Pharmacogenetics of the 5-lipoxygenase biosyntheticpathway and variable clinical response to montelukastMichael Klotsmana, Timothy P. Yorkc, Sreekumar G. Pillaia,Cristina Vargas-Irwind, Sanjay S. Sharmaa, Edwin J.C.G. van den Oordband Wayne H. Andersona Objective Interindividual clinical response to leukotriene majority of patients with the wild-type alleles had only a modifiers is highly variable, and less efficacious than inhaled corticosteroids in treating asthma. Genetic variability in 5-lipoxygenase biosynthetic and receptor Conclusions The overall mean response to montelukast pathway gene loci may influence cysteinyl-leukotriene may be skewed towards a response phenotype by a small production and subsequent response to leukotriene subset ( < 15%) of asthma patients. CYSLTR2 and ALOX5 polymorphisms may predispose a minority of individuals toexcessive cysteinyl-leukotriene concentrations, yielding a Methods Using data from two clinical trials of 12-week distinct asthma phenotype most likely to respond to duration, post-hoc analyses were performed in 174 leukotriene modifier pharmacotherapy. These findings patients randomized to montelukast. Associations between require replication to establish validity and clinical polymorphisms in 10 candidate genes (ALOX5, ALOX5AP, utility. Pharmacogenetics and Genomics 17:189–196 LTC4S, CYSLTR1, CYSLTR2, PLA2G4A, CYP2C9, CYP3A4, 2007 Lippincott Williams & Wilkins.
ADRB2, and NR3C1) and response to montelukast weremodeled using change in morning peak expiratory flow and Pharmacogenetics and Genomics 2007, 17:189–196 forced expiratory volume in 1 s (FEV1) to define the Keywords: asthma, leukotriene, montelukast, pharmacogenetics aGlaxoSmithKline, Research Triangle Park, North Carolina, bCenter for BiomarkerResearch and Personalized Medicine, Department of Pharmacy, Virginia Institute Results In our sample, eight out of 25 markers in 10 for Psychiatric and Behavioral Genetics, Medical College of Virginia of Virginia candidate genes were statistically associated with Commonwealth University, cDepartment of Human Genetics and Virginia Institute response to montelukast, with an estimated proportion of for Psychiatric and Behavioral Genetics, Virginia Commonwealth UniversitySchool of Medicine, Richmond, Virginia, USA and dDepartment of Pharmacy, false discoveries of 16%. The strongest statistical evidence Medical College of Virginia of Virginia Commonwealth University, Richmond, of clinically relevant pharmacogenetic effects peak expiratory flow were identified in CYSLTR2 (rs91227 and Correspondence and requests for reprints to Wayne H. Anderson, PhD, rs912278; P = 0.02 and P = 0.02, respectively) and ALOX5 Therapeutic Area Head, Respiratory Translational Medicine and Genetics,GlaxoSmithKline, PO Box 13398, Research Triangle Park, NC 27709-3398, (rs4987105 and rs4986832; P = 0.01 and P = 0.01, respectively). Patients with these variant genotypes, found Tel: + 1 919 483 5309; fax: + 1 919 315 0311;e-mail: [email protected] in roughly 10–13% of patients, had an 18–25% improvement in peak expiratory flow. In contrast, the Received 16 January 2006 Accepted October 13 2006 A leukotriene-driven mechanism may represent a unique Asthma is a chronic inflammatory airway disease partly asthma phenotype requiring targeted pharmacotherapy.
characterized by increased numbers of activated eosino-phils, mast cells, macrophages, and T lymphocytes in Derivatives of arachidonic acid, leukotrienes are rapidly the airway mucosa and lumen. Cysteinyl-leukotrienes generated at de novo sites of inflammation via the 5- (CysLT) (leukotriene C4, D4, and E4,), produced lipoxygenase (5-LO) biosynthetic pathway [5]. The primarily from eosinophils and mast cells in the airways, pathway begins when inflammatory stimuli trigger the are biologically active lipids that amplify and/or maintain translocation of phospholipase A2 which, in turn, releases inflammation by directing T-cell migration [1]. Leuko- arachidonic acid from cell membrane phospholipids.
