Original article 189
Pharmacogenetics of the 5-lipoxygenase biosynthetic
pathway and variable clinical response to montelukast
Michael Klotsmana, Timothy P. Yorkc, Sreekumar G. Pillaia,
Cristina Vargas-Irwind, Sanjay S. Sharmaa, Edwin J.C.G. van den Oordb
and Wayne H. Andersona
Objective Interindividual clinical response to leukotriene
modifiers is highly variable, and less efficacious than
inhaled corticosteroids in treating asthma. Genetic
variability in 5-lipoxygenase biosynthetic and receptor
pathway gene loci may influence cysteinyl-leukotriene
production and subsequent response to leukotriene
modifiers.
Methods Using data from two clinical trials of 12-week
duration, post-hoc analyses were performed in 174
patients randomized to montelukast. Associations between
polymorphisms in 10 candidate genes (ALOX5, ALOX5AP,
LTC4S, CYSLTR1, CYSLTR2, PLA2G4A, CYP2C9, CYP3A4,
ADRB2, and NR3C1) and response to montelukast were
modeled using change in morning peak expiratory flow and
forced expiratory volume in 1 s (FEV1) to define the
response phenotype.
Results In our sample, eight out of 25 markers in 10
candidate genes were statistically associated with
response to montelukast, with an estimated proportion of
false discoveries of 16%. The strongest statistical evidence
of clinically relevant pharmacogenetic effects peak
expiratory flow were identified in CYSLTR2 (rs91227 and
rs912278; P = 0.02 and P = 0.02, respectively) and ALOX5
(rs4987105 and rs4986832; P = 0.01 and P = 0.01,
respectively). Patients with these variant genotypes, found
in roughly 10–13% of patients, had an 18–25%
improvement in peak expiratory flow. In contrast, the
Introduction
Asthma is a chronic inflammatory airway disease partly
characterized by increased numbers of activated eosino-
phils, mast cells, macrophages, and T lymphocytes in
the airway mucosa and lumen. Cysteinyl-leukotrienes
(CysLT) (leukotriene C4, D4, and E4,), produced
primarily from eosinophils and mast cells in the airways,
are biologically active lipids that amplify and/or maintain
inflammation by directing T-cell migration [1]. Leuko-
trienes are also mediators and modulators in the early
phase of the asthmatic response to inhaled allergens [2].
Indeed, elevated leukotriene levels in the airways of
patients with asthma, and induction of airflow obstruction
upon leukotriene inhalation are well documented [3–5].
1744-6872

c 2007 Lippincott Williams & Wilkins
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
majority of patients with the wild-type alleles had only a
marginal (8–10%) improvement.
Conclusions The overall mean response to montelukast
may be skewed towards a response phenotype by a small
subset ( < 15%) of asthma patients. CYSLTR2 and ALOX5
polymorphisms may predispose a minority of individuals to
excessive cysteinyl-leukotriene concentrations, yielding a
distinct asthma phenotype most likely to respond to
leukotriene modifier pharmacotherapy. These findings
require replication to establish validity and clinical
utility. Pharmacogenetics and Genomics 17:189–196

