13TLRM0438_Ortega
Articles
LB

THELANCETRM-D-13-00438
S2213-2600(13)70289-3
Embargo:January 27, 2014—00:01 [GMT}

Version 2

Funding: NIH funded

This version saved: 16:06, 21-Jan-14

Effect of rare variants in ADRB2 on risk of severe
exacerbations and symptom control during longacting
β agonist treatment in a multiethnic asthma population:
a genetic study
Victor E Ortega, Gregory A Hawkins, Wendy C Moore, Annette T Hastie, Elizabeth J Ampleford, William W Busse, Mario Castro, Domingo Chardon,
Serpil C Erzurum, Elliot Israel, Federico Montealegre, Sally E Wenzel, Stephen P Peters, Deborah A Meyers, Eugene R Bleecker

Summary

Background Severe adverse life-threatening events associated with longacting β agonist (LABA) use have caused the
US Food and Drug Administration (FDA) to review the safety of these drugs, resulting in a boxed warning and a
mandatory safety study in 46 800 patients with asthma. Identification of an at-risk, susceptible subpopulation on the
basis of predictive biomarkers is crucial for understanding LABA safety. The β2-adrenergic receptor gene (ADRB2)
contains a common, non-synonymous single nucleotide polymorphism, Gly16Arg, that is unlikely to account for the
rare, life-threatening events seen with LABA use. We hypothesise that rare ADRB2 variants modulate therapeutic
responses to LABA therapy and contribute to rare, severe adverse events.
Methods In this genetic study, ADRB2 was sequenced in 197 African American, 191 non-Hispanic white, and
73 Puerto Rican patients. Sequencing identified six rare variants, which were genotyped in 1165 patients with asthma.
The primary hypothesis was that severe asthma exacerbations requiring hospital admission were associated with rare
ADRB2 variants in patients receiving LABA therapy. This outcome was assessed overall and by ethnic group [A: OK
to add?]. Replication was done in 659 non-Hispanic white patients with asthma.
Findings Patients receiving LABA with a rare ADRB2 variant had increased asthma-related hospital admissions (15 [44%]
of 34 patients with rare variant vs 121 [22%] of 553 patients with common ADRB2 alleles admitted to hospital in past
12 months; meta-analysis for all ethnic groups, p=0·0003 [A: the Editor prefers to present the p value in this way for consis­
tency throughout the journal]). Specifically, increases in hospital admission rates were recorded in LABA-treated nonHispanic white patients with the rare Ile 164 allele compared with non-Hispanic white patients with the common allele
(odds ratio [OR] 4·48, 95% CI 1·40–13·96, p=0·01) and African American patients with a 25 bp promoter polynucleotide
insertion, –del376, compared with African American patients with the common allele (OR 13·43, 95% CI 2·02–265·42,
p=0·006). The subset of non-Hispanic white and African American patients receiving LABAs with these rare variants
had increased exacerbations requiring urgent outpatient health-care visits (non-Hispanic white patients with or without
the rare Ile 164 allele, 2·6 [SD 3·5] vs 1·1 [2·1] visits, p<0·0001 [A: as above for format of p value]; and African American
patients with or without the rare insertion, 3·7 [4·6] vs 2·4 [3·4] visits, p=0·01), and more frequently were treated with
chronic systemic corticosteroids (OR 4·25, 95% CI 1·38–14·41, p=0·01, and 12·83, 1·96–251·93, p=0·006). NonHispanic white patients from the primary and replication cohorts with the rare Ile 164 allele were more than twice as
likely as Thr 164 homozygotes to have uncontrolled, persistent symptoms during LABA treatment (p=0·008–0·04).
Interpretation The rare ADRB2 variants Ile164 and –del376 are associated with adverse events during LABA therapy
and should be evaluated in large clinical trials including the current FDA-mandated safety study.
Funding US National Institutes of Health.

Introduction
Common β2-adrenergic receptor gene (ADRB2)
variation has been studied intensively including a nonsynonymous polymorphism at codon 16 encoding
either glycine or arginine, Gly16Arg, which has been
shown to affect responses to regular use of shortacting
β agonists (SABA).1,2 However, the effects of Gly16Arg
genotypes have not been observed with longacting
β agonists (LABAs).3–5
Adverse, life-threatening responses to regular SABA
therapy have also been observed during asthma mortality

epidemics in the 1960s in the UK and New Zealand related
to high-dose regimens of isoprenaline and fenoterol, but
not with salbutamol.6–8 In two large observational trials, the
Serevent Nationwide Surveillance Study9 and the SMART
study,10 the adverse outcomes of life-threatening asthma
exacerbations or asthma-related death associated with the
use of LABA were rare. Despite the rarity of these events
[A: OK to add?], the findings that severe adverse lifethreatening events are associated with LABA use have
resulted in a boxed warning from the US Food and Drug
Administration (FDA) and a mandatory ongoing LABA

www.thelancet.com/respiratory Published online January 27, 2014 http://dx.doi.org/10.1016/S2213-2600(13)70289-3 

Lancet Respir Med 2014
Published Online
January 27, 2013
http://dx.doi.org/10.1016/
S2213-2600(13)70289-3
Center for Genomics and
Personalized Medicine, Wake
Forest School of Medicine,
Winston-Salem, NC, USA
(V E Ortega MD,
G A Hawkins PhD,
W C Moore MD, A T Hastie PhD,
E J Ampleford PhD,
Prof S P Peters MD,
Prof D A Meyers PhD,
Prof E R Bleecker MD);
Department of Medicine,
University of Wisconsin,
Madison, WI, USA
(Prof W W Busse MD);
Department of Medicine,
Washington University School
of Medicine, St Louis, MO, USA
(Prof M Castro MD); Hospital
Episcopal San Lucas, Ponce
School of Medicine, Ponce,
Puerto Rico (D Chardon MD,
F Montealegre PhD);
Department of Pathobiology
and Respiratory Institute,
Lerner Research Institute,
Cleveland Clinic Foundation,
Cleveland, OH, USA
(Prof S C Erzurum MD); Brigham
and Women’s Hospital, Harvard
Medical School, Boston, MA,
USA (Prof E Israel MD); and
Asthma Institute, University of
Pittsburgh, Pittsburgh, PA,
USA (Prof S E Wenzel MD)
Correspondence to:
Prof Eugene R Bleecker, Center
for Genomics and Personalized
Medicine, Wake Forest School of
Medicine, Winston-Salem,
NC 27157, USA
ebleeck@wakehealth.edu