trienes are also mediators and modulators in the early Next, 5-LO catalyzes the conversion of arachidonic acid phase of the asthmatic response to inhaled allergens [2].
to the unstable intermediate leukotriene A4, with 5-LO- Indeed, elevated leukotriene levels in the airways of activating protein acting as a cofactor. Leukotriene A4 is patients with asthma, and induction of airflow obstruction then rapidly converted to leukotriene C4 by leukotriene upon leukotriene inhalation are well documented [3–5].
C4 synthase. Leukotriene C4 is transported extracellularly Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
where stepwise removal of amino acids results in the USA). Spirometry was performed according to the formation of leukotriene E4 and leukotriene D4. Once American Thoracic Society published guidelines [20].
formed, CysLT act through the CysLT1 and CysLT2 Spirometry measures were performed in triplicate under receptors on target cells including bronchial smooth a technician’s supervision. Before study enrollment, the muscle and inflammatory leucocytes [6]. The CysLT1 aims of these clinical trials and pharmacogenetic analyses receptor mediates bronchoconstriction, plasma exudation, were fully explained and informed consent was obtained and mucus secretion, whereas CysLT2 may play a role in from each participant. The study protocols were reviewed smooth muscle cell proliferation [7]. Additionally, the and approved by the appropriate Institutional Review relative concentration of local CysLT partly dictates the degree of receptor saturation and may alter the rate ofexogenous ligand binding. Genetic variability in 5-LO pathway genes may influence CysLT production and Eligible participants entered a 2-week run-in period, subsequent response to leukotriene modifiers [8–11].
during which all participants replaced their oral or inhaledshort-acting b2-agonist with albuterol inhalation aerosol The leukotriene modifier montelukast is a selective that was prescribed as-needed for the relief of acute antagonist of the leukotriene CysLT1 receptor [12].
asthma symptoms. Peak expiratory flow (PEF), albuterol Interindividual clinical response to leukotriene modifiers use, asthma symptoms, and nighttime awakenings were is highly variable, and less efficacious than inhaled recorded daily by the participants on a diary card. After corticosteroids in treating asthma [13–15]. The clinical, the run-in period, participants meeting randomization environmental, and genetic determinants of this hetero- criteria entered the double-blind phase of the study and geneity in response are not understood. The ability to were randomized to receive one of the following predict, by genetic means or otherwise, which patients treatments for a 12-week period: fluticasone propionate/ favorably respond to leukotriene modifiers may improve salmeterol 100/50 mg Diskus twice daily plus placebo montelukast once daily or oral montelukast 10 mg oncedaily plus placebo Diskus twice daily. The current This study was undertaken to evaluate associations analyses were restricted to participants randomized to between polymorphisms in key 5-LO biosynthetic path- way and receptor gene loci and pulmonary functionmeasures in asthma patients randomized to montelukast Information on the participant’s ethnicity, medical in a clinical trial setting. Specifically, genes encoding for condition, and treatment, medical history, and family phospholipase A2 (PLA2G4A), leukotriene C4 synthase medical history was collected and recorded according to a (LTC4S), CysLT1 receptor (CYSLTR1), CysLT2 receptor standardized protocol. Baseline data for PEF, albuterol (CYSLTR2), 5-LO-activating protein (ALOX5AP), 5-LO use, asthma symptoms, and nighttime awakenings was (ALOX5), and two cytochrome P450 isoforms (CYP3A4 defined as the mean value over the 7 days before randomization. Baseline FEV1 was defined as the b2-adrenergic receptor (ADRB2) was assessed because randomization visit FEV1 measurement. During the mechanistically, the b2-adrenergic receptor may influence study, FEV1 was measured at treatment weeks 1, 4, 8, response to leukotriene modifiers via the so-called cross- 12, and 3 days after the treatment. Weekly mean morning talk between the activation