c 2007 Lippincott Williams & Wilkins.
Pharmacogenetics and Genomics 2007, 17:189–196
Keywords: asthma, leukotriene, montelukast, pharmacogenetics
a
GlaxoSmithKline, Research Triangle Park, North Carolina, bCenter for Biomarker
Research and Personalized Medicine, Department of Pharmacy, Virginia Institute
for Psychiatric and Behavioral Genetics, Medical College of Virginia of Virginia
Commonwealth University, cDepartment of Human Genetics and Virginia Institute
for Psychiatric and Behavioral Genetics, Virginia Commonwealth University
School of Medicine, Richmond, Virginia, USA and dDepartment of Pharmacy,
Medical College of Virginia of Virginia Commonwealth University, Richmond,
Virginia, USA
Correspondence and requests for reprints to Wayne H. Anderson, PhD,
Therapeutic Area Head, Respiratory Translational Medicine and Genetics,
GlaxoSmithKline, PO Box 13398, Research Triangle Park, NC 27709-3398,
USA
Tel: + 1 919 483 5309; fax: + 1 919 315 0311;
e-mail: Wayne.h.anderson@gsk.com
Received 16 January 2006 Accepted October 13 2006
A leukotriene-driven mechanism may represent a unique
asthma phenotype requiring targeted pharmacotherapy.
Derivatives of arachidonic acid, leukotrienes are rapidly
generated at de novo sites of inflammation via the 5-
lipoxygenase (5-LO) biosynthetic pathway [5]. The
pathway begins when inflammatory stimuli trigger the
translocation of phospholipase A2 which, in turn, releases
arachidonic acid from cell membrane phospholipids.
Next, 5-LO catalyzes the conversion of arachidonic acid
to the unstable intermediate leukotriene A4, with 5-LO-
activating protein acting as a cofactor. Leukotriene A4 is
then rapidly converted to leukotriene C4 by leukotriene
C4 synthase. Leukotriene C4 is transported extracellularly
 190 Pharmacogenetics and Genomics 2007, Vol 17 No 3
where stepwise removal of amino acids results in the
formation of leukotriene E4 and leukotriene D4. Once
formed, CysLT act through the CysLT1 and CysLT2
receptors on target cells including bronchial smooth
muscle and inflammatory leucocytes [6]. The CysLT1
receptor mediates bronchoconstriction, plasma exudation,
and mucus secretion, whereas CysLT2 may play a role in
smooth muscle cell proliferation [7]. Additionally, the
relative concentration of local CysLT partly dictates the
degree of receptor saturation and may alter the rate of
exogenous ligand binding. Genetic variability in 5-LO
pathway genes may influence CysLT production and
subsequent response to leukotriene modifiers [8–11].
The leukotriene modifier montelukast is a selective
antagonist of the leukotriene CysLT1 receptor [12].
Interindividual clinical response to leukotriene modifiers
is highly variable, and less efficacious than inhaled
corticosteroids in treating asthma [13–15]. The clinical,
environmental, and genetic determinants of this hetero-
geneity in response are not understood. The ability to
predict, by genetic means or otherwise, which patients
favorably respond to leukotriene modifiers may improve
clinical treatment guidelines.
This study was undertaken to evaluate associations
between polymorphisms in key 5-LO biosynthetic path-
way and receptor gene loci and pulmonary function
measures in asthma patients randomized to montelukast
in a clinical trial setting. Specifically, genes encoding for
phospholipase A2 (PLA2G4A), leukotriene C4 synthase
(LTC4S), CysLT1 receptor (CYSLTR1), CysLT2 receptor
(CYSLTR2), 5-LO-activating protein (ALOX5AP), 5-LO
(ALOX5), and two cytochrome P450 isoforms (CYP3A4
and CYP2C9) were evaluated. Additionally, the
b2-adrenergic receptor (ADRB2) was assessed because
mechanistically, the b2-adrenergic receptor may influence
response to leukotriene modifiers via the so-called cross-
talk between the activation of Gs receptors causing airway
smooth muscle relaxation, and Gq-receptors that cause
muscle contraction [16,17]. Finally, the glucocorticoid
receptor (NR3C1) was considered because glucocorticoids
induce the upregulation of 5-LO.
Methods
Participants
Data from two identical studies in which DNA sampling
was performed were used in these post-hoc analyses. The
replicate trials, previously reported [18,19], enrolled
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
(FEV1) between 50 and 80% of the predicted normal; and
(iv) demonstrated Z 15% reversibility within 30 min
following two puffs (180 mg) of albuterol (Ventolin;
GlaxoSmithKline, Research Triangle Park, North Carolina,
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
USA). Spirometry was performed according to the
American Thoracic Society published guidelines [20].
Spirometry measures were performed in triplicate under
a technician’s supervision. Before study enrollment, the
aims of these clinical trials and pharmacogenetic analyses
were fully explained and informed consent was obtained
from each participant. The study protocols were reviewed
and approved by the appropriate Institutional Review
Boards.
Study design
Eligible participants entered a 2-week run-in period,
during which all participants replaced their oral or inhaled
short-acting b2-agonist with albuterol inhalation aerosol
that was prescribed as-needed for the relief of acute
asthma symptoms. Peak expiratory flow (PEF), albuterol
use, asthma symptoms, and nighttime awakenings were
recorded daily by the participants on a diary card. After
the run-in period, participants meeting randomization
criteria entered the double-blind phase of the study and
were randomized to receive one of the following
treatments for a 12-week period: fluticasone propionate/
salmeterol 100/50 mg Diskus twice daily plus placebo
montelukast once daily or oral montelukast 10 mg once
daily plus placebo Diskus twice daily. The current
analyses were restricted to participants randomized to
therapy with montelukast.
Information on the participant’s ethnicity, medical
condition, and treatment, medical history, and family
medical history was collected and recorded according to a
standardized protocol. Baseline data for PEF, albuterol
use, asthma symptoms, and nighttime awakenings was
defined as the mean value over the 7 days before
randomization. Baseline FEV1 was defined as the
randomization visit FEV1 measurement. During the
study, FEV1 was measured at treatment weeks 1, 4, 8,
12, and 3 days after the treatment. Weekly mean morning
PEF (AM PEF) values, averaged from the daily diary card
data, were analyzed.
Genotyping
Genotyping was performed in a blinded manner at a
central laboratory (GlaxoSmithKline) using collected
blood samples on all participants for whom a sample
was available. Previously characterized coding single
nucleotide polymorphisms (SNPs) in the b2-adrenergic
receptor gene [21] (HUGO nomenclature: ADRB2;
Sequence Accession ID: NM_000024), phospholipase A2
(PLA2G4A; NM_024420), leukotriene C4 synthase
(LTC4S; NM_000897), CysLT1 receptor (CYSLTR1;
NM_006639), CysLT2 receptor (CYSLTR2; NM_020377),
5-LO-activating protein (ALOX5AP; NM_001629), 5-LO
(ALOX5; NM_000698), glucocorticoid receptor (NR3C1;
NM_000176), and cytochrome P450 isoforms (CYP3A4;
NM_017460 and CYP2C9; NM_000771) were genotyped.

