR þ 17059 TG þ GG genotypes and either the asthma care.30 Interim analyses showed that significantly ADRB2 þ 46 AG or GG genotypes had a decreased bronch- more African Americans on salmeterol experienced respira- odilator response, with a mean change in FEV1 of 5.29% tory-related death or life-threatening experience compared (AG) and 0.54% (GG) predicted, compared with 10.04% for with those on placebo (relative risk 4.1 (1.5–10.9)). Because the other genotypes. of these preliminary findings in African Americans and Several recent studies support the role of ARG1 poly- difficulties in enrollment, the trial was stopped prematurely. morphisms in bronchodilator response. Litonjua et al.34 Overall, although the combined findings from the LABA used a novel family-based screening algorithm based on studies may be reassuring, the use of a LABA monotherapy the statistical power of each SNP to rank 844 SNPs in remains contraindicated in the treatment of asthma because 11 bronchodilator response candidate genes in a cohort of of the increased risk of severe exacerbation of asthma 209 children and their parents participating in the Child- symptoms in some patients. hood Asthma Management Program (CAMP). Genes that The majority of the pharmacogenetic studies on b2- had SNPs with median power in the highest quartile agonist response have focused on the Arg16Gly polymorph- were subsequently taken for replication analyses in three ism. However, the conflicting results from previous studies other asthma cohorts. Four SNPs from ARG1 showed the and the lack of evidence of clinical significance associated strongest evidence for association with bronchodilator with this polymorphism so far suggest that there are response, with one SNP (rs2781659) surviving Bonferroni likely other variants in partial linkage disequilibrium with correction. Three of the four SNPs were in strong linkage Arg16Gly. Drysdale et al.31 organized 13 SNPs from the disequilibrium. ADRB2 gene into 12 haplotypes with highly variable ARG1 encodes arginase, which controls L-arginine home- frequencies across different ethnic populations. Individuals ostasis. The availability of L-arginine to nitric oxide synthase homozygous for the most common haplotype had an acute determines the production of nitric oxide, a bronchodila- bronchodilator response that was twice as high as indivi- tor.35 Increased ARG1 expression has been shown in human duals homozygous for the second most common haplotype. asthma bronchoalveolar epithelial and inflammatory cells,

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supporting the regulatory role of ARG1 in airway function.36 The corticosteroid pathway Further supporting the role of ARG in bronchodilator response, Vonk et al.37 identified an association between The ICSs are considered first-line therapy and are the most the ARG1 SNP rs2781667, ARG2 SNPs rs7140310 and effective anti-inflammatory drugs for the treatment of rs10483801 and b2-agonist response in 200 adults with a persistent asthma.13 (GCs) exert their history of asthma. This ARG1 SNP was in high linkage therapeutic effects by binding and forming a complex with disequilibrium with the SNP reported by Litonjua et al.,34 the intracellular GC receptor. This complex is activated and thus replicating the previous findings. ARG1 was recently translocates into the nucleus to directly alter transcription resequenced in a population of non-Hispanic Whites, of genes involved in through binding with African Americans and Hispanics to comprehensively iden- DNA or indirectly through interaction with other transcrip- tify genetic variants and the most prevalent haplotypes.38 tion factors40 (Figure 3). For example, GC inhibits gene In addition, the authors performed a bronchodilator expression of interleukin (IL)-3, IL-5 and IL-6, which are response association study in three adult asthma popula- proinflammatory mediators involved in inflammatory cell tions. Using the 22 polymorphisms identified, 14 of which proliferation and regulation,41,42 while enhan- were