of Gs receptors causing airway PEF (AM PEF) values, averaged from the daily diary card smooth muscle relaxation, and Gq-receptors that cause muscle contraction [16,17]. Finally, the glucocorticoidreceptor (NR3C1) was considered because glucocorticoids Genotyping was performed in a blinded manner at acentral laboratory (GlaxoSmithKline) using collected blood samples on all participants for whom a sample was available. Previously characterized coding single Data from two identical studies in which DNA sampling nucleotide polymorphisms (SNPs) in the b2-adrenergic was performed were used in these post-hoc analyses. The receptor gene [21] (HUGO nomenclature: ADRB2; replicate trials, previously reported [18,19], enrolled Sequence Accession ID: NM_000024), phospholipase A2 asthma patients who: (i) were at least 15 years of age; (ii) had a history of persistent asthma of at least 6 months duration; (iii) recorded a forced expiratory volume in 1 s NM_006639), CysLT2 receptor (CYSLTR2; NM_020377), (FEV1) between 50 and 80% of the predicted normal; and 5-LO-activating protein (ALOX5AP; NM_001629), 5-LO (iv) demonstrated Z 15% reversibility within 30 min (ALOX5; NM_000698), glucocorticoid receptor (NR3C1; following two puffs (180 mg) of albuterol (Ventolin; NM_000176), and cytochrome P450 isoforms (CYP3A4; GlaxoSmithKline, Research Triangle Park, North Carolina, NM_017460 and CYP2C9; NM_000771) were genotyped.
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Pharmacogenetics of 5-lipoxygenase pathway Klotsman et al.
Markers were qualitatively selected on the basis of the false discovery rate (pFDR) [24,25]. The pFDR can available literature. A complete listing of polymorphisms either be interpreted as an estimate of the proportion of evaluated is available online on the journal’s website.
false discoveries among the markers called significant orthe probability that a significant marker is a false discovery. To calculate the pFDR, the proportion of tests Changes in AM PEF and percentage predicted FEV for which the null hypothesis is true needs to be measured over a 12-week study period, were used as estimated. For this purpose, we used the ‘lowest slope’ proxy measures to quantify clinical response to montelu- estimate, known to be conservatively biased toward one [26]. In addition to its pleasant interpretation, measures of assessing airway caliber in patients with asthma [22,23]. An overall treatment effect was calcu- effects of correlated tests in general [25,27–32]. The lated by subtracting baseline value from the week 12 estimate the ratio of false to total discoveries in a study.
Correlated tests mainly increase the variance of theseestimates. The FDR statistics themselves that are the Tests for genotype–phenotype associations were carried means of these estimates tend to, however, remain out using a total of 25 genotypic markers, against two dependent variables. Full models including additive anddominant genetic effects were estimated using twodummy variables G1 and G2, respectively. The value for In addition to the single-marker analyses, we tested for G1 corresponded to the number of rare alleles and the associations between our outcome measures and SNP value of G2 was a binary variable indicating heterozygosity haplotypes. To identify regions with low recombination status. Models with only additive genetic effects were needed for accurate haplotype analyses in samples of also estimated. P values were estimated by fitting unrelated participants, the Haploview program (version linear models and significance was initially assessed at 2.05) was used to analyze the 24 SNPs [33,34].
the 5% level. Baseline measures were corrected for Haplotype blocks were defined using the default block covariates that correlated strongly with their respective search procedure [35]. This criteria defines ‘strong outcome. Baseline FEV1 was correlated with log10 percent linkage disequilibrium (LD)’ for marker pairs if the D0 reversibility at baseline (r = – 0.34; P < 0.01). Baseline upper and lower 95% confidence bounds encompass 0.98 AM PEF was adjusted for age (r = – 0.19; P = 0.01), and 0.70, respectively. Evidence for historical recombina- tion is given when the upper bound of D0 is less than 0.9.