Markers were qualitatively selected on the basis of the
available literature. A complete listing of polymorphisms
evaluated is available online on the journal’s website.
Statistical analysis
Changes in AM PEF and percentage predicted FEV1,
measured over a 12-week study period, were used as
proxy measures to quantify clinical response to montelu-
kast. AM PEF and FEV1 are well established diagnostic
measures of assessing airway caliber in patients with
asthma [22,23]. An overall treatment effect was calcu-
lated by subtracting baseline value from the week 12
endpoint value.
Tests for genotype–phenotype associations were carried
out using a total of 25 genotypic markers, against two
dependent variables. Full models including additive and
dominant genetic effects were estimated using two
dummy variables G1 and G2, respectively. The value for
G1 corresponded to the number of rare alleles and the
value of G2 was a binary variable indicating heterozygosity
status. Models with only additive genetic effects were
also estimated. P values were estimated by fitting
linear models and significance was initially assessed at
the 5% level. Baseline measures were corrected for
covariates that correlated strongly with their respective
outcome. Baseline FEV1 was correlated with log10 percent
reversibility at baseline (r = – 0.34; P < 0.01). Baseline
AM PEF was adjusted for age (r = – 0.19; P = 0.01),
sex (r = – 0.68; P < 0.01), and height (r = 0.57;
P < 0.01). Genotype classes with frequencies less
than five were omitted. Following convention, the
microsatellite marker in ALOX5 was coded as either
the five repeat allele (wild-type) or the non-5 repeat
alleles [10].
The reduction of model error variance by the inclusion of
additional covariates in the linear regression models may
result in a more powerful test of genetic effects on
treatment if, in fact, it could be verified that these
covariates have no involvement in the effect of the
treatment itself. If the covariates are indicators of
treatment effect, then meaningful variation would be
regressed out. We therefore ran models with and without
covariate predictors including height, age, sex, reversi-
bility, short-acting albuterol use, baseline respiratory
measures, and investigator site, and observed no overall
change in significant results. To examine whether
significant results were due to the racial composition,
we fitted linear regression models that included race as a
predictor and compared them to models without race.
Observed estimates were similar with the addition of the
race in the model (results not shown).
To assess whether the SNPs with P values smaller than
0.05 were false discoveries, we estimated the positive
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Pharmacogenetics of 5-lipoxygenase pathway Klotsman et al. 191
false discovery rate (pFDR) [24,25]. The pFDR can
either be interpreted as an estimate of the proportion of
false discoveries among the markers called significant or
the probability that a significant marker is a false
discovery. To calculate the pFDR, the proportion of tests
for which the null hypothesis is true needs to be
estimated. For this purpose, we used the ‘lowest slope’
estimate, known to be conservatively biased toward
one [26]. In addition to its pleasant interpretation,
pFDR methods appear fairly robust against the
effects of correlated tests in general [25,27–32]. The
intuitive explanation is that these methods
estimate the ratio of false to total discoveries in a study.
Correlated tests mainly increase the variance of these
estimates. The FDR statistics themselves that are the
means of these estimates tend to, however, remain
similar.
In addition to the single-marker analyses, we tested for
associations between our outcome measures and SNP
haplotypes. To identify regions with low recombination
needed for accurate haplotype analyses in samples of
unrelated participants, the Haploview program (version
2.05) was used to analyze the 24 SNPs [33,34].
Haplotype blocks were defined using the default block
search procedure [35]. This criteria defines ‘strong
linkage disequilibrium (LD)’ for marker pairs if the D0
upper and lower 95% confidence bounds encompass 0.98
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
pairs in a region show evidence for historical recombina-
tion. Makers with minor allele frequencies less than 5%
were not included. Next, SNPs that were in the same
block were analyzed together to study the association of
the haplotypes within that block with each of the
outcome variables. For these analyses, we used the
UNPHASED (version 2.403) program [36]. UNPHASED
does not attempt to assign haplotypes to individual
participants, but uses an EM algorithm to estimate the
frequencies of the different haplotypes and test whether
the means differ across these haplotypes. A pooled
variance estimate is used by UNPHASED, in part, to
eliminate variance estimates on the basis of a few
observations [36]. This assumption is commonly
applied because it yields a more stable estimate of
variance, and differences in variance are not of direct
interest. Haplotypes with a frequency less than 2% were
omitted from these analyses and the baseline measures
were adjusted for correlated covariates as described
above. Permutation testing was also performed for both
single-marker and haplotype associations. The critical
region that specifies a type I error rate of 5% was
determined from the null distribution of the F statistic by
10 000 permutations of the response variable over
participants.
 192 Pharmacogenetics and Genomics 2007, Vol 17 No 3