located in the 50 region of the ARG1 gene, 3 haplotypes cing the expression of IL-10 and IL-12,43,44 whose roles with frequencies 45% were constructed, representing include suppression of macrophage activation and differ- 80–91% of the 3 asthma populations. Individuals with the entiation of T cells to the Th1 subtype.40 In vitro studies have most prevalent haplotype (haplotype 1) were up to twice as also shown that GCs can prevent and reverse the desensi- responsive to albuterol as the individuals with the least tization caused by chronic exposure to a b2-agonist.45 In the prevalent of the three haplotypes (haplotype 3). In addition, case of LABAs, this may be mediated by the GC-induced human airway epithelial cells transfected with haplotype upregulation of mitogen-activated protein kinase phospha- 1 showed greater reporter activity than the two other tase 1, which leads to decreased levels of the proinflamma- haplotypes, suggesting that ARG1 haplotypes regulate gene tory phosphodiesterase 4.46,47 expression in vitro. The evidence supports a significant role Although there is significant interindividual variability for ARG1 variants in bronchodilator response. However, in ICS responsiveness,3,48 the intraindividual response is larger prospective genotype-stratified studies are needed to highly repeatable, supporting a genetic basis for the better understand arginase regulation and the effect on heterogeneity of ICS response.3 A study of 14 candidate asthma treatment response. genes in the corticosteroid pathway revealed a significant Several candidate genes have been identified for the association between corticotropin-releasing hormone receptor 1 response to . However, few studies have (CRHR1) genotype and 8-week response to inhaled corticos- attempted to integrate the existing genetic data into a teroid in two independent populations of adult and predictive model. Recently, Himes et al.39 modeled broncho- pediatric asthmatics.12 Specifically, patients homozygous dilator response in the CAMP cohort using Bayesian for the minor allele of rs242941 (minor allele frequency of networks, which are multivariate models that are able to 30%) had a significantly higher change in percent FEV1 account for several interactions and associations among after ICS treatment, resulting in an average 2–3 times variables simultaneously. The bronchodilator response Bayesian network, constructed from 15 SNPs in 15 candidate genes, was better at predicting bronchodilator response than GC single SNPs. The integration of genetic data will be essential in the creation of accurate prediction models in pharmaco- genetics, and multivariate models such as Bayesian networks are an attractive approach. GC In summary, studies have identified several genes that Hsp90 Cytoplasm p23 p23 Hsp90 may influence the response to SABAs and LABAs. This GR Hsp90 FKBP52 Hsp90 pharmacogenetic response also varies by ethnic group. Most FKBP52 Hsp70 Nucleus Hsp70 of the literature has focused on the ADRB2 gene, especially with regard to the safety of LABAs, a widely prescribed class GC of asthma medication. Recent evidence suggests that the extensively studied Arg16Gly polymorphism does not play a GR significant role in the response to LABAs28 and is not GC associated with adverse effects. Future studies should consider the addition of other variants such as those in GR GSNOR and ARG1 in prediction models and perform Figure 3 Overview of the (GC) pathway. The binding of association studies using haplotypes rather than individual GC to the intracellular GC receptor (GR) results in activation of the SNPs. Genome-wide association studies for broncho- complex and dissociation of inhibitory proteins that usually keeps GR dilator response are also under way, which may help inactive. These include Hsp90 and Hsp70 (heat shock proteins), the p23 improve the genetic prediction of bronchodilator respon- phosphoprotein and FKBP52. The GC–GR complex translocates into the siveness. nucleus and alters transcription of genes involved in inflammation.