A haplotype block is defined when less than 5% of marker than five were omitted. Following convention, the pairs in a region show evidence for historical recombina- microsatellite marker in ALOX5 was coded as either tion. Makers with minor allele frequencies less than 5% the five repeat allele (wild-type) or the non-5 repeat were not included. Next, SNPs that were in the same block were analyzed together to study the association ofthe haplotypes within that block with each of the The reduction of model error variance by the inclusion of outcome variables. For these analyses, we used the additional covariates in the linear regression models may UNPHASED (version 2.403) program [36]. UNPHASED result in a more powerful test of genetic effects on does not attempt to assign haplotypes to individual treatment if, in fact, it could be verified that these participants, but uses an EM algorithm to estimate the covariates have no involvement in the effect of the frequencies of the different haplotypes and test whether treatment itself. If the covariates are indicators of the means differ across these haplotypes. A pooled treatment effect, then meaningful variation would be variance estimate is used by UNPHASED, in part, to regressed out. We therefore ran models with and without eliminate variance estimates on the basis of a few covariate predictors including height, age, sex, reversi- bility, short-acting albuterol use, baseline respiratory applied because it yields a more stable estimate of measures, and investigator site, and observed no overall variance, and differences in variance are not of direct change in significant results. To examine whether interest. Haplotypes with a frequency less than 2% were significant results were due to the racial composition, omitted from these analyses and the baseline measures we fitted linear regression models that included race as a were adjusted for correlated covariates as described predictor and compared them to models without race.
above. Permutation testing was also performed for both Observed estimates were similar with the addition of the single-marker and haplotype associations. The critical race in the model (results not shown).
region that specifies a type I error rate of 5% wasdetermined from the null distribution of the F statistic by To assess whether the SNPs with P values smaller than 10 000 permutations of the response variable over 0.05 were false discoveries, we estimated the positive Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
identified for rs912277 and rs912278 in CYSLTR2 (P = 0.02 and P = 0.02, respectively) and for ALOX5 Demographic and baseline clinical characteristics for markers rs4987105 and rs4986832 (P = 0.01 and P = 0.01, participants randomized to montelukast with available respectively) (Table 2). Response to montelukast was PGx sampling (n = 174; 41% of intent-to-treat sample) markedly higher in CYSLTR2 and ALOX5 genotypes with were similar to those observed in the general intent-to- the variant allele (Table 3). P values for single-marker treat sample (ITT data previously published [18,19]).
associations were also determined by permutation test- Baseline pulmonary function and other indicators of ing. The empirical critical region estimated for each asthma severity were consistent across the two protocols marker response combination did not deviate from the (Table 1). Tests of homogeneity did not identify statis- model-based estimate (data not shown).
tically significant departures for the primary endpointsand key demographics. Of the 174 participants with Admixture according to self-reported ethnicity was care- baseline data, eight individuals lacked both FEV1 and AM fully evaluated. Admixture may lead to spurious geno- PEF endpoint measures (n = 166 patients analyzed).
type-outcome associations when both treatment effectand allele frequencies differ by ethnicity. We tested for All measured genotypes were found to be in Hardy– each of these conditions and did not find any genotype- Weinberg equilibrium as assessed by a w2 goodness-of-fit outcome combinations for which both conditions were test. At baseline, no phenotype–genotype associations at satisfied. All reported associations were therefore unlikely the 5% significance level were identified (data not to be the result of the racial composition of the sample.
To further examine whether statistically significantresults were due to the racial composition, we fitted linear regression models that included race as a predictor.