1

 Articles

1
DNA sequencing of 5·3 kB
region of ADRB2 in
patients with asthma

191 non-Hispanic white patients
71 from SARP
120 from CSGA

Identification of rare
functional variants:
allele frequency <5%

One rare variant in non-Hispanic
white patients: Thr164Ile

Genotyping of five rare
ADRB2 coding SNPs

952 non-Hispanic white patients
636 from SARP
140 from CSGA
176 from severe asthma
clinical trial cohort

197 African Americans
109 from SARP
88 from CSGA
5
Five rare variants in African
Americans: –376 In-Del, Pro4His,
Asn15Ser, Thr164Ile, Ser220Cys

10

73 Puerto Ricans

Three rare variants in
Puerto Ricans: –376 In-Del,
Thr164Ile, Leu342Pro

213 African Americans
206 from SARP
7 from CSGA

15
Genotyping of rare
promoter 25 bp insertion:
–376 In-Del

316 African Americans
from SARP*

Figure 1: Sequencing and genotyping of rare ADRB2 variants in different ethnic20groups from the primary cohort
Study participants from three ethnic groups were evaluated for rare ADRB2 variants either with DNA sequencing or targeted genotyping. SARP=Severe Asthma
Research Program.17 CSGA=Collaborative Study on the Genetics of Asthma.16 SNP=single nucleotide polymorphism. *103 of 316 African American participants
genotyped for the –376 In-Del were also evaluated with sequencing of ADRB2.

safety study in 46 
800 patients with asthma.11,12 Thus, 25 LABA therapy. Additionally, 73 Puerto Rican participants
identification using genetics or other predictive biomarkers with a physician’s diagnosis of asthma were recruited
of at-risk, susceptible subpopulations is crucial in during a documented asthma exacerbation at the
emergency department of Hospital Episcopal San Lucas
understanding LABA safety.
We did a genetic study in patients with mild to severe (Ponce, Puerto Rico). This primary cohort of 1626 patients
asthma to test the hypothesis that rare ADRB2 variants are 30 was characterised with pulmonary function tests and
associated with increased hospital admission for asthma questionnaires that recorded treatment use, indices of
exacerbations in patients receiving treatment with LABAs. health-care use including hospital admissions during the
This primary endpoint is the same as that being used in preceding 12 months, and symptom control.
For replication, in a separate cohort, 3-month
the FDA-mandated LABA safety study, in which it serves
as a surrogate outcome for rare, asthma-related life- 35 questionnaire data were collected from study enrolment
threatening exacerbations or death.12,13 We hypothesise up to 24 months of longitudinal evaluation in 659 nonthat rare variant effects are independent of common gene Hispanic white patients with asthma characterised as
variation, including the Gly16Arg locus. Since previous difficult to treat by their primary care physicians from the
sequencing of ADRB2 has identified rare variants (allele TENOR study.19
frequency <5%) with allele frequencies that vary among 40 In all study cohorts, questionnaires were standardised
different ethnic groups, study of patients from different across all study sites and administered by centrally
ancestral backgrounds is important.14,15
trained clinical staff.20 Questions regarding health-care
use specifically queried whether these events occurred
because of an asthma attack or breathing problems.
Methods
Study populations
45 Symptom control was recorded for the previous 3 months
For this genetic study the primary cohort included in the primary and replication cohorts based on National
355 participants from the National Heart Lung and Blood Asthma Education and Prevention Program (NAEPP)
Institute (NHLBI) Collaborative Study on the Genetics of guidelines.21
Asthma (CSGA)16 and 1022 participants from the NHLBI
These studies were approved by the institutional review
Severe Asthma Research Program (SARP)17 in which 50 boards at all sites and informed consent was obtained
diagnosis of asthma was based on the presence of either from all participants.
methacholine bronchial hyper-responsiveness or
bronchodilator reversibility, less than 5 pack-years of Procedures
tobacco exposure, and asthma symptoms. Baseline data Figure 1 summarises sequencing and genotyping
were also analysed from a clinical trial18 of 176 white 55 undertaken in the primary cohort. A 5350 bp region [A:
patients with uncontrolled, persistent, severe asthma please confirm number (–3470 to 1886 = 5356?] of
while receiving high-dose inhaled corticosteroids and ADRB2 (–3470 bp 5ʹ of the ATG start site to +1886 bp
2 

www.thelancet.com/respiratory Published online January 27, 2014 http://dx.doi.org/10.1016/S2213-2600(13)70289-3

 Articles

1
Analysis of six ADRB2 rare variants in 1209 patients with asthma for
primary outcome of hospital admission: meta-analysis, p=0·002

5
LABA subgroup analyses*

587 LABA-treated patients:
meta-analysis, p=0·0003

418 non-LABA-treated patients:
meta-analysis, p=0·41

10
397 LABA-treated non-Hispanic
white patients: p=0·005

186 LABA-treated African American
patients: p=0·02

Single variant regression-based analyses

Single rare variant in LABA-treated
non-Hispanic white patients: Thr164Ile,
OR 4·48, p=0·01
15

Two rare variants in >3 LABA-treated
African Americans: Ser220Cys, OR 0·87,
p=0·83; –376 In-Del OR 13·43, p=0·006

Analysis of health-care-related outcomes

Thr164Ile in LABA-treated non-Hispanic
white patients: urgent+ED visits,
p<0·0001; systemic steroid use, p=0·01

–376 In-Del in LABA-treated African
Americans: urgent+ED visits, p=0·01;
systemic steroid use, p=0·006

Analysis of symptom-related outcomes

Thr164Ile in LABA-treated
non-Hispanic white patients:
NAEPP symptoms, p=0·008–0·04

Ethnic subgroup analyses†

20
–376 In-Del in LABA-treated African
Americans: NAEPP symptoms, p=0·55

25
Validation
of variant effects

Thr164Ile replication in 516 LABA-treated
non-Hispanic white patients
from TENOR: NAEPP symptoms at
12–24 months, p=0·02–0·02