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improvement compared with those without the variant. FCER2 encodes for CD23, a low-affinity IgE receptor, and SNPs in CRHR1 were also organized into four haplotypes, GCs have been shown to decrease FCER2 expression and distinguished by three haplotype-tag SNPs, rs1876828, CD23 production.58 However, a variant in FCER2, T2206C, rs242939 and rs242941. Homozygozity for the GAT haplo- has been recently associated with increased IgE levels and type (frequency 27%) was associated with greater improve- severe asthma exacerbations in asthmatic children despite 59 ment in FEV1 after ICS treatment. Corticotropin-releasing ICS use. This SNP was not infrequent in white subjects, hormone (CRH) is an important mediator of the responses with a minor allele frequency of 26%. White children of endocrine, autonomic and immune systems to stress. homozygous for the T2206C mutant allele were 3.95 times It binds to CRHR1 in the pituitary gland and modulates the (95% confidence interval 1.64–9.51) more likely to have a release of adrenocorticotropic hormone-induced cortisol severe exacerbation compared with all other T2206C production. Peripherally, CRH may exert a proinflammatory genotypes. African-American children demonstrated a simi- effect through degranulation,49 and alterations in lar trend with 3.08 (95% confidence interval 1.00–9.47) the CRH pathway have the potential to influence the increased risk of exacerbations for those homozygous for the pathogenesis of asthma. In fact, CRH-knockout mice sub- mutant allele. Of further interest, in both White and jected to ovalbumin-induced airway inflammation showed African-American children, the variant demonstrated evidence of lung mechanical dysfunction, increase airway no effect on the risk of exacerbation in those children not inflammation and goblet cell hyperplasia.50 In contrast to taking inhaled corticosteroid therapy. Incorporating this Tantisira’s study,12 Dijkstra et al. studied the 3 haplotype-tag variant into a prognostic model may identify those subjects SNPs in CRHR1 and found no association between CRHR1 at risk of severe exacerbations and who may benefit from genotype and long-term lung function decrease in adults, therapies other than ICS. independent of treatment with ICS,51 suggesting that there Because of issues with population stratification, most are other factors responsible for the ICS response variability. studies in ICS pharmacogenetics have examined associa- Recently, our group demonstrated that the association tions in white subjects. A recent study looked at the between CRHR1 and lung function response to ICS may be association between SNPs in the DUSP1 (dual-specificity mediated by a large structural inversion on chromosome 17, phosphatase 1) gene and effect modification by ICS on with which CRHR1 is in strong linkage disequilibrium.52 bronchodilator responsiveness in Latinos (GALA study). An obvious candidate to explain ICS response variability The findings were subsequently replicated in African Amer- is the GC receptor. GC receptor is part of a large hetero- icans (SAGE and SAPPHIRE studies).60 DUSP1 is also known complex of proteins, and variations in genes encoding as mitogen-activated protein kinase phosphatase 1 and is any of these components might contribute to variable induced by corticosteroids, resulting in inhibition of mito- interindividual steroid response. Hawkins et al.53 recently gen-activated protein kinase and hence the production of genotyped SNPs in 8 GC-complex genes in 382 white proinflammatory cytokines. In the above study, rs881152 in subjects on inhaled corticosteroids and found that DUSP1 (GG genotype) was associated with greater broncho- several SNPs in the STIP1 gene were associated with percent dilator response when ICS was used concurrently among the change in FEV1 after 4 and 8 weeks of treatment. rs6591838 GALA and SAPPHIRE populations. Among patients who was associated with the largest increase in FEV1 (20.70± were treated with ICS for 6 weeks prospectively (SAPPHIRE), 28.29%) after 8 weeks, with the rare GG allele. Haplotype the GG genotype at rs881152 was also associated with analysis also showed promising positive correlations improved self-reported asthma control compared with AG between haplotypes and percent change in FEV1. However, or AA genotypes. replication of these findings is needed to confirm these Genomics studies have also begun to focus on differential observations. expression of genes associated with ICS treatment response. TBX21, another candidate gene, encodes for the nuclear For example, corticosteroid treatment downregulated transcription factor T-bet. T-bet is responsible for the expression of CLCA1, periostin and serpinB2 while upregulat- differentiation of naive T lymphocytes into Th1 cells while ing FKBP51 expression in airway epithelial cells from simultaneously repressing Th2 cells.54 Compared