Regression modeling was performed to test for associa- After statistically adjusting for ethnicity, all of our tions between individual genotypes and response to findings remained significant. Finally, we repeated our montelukast, as defined by change from baseline in FEV1 analysis in Caucasians only (79% of our total sample).
percentage predicted, and AM PEF (Table 2). Associa- In Caucasians, six of nine associations remained statisti- tions between at least one selected marker in ADRB2, cally significant under the full model (Supplemental NR3C1, ALOX5, and CYSLTR2 and at least one respiratory outcome measure were identified (Table 3). Of note,several markers at each of these loci were found to be in LD, thus partially explaining the consistency of geno- Focusing on the two main outcome measures (percent type–phenotype associations observed at each locus change FEV1 and AM PEF), nine of the 50 association tests resulted in P values smaller than 0.05. The pFDRwas calculated to estimate the proportion of false Using change in AM PEF to estimate treatment effects, discoveries (using an a = 0.05, 2.5 false positives would evidence for genotype–phenotype associations were be expected by chance). Using the conservative ‘lowestslope’ method [26], the estimated number of true nullhypotheses was 0.92 with a corresponding pFDR of 0.16. Thus, the expected proportion of false discoveries among the nine tests with P values less than 0.05 is Seven haplotype blocks, consistent with the observed LD patterns, were identified (Table 4). Haplotype frequen- cies less than 2% were omitted. All analyses were repeated in self-identified Caucasians with similar haplotype block structures identified (data not shown).
Using the determined block structure, additional sig- nificance testing was performed to detect haplotype– phenotype associations (Table 4). Consistent with the single-marker results, ALOX5 and CYSLTR2 haplotypes were also associated with AM PEF. The observed ALOX5 haplotype was composed of a common haplotype (CG) AM PEF, weekly mean morning peak expiratory flow.
and a relatively less frequent haplotype (TA). The less Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Pharmacogenetics of 5-lipoxygenase pathway Klotsman et al.
P values for single marker polymorphisms associated with response to montelukast characterized by change in FEV1 and AM PEF AM PEF, Weekly mean morning peak expiratory flow.
aP values r 0.05 in bold. Additive and dominant models as specified.
bLinkage disequilibrium r2 = 0.99.
cLinkage disequilibrium r2 = 0.31.
dLinkage disequilibrium r2 = 0.98.
eOnly microsatellite marker tested. Minor allele frequency is for all non-5 repeats.
fMarker (referenced as rs-gsk7352707) is located 208 bp upstream of exon 2 (or at position 179154983 on chromosome 5; NCBI Build 36.1).
gLinkage disequilibrium r2 = 0.10.
Mean clinical responses to montelukast for statistically frequent haplotype (B19%) was associated with greater response defined by change in AM PEF from baseline (P = 0.003). Mean improvement in AM PEF was greatestamong patients with the less frequent ( haplotype (P = 0.02). P values empirically estimated from 10 000 permutations of participant responses remained essentially the same as theoretical values.
Additionally, for haplotypes with more than two alleles, we tested whether individual haplotypes differed from all other haplotypes grouped together within a block. The infrequent NR3C1 TT haplotype was found to have a significantly attentuated mean change in PEF as compared with the other haplotypes (P = 0.05), whereas the single- marker analysis showed variant NR3C1 homozygotes to have the largest improvement in percent predicted FEV1.
The common CYSLTR2 TT and TC haplotypes were each shown to have a significantly lower mean change in AM PEF (P = 0.007 and P = 0.04), observations that are consistent with the single-marker results.
Our results identify genetic variants in the 5-LO pathway aNumber of ALOX5 alleles with five repeats of the Sp1-binding motif GGGCGG.
associated with therapeutic response to montelukast. In Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
P values for associations of haplotypes with respiratory with asthma did not identify any pharmacogenetic associations between ALOX5 and response to leukotriene modifier therapy [38]. The ALOX5 microsatellite wasassociated with response in the present study. We also observed the variant rs4987105 and rs4986832 homo-zygotes to be associated with improved AM PEF. The underlying mechanisms to explain these clinical observa- Although results were not as robust as for ALOX5, CYSLTR2 remains a plausible candidate from a biological perspective. CYSLTR2 encodes for a 346 amino acid protein with 38% amino acid identity to the CysLT ALOX5AP (rs3803278, rs12721458, rs3803277) receptor [39]. It is believed that both CysLT1 and CysLT2 may be potent biological mediators in the pathophysiology of asthma by predisposing to broncho- constriction, vascular hyperpermeability, and mucus hypersecretion in asthmatic patients. As such, it is hypothesized that leukotriene modifier therapy may be more efficacious among asthma patients with concen- trated leukotriene activity [12,40]. Mapped to 13q14, a chromosomal region linked to asthma, it has been shown that CYSLT2 is associated with: (i) susceptibility to asthma in Caucasians [8] and Japanese [41]; (ii) atopic asthma in a founder population with a high prevalence of There were no significant associations for baseline percent predicted FEV1 and atopy [9]; and (iii) aspirin intolerance in Korean patients with asthma [42]. Moreover, in comparison to wild-type AM PEF, weekly mean morning peak expiratory flow.