Thr164Ile replication in 143
non-LABA-treated non-Hispanic
white patients from TENOR: NAEPP
symptoms at 12–24 months, p=0·52–0·63
30

Figure 2: Hierarchical analysis of rare ADRB2 variants and hospital admissions
A hierarchical analysis of rare ADRB2 variants and risk of hospital admission. The first step analysed each ethnic group for rare variant effects on risk of hospital
admission to obtain a gene-level p value for all rare variants in the multiethnic population (p=0·002); this analysis included only patients for whom data were
available for hospital admission (1209 of 1626 in the primary cohort). [A: legend shortened to reduce repetition of data from figure and main text] The first
subgroup analysis was stratified by LABA treatment, followed by analyses by ethnic group. Regression-based analysis of single rare variants in each ethnic group
further identified variants accounting for increased risk of hospital admission. Subsequent, regression-based analyses assessed effects on additional health-care35
related and symptom-related outcomes. The adverse effect of the Thr164Ile variant on symptom control was validated in LABA-treated non-Hispanic white patients
from an independent cohort. LABA=longacting β agonist. OR=odds ratio. ED=emergency department. NAEPP=National Asthma Education and Prevention Program.
TENOR=The Epidemiology and Natural History of Asthma: Outcomes and Treatment Regimes cohort.19 *A subgroup of patients from the Collaborative Study on the
Genetics of Asthma16 were recruited before or soon after LABAs were approved for use in the USA for management of asthma; therefore, data for LABA treatment
were not obtained in these patients. †Four LABA-treated Puerto Rican patients were not analysed owing to the small number.
40

after the ATG start site) including the 413-aminoacid CA, USA). Genotypes for eight common SNPs were
(1239 bp) intronless coding region was sequenced in obtained, including Gly16Arg (rs1042713).
772 control participants without asthma from CSGA
191 non-Hispanic white and 197 African American
patients from CSGA and SARP and 73 Puerto Rican and SARP were also sequenced or genotyped for rare
asthma cases using previously described methods.14,15 45 variants (appendix). Thr164Ile was genotyped in 659 nonRare genetic variants were defined by an allele frequency Hispanic white patients from the TENOR study for
of less than 0·05. Five rare non-synonymous single replication. The reference genomic sequence for
nucleotide polymorphisms (SNPs) of the ADRB2 gene, ADRB2 in human beings was compared with nonidentified after sequencing and predicted to have human primates using the University of California,
functional effects, were genotyped with the MassARRAY 50 Santa Cruz (CA, USA) genome browser to determine the
genotyping system (Sequenom Inc, San Diego, CA, USA) ancestral alleles of common ADRB2 variants.
in the remaining participants from the primary cohort
(952 non-Hispanic white and 213 African American Statistical analysis
patients). We genotyped a 25 bp insertion-deletion within Rare variants identified with sequencing that had a
the promoter region in 316 African American patients 55 predicted effect on protein structure and function (based
from SARP with fragment analysis using an on TFsearch, FastSNP, SIFT, and Polymorphism
ABI3700 DNA Analyzer (Applied Biosystems, Foster City, Phenotyping v2 [Poly-Phen 2]) scores were analysed with
www.thelancet.com/respiratory Published online January 27, 2014 http://dx.doi.org/10.1016/S2213-2600(13)70289-3 

See Online for appendix

3

 Articles

Non-Hispanic white patients

1

African American patients

SARP
(n=707)

CSGA
(n=260)

Severe asthma Combined
(n=176)
(n=1143)

Female (%)

445 (63%)

162 (62%)

95 (54%)

702 (61%)

185 (59%)

59 (62%)

244 (60%)

60 (82%)

Age (years)

37 (15)

27 (14)

51 (12)

37 (16)

30 (15)

28 (13)

30 (14)

38 (19)

74 (23)

82 (18)

60 (12)

74 (21)

77 (20)

74 (21)

76 (21)

NA

5

SARP
(n=315)

CSGA
(n=95)

Puerto Rican
patients (n=73)
Combined
(n=410)

Pulmonary function
% predicted baseline FEV1
FEV1/FVC ratio

0·70 (0·12)

FEV1 % reversibility from baseline
Methacholine PC20
Cases with a rare variant

13 (15)
3·8 (6·0)
26 (4%)

0·77 (0·12)
12 (16)
4·0 (6·2)
7 (3%)

0·61 (0·10)
17 (16)

10

NA

0·70 (0·13)
13 (15)*
3·9 (6·1)

5 (3%)

38 (3%)†

0·72 (0·12)
14 (15)
2·5 (4·3)
36 (11%)

0·71 (0·13)
19 (17)

0·72 (0·12) NA
15 (15)*

4·6 (13)

3·0 (7·2)

19 (20%)

55 (13%)†‡

NA
NA
4 (5%)‡

Data are n (%) or mean (SD). SARP=National Heart Lung and Blood Institute Severe Asthma Research Program.17 CSGA=National Heart Lung and Blood Institute Collaborative
18
FEV1=forced expiratory volume in 1 s. NA=not available. FVC=forced vital capacity.
Study on the Genetics of Asthma.16 Severe asthma=Severe Asthma Clinical Trial Cohort.15
PC20=provocation concentration of inhaled methacholine needed to reduce FEV1 by 20%. *p=0·02 between African American and non-Hispanic white patients. †p<0·0001
between African American and non-Hispanic white patients. ‡p=0·04 between African American and Puerto Rican patients.