with asthmatic patients.61 High baseline expression of CLCA1, controls, asthmatic subjects have significantly decreased periostin and serpinB2 was correlated with improvement in expression of T-bet expression in their peribronchial CD4 þ FEV1 after 4 weeks of ICS treatment, whereas high expres- lymphocytes.55 rs2240017, a nonsynonymous variation sion of FKBP51 was associated with a poor response. Another coding for replacement of histidine 33 with glutamine study looking at genome-wide gene expression profiles in (H33Q, minor allele frequency of 4.5% in Caucasians), has peripheral blood mononuclear cells derived a prediction been associated with marked improvement in airway model comprising 11 genes that could discriminate GC- hyperresponsiveness in children on ICS therapy.56 On the sensitive and GC-resistant asthmatic patients with 84% other hand, subjects on ICS who were homozygous for accuracy.62 Among these 11 genes, the NFKB gene alone histidine (H33H) demonstrated only a slight improvement predicted GC resistance with 81.25% accuracy. The findings in PC20 compared with the placebo group. Resequencing from these studies require replication. Nonetheless, they of the T-bet locus identified 7 haplotypes from 24 SNPs, identified novel candidates for further investigation. several of which were associated with airway hyperrespon- The breadth of research on the genetics and genomics of siveness.57 ICS response is less extensive compared with the studies in

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bronchodilator response. Several candidate genes and their 5-LO inhibitor, ABT-761, there was an improvement of functional variants have been identified, and although 19–23% in FEV1 at the end of the treatment period for promising, most results need replication in future studies. asthmatics who were homozygous or heterozygous for the wild-type allele (homozygous or heterozygous for five

repeats), whereas no change in FEV1 was observed among The leukotriene antagonist pathway those homozygous for mutant alleles (combinations of three, four or six repeats).69 These findings were supported play a central role in a variety of inflammatory by a more recent study by Telleria et al.70 that reported diseases such as asthma. In particular, increased levels of decreased number of asthma exacerbations, improvement of urinary (LTE4) have been associated with FEV1 and decreased use of b2-agonists following treatment 63 aspirin-exacerbated respiratory disease and exercise- with in asthmatics with at least one wild-type 64 induced . The leukotriene pathway allele. begins with the conversion of to leuko- Klotsman et al.71 investigated 25 polymorphisms in triene A4 (LTA4), a reaction catalyzed by the 10 leukotriene pathway candidate genes in a group of 65 5-lipoxygenase (5-LO) (Figure 4). LTA4 is subsequently asthmatics randomized to montelukast for 12 weeks. converted to under the influence of Analyses were performed on ethnically diverse subjects, (LTC4S), which is transported with 79% Caucasians, 22% Hispanics and 10% African extracellularly. Sequential cleavage of glutamate and glycine Americans. Variants in CYSLTR2 (rs912277 and rs912278) residues results in the formation of leukotriene E4 and D4. and ALOX5 (rs4986832) were associated with an 18–25% Leukotrienes bind to receptors present on leukocytes and improvement in PEFRs, in contrast to 8–10% in subjects lung smooth muscle cells, such as cysteinyl leukotriene with the wild-type alleles. Lima et al.72 genotyped 28 SNPs receptor 1 (cysLTR1), to cause smooth muscle contraction from genes involved in the leukotriene pathway and 66 and mucus secretion. Current therapies performed association studies on 61 subjects taking mon- include cysteinyl blockers (for example, telukast as part of a larger clinical study. In contrast to montelukast, and ) and inhibitors of the study of Klotsman et al.,71 this analysis was restricted to 5-LO (for example, ). However, the response white subjects. Two outcomes were analyzed: change in to treatment with leukotriene modifiers among asthmatics FEV1 after 6 months of montelukast therapy and presence of 67 is highly heterogeneous. asthma exacerbations. Two SNPs were associated with ALOX5, the gene coding for 5-LO, has a tandem repeat change in FEV1, one in the ALOX5 gene (rs2115819), which polymorphism (factor Sp1-binding motif) within its promo- was later replicated in a zileuton treatment response study,73 ter region that has been associated with diminished and the other in the MRP1 (multidrug resistance protein 1) 68 promoter-reporter activity. In a clinical drug trial of a gene (rs119774). MRP1 transports LTC4 into the extracel- lular space and is highly expressed in human bronchial 74 Zileuton epithelial cells. A variation in LTA4H (rs2660845) was Arachidonic Leukotriene associated with a fourfold increase in the risk of asthma Acid 5-LO and A4 exacerbation over the treatment