a CYSLTR2, CYSLTR2 coding variants at positions A601G bEach block is represented by a set of markers in parenthesis. The stated order of [13] and M202V [12] demonstrated reduced leukotriene marker alleles corresponds to the order of markers listed for each block.
potency as measured by calcium flux assays. If CYLTR2polymorphisms do indeed predispose individuals to a our sample of 166 asthma patients, eight out of 25 leukotriene-based asthma phenotype, then our results are markers in 10 candidate genes were statistically asso- consistent with the notion that leukotriene modifier ciated with response to montelukast, with an estimated therapies are more efficacious among a small subset of proportion of false discoveries of 31%. Specifically, using patients with concentrated leukotriene activity (i.e. those patients harboring CYSLTR2 variants). This is also period) to approximate the montelukast response phe- consistent with the observations of Szefler and colleagues notype, the strongest statistical evidence for clinically [43] who report that some asthmatic children (B22% of relevant associations were identified in CYSLTR2 and sample) who demonstrated an improvement in FEV1 of 7.5% or greater had elevated median leukotriene con-centration. The direct functional significance of theCYSLTR2 SNPs we evaluated is unknown. Systematic It has previously been shown that polymorphisms in analysis of the haplotype structure and sequence variation ALOX5 are associated with clinical response to leuko- of CYSLTR2 will be needed to identify the actual casual triene modifier therapy. In one study of 221 asthma variant(s) predisposing to asthma and/or influencing patients, the ALOX5 microsatellite was tested against response to ABT-761, an experimental leukotrienemodifier [10]. Among predominant homozygotes (repeatlength = 5; n = 64) and heterozygotes (n = 40), mean The net effect of signaling events from Gs (e.g. b2- FEV1 improved by approximately 18.8 ± 3.6 and 23.3 ± 6%, adrenergic receptor activation ultimately results in a respectively. In comparison, variant homozygotes (non-5 decrease of intracellular Ca2 + ) and Gq (e.g. cysteinyl repeats; n = 10) demonstrated a – 1.2 ± 2.9% change.
leukotriene receptor activation increase intracellular In a more recent study, the ALOX5 rs2115819 SNP Ca2 + stores) on airway smooth muscle cells, muscle was associated with change in FEV1 and non-5 repeat tone, and airway responsiveness is a critical and dynamic carriers had higher rates of exacerbations among 61 process responsible for maintaining homeostasis. This Caucasian patients monitored for 6 months [37].
study is supported by emerging in-vitro data, which Conversely, a smaller study conducted in 52 patients suggests that the propensity for one G-protein coupled Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Pharmacogenetics of 5-lipoxygenase pathway Klotsman et al.
receptor signal to alter another, or the so-called cross-talk with long-acting b-agonists are, on average, more between Gas-receptors and Gaq-receptors, may partly efficacious than montelukast in treating asthma patients underly pharmacogenetic variation of b-agonists and [43,47–51], further elucidation of 5-LO pharmaco- ADRB2 polymorphisms [44,45]. Considering the postu- genetics may serve to identify the subset of asthma lated interdependence of Gas and Gaq-receptor signaling, patients most likely to benefit from leukotriene modifier genetic variability influencing the Ga signaling may also hold clinical relevance for leukotriene modifier therapy.
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