Table 1: Characteristics of the asthma cohorts

We combined tests done in each ethnic group using
meta-analysis to account for the genetic heterogeneity
and population substructure in participants from
n
MAF
n
MAF
n
MAF
different ancestral backgrounds.29 For SKAT, p values
Insertion-deletion
–376
0
0
15
0·02
2
0·01
from each ethnic group were combined with Stouffer’s
Pro4His
11
0
0
1
0·001
0
0
25 Z score method.30 To account for rare variant associations
Asn15Ser
44
0
0
2
0·002
0
0
related to ancestry, we analysed a subset of 364 African
Thr164Ile
491
38
0·02
5
0·006
1
0·007
Americans for estimates of global African ancestry using
Ser220Cys
659
0
0
32
0·04
0
0
previous genome-wide genotyping chip data, and
Leu342Pro
1025
0
0
0
0
1
0·007
genotypes from 225 HapMap founders with the
All rare variants
··
38
0·02
55
0·07
4
0·03
30 ADMIXTURE program.31
Data for the outcome of symptom control based on
Rare variant frequency is denoted in terms of total number of patients with asthma from the study population with a
NAEPP guidelines were collected for the past 3 months
rare variant and minor allele frequencies (MAF).
and analysed in both the primary and replication cohorts
Table 2: Rare variants identified within ADRB2 in asthma cases by ethnic group
to validate the rare variant effects observed in the primary
Haplotypes, linkage disequilibrium, and
35 cohort.21
the sequence kernel association test (SKAT) for the haplotype associations with the primary endpoint using
primary outcome of hospital admission for a severe logistic regression were calculated with standard methods
asthma exacerbation to identify gene-level rare variant (PLINK v1·07).32 Haplotypes were designated by a
associations.22–26 All analyses were adjusted for study numerical nomenclature initially described by Drysdale
cohort, sex, and age. Since the primary outcome was 40 and coworkers14 and confirmed in a larger population.15
significant, further analyses were done with a stratified,
hierarchical analysis (figure 2) to test for individual rare Role of the funding source
variant effects on the primary outcome of hospital The sponsors of the study had no role in study design,
admission and other secondary outcomes related to data collection, analysis, and interpretation, or writing of
health-care use and asthma severity including urgent 45 the report. ERB, DAM, and VEO had full access to all
outpatient health care or emergency department visits data in the study and had final responsibility for the
during the past year, level of corticosteroid use from low- decision to submit for publication.
dose to high-dose inhaled corticosteroids or chronic
systemic (oral and injectable) corticosteroids, and Results
symptom control based on the NAEPP guidelines.21,27 We 50 Table 1 shows baseline characteristics of the asthma
analysed urgent outpatient health care or emergency cohorts. Table 2 shows rare variants identified within
department visits as a continuous variable using a ADRB2 in asthma cases in each ethnic group. Six rare
generalised linear model with a Poisson distribution and ADRB2 polymorphisms with predicted functional effects
a log link function. Remaining outcomes were analysed were identified: a 25 bp insertion-deletion at nucleotide
with logistic regression. This hypothesis-driven stratified, 55 –376 relative to the ATG start site (–376 In-Del), Pro4His
hierarchical analysis (figure 2) tested for gene-by- (rs148459047), Asn15Ser (rs33973603), Thr164Ile
environment interactions with LABA exposure.28
(rs1800888), Ser220Cys (rs3729943), and Leu342Pro. All
Nucleotide
position

4 

Non-Hispanic
white patients

African American
patients

Puerto Rican
patients

20

www.thelancet.com/respiratory Published online January 27, 2014 http://dx.doi.org/10.1016/S2213-2600(13)70289-3

 Articles

1

Non-Hispanic white patients
SARP
(n=707)
Patients with available data
Asthma-related hospital admission in past 12 months (%)
Urgent visits in past 12 months (mean visits per year)

627

CSGA
(n=260)
5

73 (12%)*

151
8 (5%)

1·1 (2·2)

0·8 (2·2)

LABA use (%)

398 (64%)

High-dose ICS use (%)

246 (40%) 10 NA
127 (20%)
1 (1%)

Regular systemic steroid use (%)

37 (36%)§

African American patients

Puerto Rican
patients (n=73)

Severe asthma
(n=176)

Combined
(n=1143)

SARP
(n=315)

CSGA
(n=95)

Combined
(n=410)

176

954

314

49

363

NA†

81 (10%)‡

76 (24%)*

13 (27%)
1·5 (2·2)

0·6 (1·2)

0·9 (2·1)‡

1·7 (2·9)

176 (100%)

611 (68%)¶

189 (60%)

176 (100%)

422 (53%)

143 (46%)

NA

128 (16%)

60 (19%)

6 (13%)§
NA
2 (4%)

73

89 (25%)‡

18 (25%)*

1·7 (2·8)‡

5·1 (12·4)

195 (54%)¶
143 (46%)
62 (17%)

5 (7%)
NA
4 (6%)

Data are n (%) or mean (SD). Data are missing for a subset of patients: in SARP, medication use and health-care use data were not obtained for 81 patients; CSGA participants were recruited in different phases
during which time the questionnaire data were changed, thus data were not available for 155 patients. SARP=National Heart Lung and Blood Institute Severe Asthma Research Program.17 CSGA=National Heart
Lung and Blood Institute Collaborative Study on the Genetics of Asthma.16 Severe asthma=Severe Asthma Clinical Trial Cohort.18 NA=not available. LABA=longacting β agonist. ICS=inhaled corticosteroid. *Data
for systemic steroid use and urgent outpatient visits were evaluated in one non-Hispanic white patient and three African Americans from SARP and one Puerto Rican patient, but hospital admission data were
15
not available for these five participants. †In the severe asthma cohort, urgent outpatient visits were assessed whereas hospital admission was not. ‡p<0·0001 between African American and non-Hispanic white
patients. §The first LABA, salmeterol, was approved for management of asthma after recruitment for CSGA was started; therefore, only a subgroup of 102 non-Hispanic white and 46 African American patients
were evaluated for LABA treatment. ¶p=0·02 between African American and non-Hispanic white patients.