period, whereas LTC4S 5-LO activating Leukotriene C4 synthase rs730012 and the mutant ALOX5 repeat polymorphisms protein were associated with over 70% reductions in exacerbations. Leukotriene C4 This last finding seems to contradict the results from the study of Telleria et al.,70 which may be explained by the Intracellular space small sample sizes of the studies. However, taken together, these data suggest that the ALOX5 repeat polymorphism is Extracellular space an important pharmacogenetic locus. Leukotriene Leukotriene Leukotriene Another polymorphism in the leukotriene pathway C4 D4 E4 associated with response to leukotriene modifiers is the LTC4S C-444A locus. Several studies have shown that Montelukast Zafirlukast asthmatic patients with at least one variant allele (C/C or Pranlukast C/A) had a significantly better response to zafirlukast,75 montelukast76 and pranlukast77 compared with the patients with the two wild-type alleles, as evidenced by increased

FEV1, FVC and PEFR. Leukotriene modifiers are the only class of commonly CysLTR1 CysLTR2 prescribed asthma medications that are administered orally and are subject to first-pass effects. Although few studies Bronchoconstriction have focused on genetic variants involved in leukotriene Mucus secretion modifier-metabolizing , it has been shown that Figure 4 Overview of the leukotriene pathway, demonstrating the site there are significant interindividual differences in plasma of action of antileukotriene therapies. levels of montelukast.67 Variants in SLCO2B1, a gene that

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encodes for the organic anion transporter OATP2B1, may Advances in statistical modeling will also aid in the partly mediate this variability. In fact, a nonsynonymous creation of better predictive models. Asthma treatment SNP in SLCO2B1 has been associated with plasma concen- response is a complex trait that is the result of many trations of montelukast after 1 and 6 months of treatment.78 variables and genetic variants. Thus, integration of large- Heterozygotes for the mutant allele demonstrated up to 30% volume genetic data is needed in order to predict therapeu- reduction in their plasma montelukast levels compared with tic response. The use of machine learning techniques such homozygotes for the wild-type alleles. as Bayesian networks for data analysis may be useful in The existing pharmacogenetic studies on leukotriene constructing future prediction models.39 modifiers are limited by their small sample sizes and lack of replication of their findings. Current data suggest that variants in ALOX5 and LTC4S likely modulate a portion of Conclusion the variability in responses to these agents. Additional recently proposed candidates in the leukotriene pathway The field of pharmacogenetics has come a long way since include TBXA2,79 PTGDR80 and IL-13.81 However, replica- Garrod developed the concept of ‘chemical individuality’ at 85 tions of these findings in larger cohorts of asthmatics are the beginning of the twentieth century, and so has warranted. the field of asthma pharmacogenetics. High-throughput technologies have also allowed the progression of candidate Future directions gene studies to large-scale genetic investigations. The integration of emergent fields of asthma pharmacogenomics Many current studies are limited by their small sample size, such as epigenetics and copy number variations in future imprecise phenotypic definitions, population stratification studies for a more comprehensive pharmacogenomic app- issues, multiple comparisons and the lack of replication of roach may help in the formulation of improved predictive the results. In addition, each genetic variant accounts for a models. This forms the basis for personalized asthma minimal portion of the interindividual variability. Future treatment, which will lead to improved health outcomes studies should focus on these limitations. With the decreas- and more cost-effective care. ing cost of high-throughput technologies, continued colla- borations between genomic investigators are needed to Conflict of interest perform and replicate large-scale genotyping and microarray studies, which will allow the identification of relevant and The authors declare no conflict of interest. true association. Furthermore, future studies should incor- porate multiple genetic variants and haplotype analyses to Acknowledgments formulate a better prognostic model for response to asthma therapy. This work was funded by National Institutes of Health Grants U01 Emergent areas in asthma pharmacogenomics include HL65899 and R01 HL92197. epigenetics and copy number variation. Epigenetics is the study of changes in gene expression patterns that are caused References by mechanism other than changes in the nucleotide sequence of the genetic code itself and include DNA 1 Akinbami LJ, Moorman JE, Liu X. 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