Results identified for rs912277 and rs912278 in CYSLTR2

Baseline distributions (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 endpoints
and 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 effect
and 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.
shown). To further examine whether statistically significant
results were due to the racial composition, we fitted
Single-marker associations 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 Table 1).
outcome measure were identified (Table 3). Of note,
several markers at each of these loci were found to be in Control of false discoveries
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
(Table 2). tests resulted in P values smaller than 0.05. The pFDR
was 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 ‘lowest
slope’ method [26], the estimated number of true null
hypotheses was 0.92 with a corresponding pFDR of
Table 1 Baseline clinical characteristics 0.16. Thus, the expected proportion of false discoveries
Characteristic Protocol Protocol Total among the nine tests with P values less than 0.05 is
SAS40020 SAS40021 16% [32].
N 94 80 174
Age (SD) 37.9 (13.4) 37.2 (12.9) 37.5 (13.2)
Sex, n (%)
Haplotype associations
Male 40 (42.6) 41 (51.3) 81 (46.6) Seven haplotype blocks, consistent with the observed LD
Female 54 (57.4) 39 (48.8) 93 (53.4) patterns, were identified (Table 4). Haplotype frequen-
Race, n (%)
Caucasian 78 (83.0) 60 (75.0) 138 (79.3) cies less than 2% were omitted. All analyses were
Hispanic 7 (7.4) 15 (18.8) 22 (12.6) repeated in self-identified Caucasians with similar
African-American 5 (5.3) 5 (6.3) 10 (5.8)
Other 4 (4.3) 0 4 (2.3)
haplotype block structures identified (data not shown).
FEV1 % reversibility (SD) 25.1 (10.9) 25.4 (13.9) 25.2 (12.3)
FEV1 % predicted (SD) 66.8 (8.2) 65.8 (9.0) 66.3 (8.5)
Night-time awakenings 0.53 (0.57) 0.46 (0.56) 0.50 (0.57)
Using the determined block structure, additional sig-
(SD) nificance testing was performed to detect haplotype–
Albuterol use, total puffs 4.8 (3.6) 4.6 (2.5) 4.7 (3.1) phenotype associations (Table 4). Consistent with the
(SD)
Asthma symptom scores 30.2 (14.0) 33.0 (16.4) 31.5 (15.2) single-marker results, ALOX5 and CYSLTR2 haplotypes
(SD) were also associated with AM PEF. The observed ALOX5
AM PEF (SD) 357.0 (121.8) 361.8 (117.5) 359.2 (119.5) 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