Table 3: Asthma-related health-care use
20

rare SNPs had FastSNP risk scores ranging from two population as possible. Patients with asthma from CSGA
(low predicted effect on protein function) to four (high and SARP were recruited either at different times or from
predicted effect; appendix) and four SNPs were predicted different study sites; therefore, LABA use and health-care
to be damaging by a SIFT score less than 0·05.24 The use were not evaluated in a subset of these patients
–376 In-Del rare variant is a 25 bp polynucleotide 25 (n=236). Table 3 summarises asthma-related medication
insertion located 5ʹ upstream of the coding region within and health-care use in these cohorts. Data for hospital
a SP1 transcription factor binding site (TFsearch score of admission were not obtained from the 176 participants
87·7).23,26 Thr164Ile and Ser220Cys were identified in from the severe asthma trial cohort, a further four
controls as well as two novel rare variants, Phe240Leu participants from SARP, and one Puerto Rican patient;
and Gly383Arg, each in one patient (appendix). Rare 30 thus, of the primary cohort (n=1626), data for 1209
variants were not associated with asthma susceptibility patients were available for analysis of hospital admission.
(appendix). African American patients with asthma had a A meta-analysis of the combined multiethnic asthma
significantly higher frequency of rare variants compared populations of non-Hispanic white, African American,
with non-Hispanic white (p<0·0001) and Puerto Rican and Puerto Rican patients with rare ADRB2 variants
patients (p=0·04; table 1). With one exception, all rare 35 (n=75) showed an increased likelihood of a severe asthma
variants occurred on a haplotype background containing exacerbation requiring hospital admission in the past
12 months compared with those without rare variants
the ancestral Gly 16 allele (appendix).
Table 3 shows asthma-related health-care use. African (n=1134; p=0·002; table 4). In the subset treated with a
American participants had a greater frequency of hospital LABA, 15 (44%) of 34 patients with rare variants had an
admissions (p<0·0001, table 3) and urgent health care 40 asthma-related hospital admission during the past year
including emergency room visits for asthma exacerbation compared with 121 (22%) of 553 without a rare variant
(p<0·0001) during the preceding year, but were less likely (p=0·0003; table 4). This increased frequency of hospital
to be treated with a LABA compared with non-Hispanic admission was primarily observed in LABA-treated nonwhite participants (odds ratio [OR] 0·73, 95% CI Hispanic white patients (p=0·005; table 4) and LABA0·56–0·96, p=0·02; table 3). Non-Hispanic white patients 45 treated African Americans (p=0·02; table 4). These
with asthma selected for sequencing had a higher associations were due to the Ile 164 rare variant in LABAfrequency of urgent physician visits in the past year treated non-Hispanic white patients (OR 4·48, 95% CI
compared with those who were genotyped (p<0·0001; 1·40–13·96, p=0·01; figure 3A) and the rare –376 promoter
appendix). African American patients selected for insertion variant (OR 13·43, 2·02–265·42, p=0·006;
sequencing had significantly lower baseline lung 50 figure 3B) in LABA-treated African Americans. Ser220Cys,
function and a greater degree of airflow obstruction the remaining frequent rare variant in the asthma cohort,
compared with those who were genotyped (p<0·0001 for was not associated with hospital admission in LABAforced expiratory volume in 1 s [FEV1] percentage of treated African Americans (OR 0·87, 95% CI 0·22–2·98,
predicted and p<0·0001 for FEV1/forced vital capacity p=0·83). In patients not on LABAs, rare ADRB2 variants
ratio; appendix).
55 were not associated with hospital admission (table 4).
Analysis of haplotypes with common coding variants
In the search for rare variants and rare variant
frequencies it was important to evaluate as large a (Gly16Arg, Gln27Glu) identified only an association of one
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5