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 Pharmacogenetics of 5-lipoxygenase pathway Klotsman et al. 193

Table 2 P values for single marker polymorphisms associated with response to montelukast characterized by change in FEV1 and AM PEF
measuresa
FEV1 PEF

Gene symbol Marker Minor allele frequency Additive Full-model Additive Full-model

CYP2C9 rs1799853 0.10 0.846 0.857 0.935 0.439

CYP3A4 rs1057910 0.05 0.853 0.853 0.731 0.731
rs2740574 0.05 0.987 0.987 0.837 0.837
ADRB2 rs1042713 0.41 0.372 0.306 0.226 0.417
rs1042714b 0.39 0.458 0.021 0.704 0.622
rs1042711b 0.39 0.431 0.018 0.702 0.621
NR3C1 rs6188c 0.29 0.592 0.013 0.462 0.613
rs6196c 0.14 0.810 0.026 0.132 0.085
rs6190 0.05 0.206 0.206 0.128 0.128
rs6195 0.02 0.229 0.229 0.754 0.754
ALOX5 rs4987105d 0.19 0.973 0.776 0.003 0.012
rs4986832d 0.19 0.779 0.736 0.005 0.012
rs2229136 0.07 0.764 0.764 0.842 0.842
rs2228064 0.02 0.807 0.737 0.208 0.438
Sp1 motife 0.24 0.845 0.817 0.016 0.052
LTC4S rs730012 0.31 0.383 0.253 0.859 0.917
Unassignedf 0.30 0.467 0.229 0.749 0.805
CYSLTR1 rs320995 0.23 0.679 0.679 0.988 0.988
ALOX5AP rs3803278 0.24 0.282 0.362 0.647 0.782
rs12721458 0.29 0.653 0.568 0.315 0.521
rs3803277 0.48 0.948 0.985 0.847 0.893
PLA2G4A rs3736741 0.27 0.964 0.894 0.632 0.615
rs2307200 0.22 0.620 0.422 0.547 0.328
CYSLTR2 rs912278g 0.41 0.476 0.269 0.008 0.020
rs912277g 0.07 0.574 0.574 0.021 0.021

AM PEF, Weekly mean morning peak expiratory flow.

a
P values r 0.05 in bold. Additive and dominant models as specified.
b
Linkage disequilibrium r2 = 0.99.
c
Linkage disequilibrium r2 = 0.31.
d
Linkage disequilibrium r2 = 0.98.
e
Only microsatellite marker tested. Minor allele frequency is for all non-5 repeats.
f
Marker (referenced as rs-gsk7352707) is located 208 bp upstream of exon 2 (or at position 179154983 on chromosome 5; NCBI Build 36.1).
g
Linkage disequilibrium r2 = 0.10.