 Articles

1 year. Thus, the population attributable risk for hospital
admission during LABA treatment due to these rare
Rare
Common
Rare variant, Common alleles, SKAT
ADRB2 variants was 0·07 for Thr164Ile in non-Hispanic
variant
alleles
n (%)
n (%)
p value
white people and 0·09 for the –376 insertion variant in
All patients with asthma
75
1134
22 (29%)
166 (15%)
0·002
5 African Americans.
Non-Hispanic white
27
750
7 (26%)
74 (10%)
0·02
The analysis of urgent outpatient visits included data
African American
44
316
14 (32%)
75 (24%)
0·07
from the severe asthma clinical trial cohort18 (n=176, table
Puerto Rican
4
68
1 (25%)
17 (25%)
0·4
3), including five additional LABA-treated patients with
Treatment with LABA*
34
553
15 (44%)
121 (22%)
0·0003
the rare Ile 164 variant (table 1). Non-Hispanic white
Non-Hispanic white
13
384
6 (46%)
61 (16%)
0·005
10 patients on LABAs with the rare Ile 164 variant
African American
21
165
9 (43%)
59 (36%)
0·02
(n=18 Thr164Ile heterozygotes) had a greater number of
Puerto Rican
0
4
NA†
1 (25%)
 NA
urgent outpatient health-care or emergency department
No treatment with LABA*
28
390
2 (7%)
29 (7%)
0·41
visits for asthma exacerbations during the past year
Non-Hispanic white
10
218
0
6 (3%)
0·51
compared with homozygotes for the common allele
African American
15
109
1 (7%)
7 (6%)
0·98
15 (n=553 Thr 164 homozygotes; 2·6 [SD 3·5] vs
1·1 [2·1] visits, p<0·0001). A similar association with
Puerto Rican
3
63
1 (33%)
16 (25%)
0·42
increased urgent outpatient health-care or emergency
SKAT=Sequence Kernel Association Test adjusted for age, sex, and study cohort. LABA=longacting β agonist. NA=not
department visits was observed for African Americans
available. *Data for LABA use were not available for a subgroup of patients from the Collaborative Study on the Genetics of
with the rare –376 insertion (n=6) compared with
Asthma recruited before or soon after LABAs were approved for use in the USA for management of asthma. †Not
reported individually for hospital admissions owing to small numbers for comparisons.
20 homozygotes for the common allele (n=181; 3·7 [SD 4·6] vs
2·4 [3·4] visits, p=0·01). Of these six LABA-treated
Table 4: Rare ADRB2 variant associations with asthma-related hospital admission
African Americans with the rare insertion, five (83%)
needed two or more urgent outpatient health-care or
emergency department visits in the past year. In patients
A Non-Hispanic white patients
B African-American patients
90
90
25 not on LABAs, the rare Ile 164 variant was associated
–376 In-Del
Thr164Ile
n=6
Rare insertion
Rare genotype (CT)
with reduced urgent visits in non-Hispanic white patients
80
80
Common genotype
Common genotype (CC)
(nine Thr164Ile patients vs 216 Thr 164 homozygotes:
OR 4·48 (95% CI 1·40–13·96)
70
70
OR 13·43 (95% CI 2·02–265·42)
0·1 [SD 0·3] vs 0·5 [1·6] visits, p=0·01) and not associated
p=0·01
p=0·006
in African Americans (five patients with –376 insertion vs
60
60
30 119 without insertion: 1·2 [2·2] vs 0·7 [1·2] visits, p=0·18).
50
50
n=13
Detailed data for the use of inhaled and oral, systemic
40
40
corticosteroid treatment were not available in some
n=179
cohorts; however, non-Hispanic white patients on LABAs
30
30
from SARP with the rare Ile 164 variant needed higher
20
20
n=384
35 levels of corticosteroid treatment compared with those
n=5
n=119
n=10 n=218
with the common allele (p=0·003; appendix). For
10
10
example, eight (62%) of 13 non-Hispanic white patients
0
0
LABA treatment
No LABA treatment
on LABA therapy with the rare Ile 164 variant were
LABA treatment
No LABA treatment
treated with chronic systemic corticosteroids compared
Figure 3: Two rare ADRB2 variants and hospital admission for a severe asthma exacerbation with longacting
40 with 104 (27%) of 385 Thr 164 homozygotes (OR 4·25,
β agonist (LABA) treatment
95% CI 1·38–14·41, p=0·01). Additionally, five (83%) of
Red bars show the percentage of patients with Thr164Ile variant or the –376 In-Del variant and blue bars show
the percentage in those without these rare variants (common genotypes). Thr164Ile was the only rare variant
six African Americans on LABA therapy with the
identified in non-Hispanic white patients and a 25 bp promoter insertion deletion (–376 In-Del) was identified in
–376 promoter insertion were treated with chronic
African American but not non-Hispanic white patients.
systemic cortico­
steroids compared with 52 (29%) of
45 180 homozygotes for the common allele without the
ADRB2 haplotype containing the rare Ile 164 variant with insertion (OR 12·83, 95% CI 1·96–251·93, p=0·006). In
hospital admission in LABA-treated non-Hispanic white patients not on LABAs, rare ADRB2 variants were not
patients (p=0·02; appendix).14,15 This Ile 164-containing associated with systemic corticosteroid treatment
ADRB2 haplotype number seven (based on Drysdale and (Thr164Ile in non-Hispanic white patients, p=0·13;
coworkers) contains the Gly 16 allele of Gly16Arg 50 –376 In-Del in African Americans, p=0·69).
All rare ADRB2 variants remained significantly
(appendix).14 On the basis of the observed rare variant
effects in patients with asthma treated with a LABA in this associated with hospital admission in African Americans
study, we estimate that 150 non-Hispanic white people treated with LABAs when the covariate ancestry was
would need to be genotyped to identify three people with included (p=0·01). Additionally, the rare –376 In-Del
the rare Ile 164 allele and 100 African Americans genotyped 55 remained significantly associated with hospital
to identify two people with the –376 rare insertion to admission (p=0·006), urgent outpatient health-care visits
prevent one hospital admission during LABA treatment in (p=0·02), and chronic systemic corticosteroid use
n

Patients admitted to hospital in past 12 months

1

Participants admitted to hospital in past year (%)

16

6 

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 Articles

(p=0·007) in African Americans treated with LABAs 1 genetic background of the Gly 16 allele at the Gly16Arg
locus. Findings from sequence analysis in primates
when ancestry was used as a covariate.
In non-Hispanic white patients treated with LABAs from indicate that Gly 16 is the sole or ancestral allele at the
the primary cohort, seven (54%) of 13 with the rare Gly16Arg locus. Therefore, changes in β agonist
Ile 164 variant needed treatment with a rescue inhaler for 5 responsiveness previously observed during SABA
uncontrolled symptoms more than twice a week compared therapy in Arg 16 homozygotes are not likely to be due to
with 100 (26%) of 379 Thr 164 homozygotes (p=0·02; a rare ADRB2 variant.1,2
appendix). Significant associations at this locus were also
Rare ADRB2 variants were associated with increased
identified for four additional measures of symptom control likelihood of a severe asthma exacerbation requiring
in non-Hispanic white patients treated with LABAs 10 hospital admission, specifically in LABA-treated white
including dyspnoea (p=0·02), chest tightness (p=0·03), patients with the Thr164Ile rare variant and LABA-treated
wheezing (p=0·008), and nocturnal symptoms (p=0·04; African Americans with the –376 In-Del rare variant. The
appendix). Significant associations were not identified for effect of these rare variants on asthma control in patients
any measure of symptom control in non-Hispanic white exposed to LABAs is further supported by its association
patients not treated with a LABA (rescue inhaler use, 15
p=0·97; dyspnoea, p=0·82; chest tightness, p=0·33;
Panel: Research in context
wheezing, p=0·26; nocturnal symptoms, p=0·75). In
Systematic review
African Americans on LABA therapy, three (50%) of six
We searched PubMed for articles published in any language before Sept 27, 2013, using the
patients with the rare –376 insertion variant needed
treatment with a rescue inhaler for uncontrolled symptoms 20 terms “asthma”, “beta agonist”, and “ADRB2.” This search identified pharmacogenetic
studies of the ADRB2 locus that reported associations with common variants, particularly
more than twice a week compared with 55 (31%) of
Gly16Arg, and inhaled β agonist response. These studies consistently reported that
179 homozygotes for the common allele, although this
Gly16Arg genotypes were associated with clinical responses to shortacting β agonists
difference was not significant (p=0·55).
(SABA) during acute and chronic exposure in patients with asthma, but was not associated
In the replication cohort, symptom severity based on
NAEPP guidelines was greater in LABA-treated non- 25 with clinical response to longacting β agonist (LABA) treatment. None of these studies
genotyped functional rare variants based on sequencing data from patients with asthma
Hispanic white patients (n=13) with the rare Ile 164
from different ethnic groups or tested for rare variant associations.1–5,15 We did another
variant compared with Thr 164 homozygotes (n=446;
search
of PubMed with the terms “asthma” and “ADRB2” to identify previous studies
p=0·02; appendix). Five (38%) of 13 non-Hispanic white
testing the association of rare ADRB2 variants with asthma severity. This search identified a
patients on LABA therapy with the rare Ile 164 variant had
severe, persistent symptoms compared with 68 (15%) of 30 population-based study of 8018 participants from the British 1958 birth cohort in which
Thr164Ile was not associated with asthma, wheezing, or lung function measures; however,
446 Thr 164 homozygotes at the 12-month follow-up visit
treatment use and health-care use outcomes were not reported.33 Another population(appendix). This rare variant association was observed
based study34 of the Copenhagen City Heart Study and the Copenhagen General Population
only in patients treated with LABAs at the 12-month and
Study cohorts genotyped Thr164Ile in 62 748 participants including a subset of
24-month (n=430, ten patients with Ile 164) follow-up
visits (p=0·02 at each visit; appendix). Significant 35 1300 people with self-reported asthma, which was not reported individually for lung
function measures, treatment use, or health-care use outcomes. A final search of PubMed
associations were not identified in those not treated with
with the terms “asthma” and “beta2” identified a study of 251 patients35 with asthma of
a LABA at the 12-month and 24-month follow-up visits
whom 81 had severe, life-threatening asthma. Thr164Ile was not associated with
(p=0·52 and p=0·63). The baseline characteristics of the
life-threatening asthma in this cohort; however, LABA use was not evaluated.
replication cohort are summarised in the appendix.