Table 3 Mean clinical responses to montelukast for statistically frequent haplotype (B19%) was associated with greater
significant single marker associations response defined by change in AM PEF from baseline
Gene symbol/marker Genotype No. Mean ( ± SD) R2 (P = 0.003). Mean improvement in AM PEF was greatest
FEV1 among patients with the less frequent (B6%) CYSLTR2
ADRB2/rs1042714 C/C 61 5.7 (10.4) 0.048 haplotype (P = 0.02). P values empirically estimated
C/G 75 10.4 (11.1)
G/G 25 5.5 (9.6)
from 10 000 permutations of participant responses
ADRB2/rs1042711 T/T 60 5.6 (10.5) 0.050 remained essentially the same as theoretical values.
T/C 76 10.4 (11.0)
C/C 25 5.5 (9.6)
NR3C1/rs6188 G/G 81 8.5 (10.5) 0.052 Additionally, for haplotypes with more than two alleles, we
G/T 66 5.7 (10.7)
T/T 14 14.7 (11.0) tested whether individual haplotypes differed from all
NR3C1/rs6196 T/T 118 8.4 (10.3) 0.045 other haplotypes grouped together within a block. The
T/C 38 4.9 (11.3)
C/C 5 17.5 (13.2)
infrequent NR3C1 TT haplotype was found to have a
Peak expiratory flow significantly attentuated mean change in PEF as compared
ALOX5/rs4987105 C/C 110 33.7 (60.7) 0.054 with the other haplotypes (P = 0.05), whereas the single-
C/T 42 55.3 (76.1)
T/T 10 94.8 (108.3) marker analysis showed variant NR3C1 homozygotes to
ALOX5/rs4986832 G/G 110 34.9 (59.8) 0.054 have the largest improvement in percent predicted FEV1.
G/A 46 52.9 (78.5) The common CYSLTR2 TT and TC haplotypes were each
A/A 9 102.4 (112.1)
ALOX5/microsatellitea 2 101 34.8 (62.3) 0.036 shown to have a significantly lower mean change in AM
1 46 51.1 (74.7) PEF (P = 0.007 and P = 0.04), observations that are
0 17 76.6 (93.5)
CYSLTR2/rs912278 T/T 60 29.1 (61.9) 0.048
consistent with the single-marker results.
T/C 75 41.3 (64.1)
C/C 28 71.6 (77.3)
CYSLTR2/rs912277 T/T 144 38.8 (68.3) 0.032 Discussion
T/C 21 76.5 (76.7) Our results identify genetic variants in the 5-LO pathway
a
Number of ALOX5 alleles with five repeats of the Sp1-binding motif GGGCGG. associated with therapeutic response to montelukast. In

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 194 Pharmacogenetics and Genomics 2007, Vol 17 No 3

Table 4 P values for associations of haplotypes with respiratory with asthma did not identify any pharmacogenetic
measuresa associations between ALOX5 and response to leukotriene
FEV1 PEF modifier therapy [38]. The ALOX5 microsatellite was
Haplotype Frequency Mean (SD) P value Mean (SD) P value associated with response in the present study. We also
blockb observed the variant rs4987105 and rs4986832 homo-
ADRB2 (rs1042713, rs1042714, rs1042711) zygotes to be associated with improved AM PEF. The
G,C,T 0.21 8.1 (10.8) 28.7 (69.7) underlying mechanisms to explain these clinical observa-
G,G,C 0.39 8.4 (10.8) 45.3 (69.7)
A,C,T 0.40 7.1 (10.8) 0.61 49.4 (69.7) 0.11
tions remain poorly defined.
LTC4S (rs730012, rs-gsk7352707)
A,C 0.70 7.5 (10.8) 43.5 (69.8)
C,T 0.30 8.6 (10.8) 0.40 41.6 (69.8) 0.82 Although results were not as robust as for ALOX5,
ALOX5 (rs4987105, rs4986832) CYSLTR2 remains a plausible candidate from a biological
C,G 0.81 7.6 (10.9) 38.1 (68.2) perspective. CYSLTR2 encodes for a 346 amino acid
T,A 0.19 8.1 (10.9) 0.84 69.4 (68.2) 0.003
ALOX5AP (rs3803278, rs12721458, rs3803277) protein with 38% amino acid identity to the CysLT1
T,G,C 0.24 8.4 (10.7) 49.9 (69.6) receptor [39]. It is believed that both CysLT1 and
T,G,A 0.23 6.9 (10.7) 42.8 (69.6)
T,C,C 0.29 7.7 (10.7) 39.1 (69.6)
CysLT2 may be potent biological mediators in the
C,G,A 0.25 9.3 (10.7) 0.57 47.8 (69.6) 0.74 pathophysiology of asthma by predisposing to broncho-
NR3C1 (rs6188, rs6196) constriction, vascular hyperpermeability, and mucus
G,T 0.70 7.9 (10.7) 42.5 (69.3)
T,T 0.16 8.6 (10.7) 62.6 (69.3) hypersecretion in asthmatic patients. As such, it is
T,C 0.14 8.3 (10.7) 0.91 30.9 (69.3) 0.08 hypothesized that leukotriene modifier therapy may be
PLA2G4A (rs3736741, rs2307200) more efficacious among asthma patients with concen-
A,C 0.52 8.1 (10.8) 44.1 (70.0)
A,T 0.21 7.3 (10.8) 39.0 (70.0) trated leukotriene activity [12,40]. Mapped to 13q14, a
G,C 0.27 7.9 (10.8) 0.87 46.8 (70.0) 0.78 chromosomal region linked to asthma, it has been shown
CYSLTR2 (rs912278, rs912277)
T,T 0.60 7.5 (10.8) 33.8 (65.9)
that CYSLT2 is associated with: (i) susceptibility to
T,C 0.34 8.7 (10.8) 52.5 (65.9) asthma in Caucasians [8] and Japanese [41]; (ii) atopic
C,C 0.06 7.1 (10.8) 0.64 63.7 (65.9) 0.02 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
baseline AM PEF. with asthma [42]. Moreover, in comparison to wild-type
AM PEF, weekly mean morning peak expiratory flow.
a
P values r 0.05 in bold. CYSLTR2, CYSLTR2 coding variants at positions A601G
b
Each 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 CYLTR2
polymorphisms 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
PEF and FEV1 measures (tracked over a 12-week study 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
ALOX5 variants. 7.5% or greater had elevated median leukotriene con-
centration. The direct functional significance of the
CYSLTR2 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 pharmacotherapy.
response to ABT-761, an experimental leukotriene
modifier [10]. Among predominant homozygotes (repeat
length = 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.

receptor signal to alter another, or the so-called cross-talk
between Gas-receptors and Gaq-receptors, may partly
underly pharmacogenetic variation of b-agonists and
ADRB2 polymorphisms [44,45]. Considering the postu-
lated interdependence of Gas and Gaq-receptor signaling,
genetic variability influencing the Ga signaling may also
hold clinical relevance for leukotriene modifier therapy.
Although statistical associations between ADRB2 poly-
morphisms and response to montelukast were identified,
these observations did not appear to have clinical
significance.
LTC4S has been evaluated as a pharmacogenetic locus
because variability in this gene may be associated with
enhanced production of CysLTs, thus sustaining airway
inflammation and bronchoconstriction in patients with
asthma [11]. In contrast to previous reports, we did not
find evidence for association between LTC4S and
response to montelukast.
A potential limitation of this study includes the
incomplete coverage of polymorphisms in the candidate
gene loci evaluated. Untyped polymorphisms, or haplo-
types, may have functional effects on gene function, and
thus be more informative in characterizing pharmacoge-
netic associations. Consequently, we cannot rule out a
gene if no association was observed in our sample. A
second potential limitation is multiple comparisons. As
traditional multiple testing corrections tend to be overly
conservative, the FDR was calculated. The estimated
proportion of false discoveries in our study is 16%,
suggesting that at least six of the eight genotype–
phenotype associations are not spurious statistical asso-
ciations owing to multiple comparisons. Larger replication
studies, including asthma patients with a more broad
representative spectrum of asthma severity and control,
will be needed to confirm our findings. The underlying
drug mechanism(s) of action are complex, and it is likely
that polymorphisms in other genes contribute to the
heterogeneity of response. Given the many gene products
involved in the pharmacodynamic and pharmacokinetic
pathways of asthma drugs [46], the modest effect sizes
reported herein are consistent with what would be
expected for a polygenic trait. Once a panel of
pharmacogenetic loci are identified, potential clinical
applications of PGx for asthma management strategies
will require prospective testing.
In summary, our results suggest that variant CYSLTR2
and ALOX5 polymorphisms may enhance response to
montelukast. These markers were found in roughly 10–
13% of patients enrolled on our trials. Asthma genetic
studies, animal models, and in-vitro findings suggest that
these loci predispose to allergic respiratory disorders
and may correlate with an asthma phenotype most likely
to respond to leukotriene modifier pharmacotherapy.
Although inhaled corticosteroids alone or coadministered
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Pharmacogenetics of 5-lipoxygenase pathway Klotsman et al. 195
with long-acting b-agonists are, on average, more
efficacious than montelukast in treating asthma patients
[43,47–51], further elucidation of 5-LO pharmaco-
genetics may serve to identify the subset of asthma
patients most likely to benefit from leukotriene modifier
therapy.
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