Discussion

40

This sequencing and genotyping analysis of
ADRB2 represents the largest collection of well
characterised, multiethnic patients with asthma studied
for rare ADRB2 variants, many of whom have been 45
treated with LABAs (panel). Patients with asthma
analysed with sequencing had more severe disease
compared with those who were genotyped, which
provided the potential to identify rare variants enriched in
severe asthma. The rare variants identified in these 50
asthma cohorts represent all rare non-synonymous
polymorphisms with an allele frequency greater than
0·0006 from the US National Institutes of Health (NIH)
NHLBI GO exome sequencing project (appendix).36
Six rare ADRB2 variants were identified with varying 55
frequencies between ethnic groups. These rare variants
were exclusively found (with one exception) on the

Interpretation
To the best of our knowledge, our study is the first to identify a pharmacogenetic
interaction between rare genetic variants and asthma severity during LABA therapy.
The probable effect of gene variant discoveries at a population level is predicted by the
population attributable risk, which depends on the genetic risk and allele frequency
observed within a study cohort. The population attributable risks identified for rare
variants in this LABA-treated asthma population were low, which reflects low allele
frequencies; however, these risks were three times greater than the frequency of these
rare variants. Thus, rare ADRB2 variants are more likely to account for the rare,
life-threatening events reported in the SMART LABA surveillance study than a common
variant such as Gly16Arg. The identification of an asthma subpopulation at risk of rare,
adverse events associated with LABA therapy will help to clarify concerns over LABA
safety. The rare Ile 164 variant was also associated with poor symptom control during
LABA treatment in two, independent cohorts. Thus, Thr164Ile is a potential biomarker for
more personalised and precise guideline-based management approaches in the small
subset of patients with asthma who are unresponsive to the combination therapy of a
LABA with inhaled corticosteroids.

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7

 Articles

with increased urgent physician or emergency 1 Copenhagen City Heart Study and the Copenhagen
department visits, systemic corticosteroid use, and poor General Population Study, which included a subset with
symptom control. We were able to replicate the genetic self-reported asthma (n=1300).34
effects of the Thr164Ile variant on symptom control
By contrast with non-Hispanic white patients with
during LABA treatment in an independent, non- 5 asthma who primarily have one rare ADRB2 variant,
Hispanic white asthma cohort. These findings suggest African Americans are an admixed ethnic group who
changes in therapeutic response to LABAs in these had a significantly greater frequency of different rare
asthma genetic subpopulations resulting from rare variants. The relatively higher frequency of different rare
ADRB2 variants that have rendered asthmatic carriers variants in African Americans provides a testable
less responsive to the beneficial bronchodilatory and 10 hypothesis that could partly account for the differences
bronchoprotective effects of LABA therapy.
in asthma morbidity and responses to LABA therapy
As expected, rare variants differ between different observed between these ethnic groups.10,37 In African
ethnic groups but resulted in similar effects on three American patients, we identified a rare 25 bp
different health-care-related outcomes during LABA polynucleotide insertion with similar effects on healthtreatment, which is an example of gene-level 15 care use in those treated with a LABA to that observed
replication.34,37 The heterogeneity of rare functional with Thr164Ile in non-Hispanic white patients. The
variants at specific loci has been shown to collectively association of the –376 insertion variant with hospital
determine susceptibility to diseases such as breast cancer admission and other health-care-related outcomes in
and idiopathic pulmonary hypertension.38,39 Gene-level African Americans remained equally significant after
replication is crucial to understanding of rare variant 20 adjustment for ancestry. Thus, the effects of rare variants
effects in human disease because populations with on health-care use in African Americans are not due to
different ancestries will each have unique rare variants specific African or European ancestry. Although there
are no in-vitro studies of the –376 In-Del, this promoter
that might affect human disease in a similar way.
Thr164Ile is a rare ADRB2 variant that results in an polynucleotide insertion is located within an SP1
aminoacid coding change within the fourth trans­ 25 transcription factor binding site and predicted to affect
membrane domain of the β2-adrenergic receptor. In vitro, gene expression.23,48
the rare Ile 164 variant decreases agonist-promoted
The ongoing concern for increased risk of asthmareceptor sequestration and reduces receptor ligand related life-threatening events and death associated with
binding affinity and coupling to Gs protein in response to LABA therapy is based on surveillance trials and metadifferent SABAs such as isoprenaline and salbutamol.40,41 30 analyses in which LABAs were not always used with
These in-vitro effects have also been observed for concomitant inhaled corticosteroid therapy.9,10 In our
formoterol and salmeterol and also include impaired multiethnic asthma populations, LABA and inhaled
binding of salmeterol to its exosite in receptors expressing corticosteroid therapy was the standard of care making
this rare variant.41
detection of the risk of rare, life-threatening events in
On the basis of these in-vitro observations, there are 35 participants treated with a LABA alone impossible.49,50
several possible mechanisms through which the rare Patients with severe asthma characterised by frequent
Ile 164 variant might affect LABA efficacy. First, the health-care use were well represented in this cohort.
deleterious effects of the Ile 164 variant on receptor ligand Recent hospital admission is usually an exclusion
binding or coupling to Gs protein results in attenuation of criterion for most clinical trials, making replication of
the bronchodilator or bronchoprotective effects of LABAs. 40 these findings more difficult.11,12
A second mechanism is that increased airway
We show gene-level replication for two different rare
inflammation occurs in response to LABA treatment in ADRB2 variants on severe exacerbations requiring
individuals with the rare Ile 164 variant as a result of hospital admission and two related, secondary outcomes
decreased receptor sequestration and a subsequent in our primary cohort. We could not replicate the findings
increase in T-helper-2-type inflammatory cytokine 45 for each rare variant [A: edit OK?] for hospital admission
production from continued receptor stimulation.42–44 in an independent, LABA-treated cohort. Further
Third, the rare Ile 164 variant might be related to impaired replication of the association between Thr164Ile in noncardiovascular reserve.45,46 This rare variant has been Hispanic white patients or the –376 insertion variant in
associated with reduced β2-adrenergic-mediated myo­ African Americans and this primary outcome remains to
cardial contractile response, cardiac inotropic reserve, 50 be done in another large multiethnic asthma population.
and chronotropic reserve in controls and an increased Despite this limitation, we replicated associations for a
relative risk of death or the need for cardiac transplantation secondary outcome related to asthma severity (symptom
in individuals with congestive heart failure from control) at the Thr164Ile locus during LABA treatment in
ischaemic or dilated cardiomyopathy.45–47 Interestingly, the the primary cohort and an independent cohort. Thus, the
rare Ile 164 variant was associated with reduced baseline 55 Thr164Ile genotype could be applied to standard
lung function and baseline airflow obstruction in a cross- guideline-based approaches for asthma management to
sectional analysis of 62 
748 participants from the determine the best therapeutic option for a small subset
8 

www.thelancet.com/respiratory Published online January 27, 2014 http://dx.doi.org/10.1016/S2213-2600(13)70289-3

 Articles

of Ile 164 heterozygotes with poor symptom control 1 Airsonnet, Boehringer-Ingelheim, GlaxoSmithKline, Merck, Pfizer, PPD
despite combination therapy with a LABA and inhaled Incorporated, Quintiles, Teva Pharmaceuticals, and Targacept, lecture
fees from Integrity and Merck, is a member of speakers’ bureaus funded
corticosteroids.49,50 In coming years, pharmacogenetic by Integrity CE and Merck, and reports receiving fees from UptoDate for
data—such as those described in this study—may his contribution to a chapter on asthma management. ERB reports
become readily available through genetic panels or 5 receiving funding from NHLBI to support this study (Severe Asthma
complete genome sequencing and could be used for Research Program U10 HL109164) and consultancies with Aerovance,
AstraZeneca, Boehringer, Centocor, GlaxoSmithKline, Genentech, Merck,
personalised medicine.
Novartis, Pfizer, and Roche; he performs clinical trials at Wake Forest
The occurrence of life-threatening events in the University supported by Boehringer, Centocor, GlaxoSmithKline,
SMART LABA surveillance study was very rare but these MedImmune, Genetech, Aerovance, Ception, AstraZeneca, Novartis,
Pfizer, Forest, and Sanofi-Aventis; and is also supported by
rare, serious findings with LABA therapy have caused 10 Amgen,
grants from Spiromics (HHSN 268200900019C), AsthmaNet
the FDA to issue a boxed safety warning and initiate a (U10 HL098103), and the Pharmacogenetics Network (U01 HL65899).
large LABA surveillance study in 46 800 participants.10,11,13 DNA and baseline patient data from Johnson and Johnson (previously
Identification of a susceptible asthma subpopulation at known as Centocor) was obtained from patients with severe asthma who
participated in a randomised clinical trial of golimumab for which ERB
increased risk of LABA-related adverse events will not was principal investigator of the Wake Forest clinical site. These patients
only elucidate major pharmacogenetic mechanisms, but 15 were genotyped and analysed in a pharmacogenetic study of the tumour
could serve as an important predictive biomarker for necrosis factor α gene that was supported by Johnson and Johnson and
presented in abstract form. GAH, ATH, EJA, DC, SCE, FM, and DAM
these rare, serious side-effects.
18

51

declare that they have no conflicts of interest.

Contributors
Acknowledgments
VEO, DAM, and ERB did the literature search. VEO, GAH, WCM, SPP,
This research was supported by research funds from the NIH NHLBI
DAM, and ERB designed the study. VEO, WCM, WWB, MC, DC, SCE,
20
(grants U10 HL109164, RC2 HL 101487, U01 HL65899, R01 HL76285,
EI, FM, SEW, SPP, and ERB enrolled patients in the studies. VEO, GAH,
K12 HL 89992, U10 HL098103, and NR 013700). We thank all the
WCM, EJA, WWB, MC, DC, SCE, EI, FM, SEW, SPP, DAM, and ERB
patients who participated in these studies and all faculty and staff; Elliot
were involved in the acquisition of data. VEO, GAH, EJA, SPP, DAM,
Barnathan (Johnson and Johnson, Malvern, PA, USA) for providing
and ERB analysed data. VEO, GAH, WCM, WWB, MC, DC, SCE, EI, FM,
baseline patient data; Tmirah Haselkorn (Genentech Inc, South San
SEW, SPP, and ERB contributed to oversight of the study. VEO, EJA,
Francisco, CA, USA); and Novartis Pharmaceuticals Corporation, East
DAM, and ERB provided statistical expertise. VEO, GAH, ATH, EJA,
25 Hanover, NJ, USA, for providing patient data from the TENOR cohort.
SCE, EI, SEW, SPP, DAM, and ERB participated in data interpretation.
The report was drafted by VEO, GAH, DAM, and ERB. All authors have
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www.thelancet.com/respiratory Published online January 27, 2014 http://dx.doi.org/10.1016/S2213-2600(13)70289-3