Role ofADRB2 gene polymorphism in asthma and response to β2-agonists in Polish children

Abstract

The aims of this study were: (1) to find associations of asthma with single-nucleotide polymorphisms (SNPs) within theADRB2 gene: Arg16Gly, Gln27Glu, −1023 G/A, −367 T/C, −47 C/T ; (2) to define linkage disequilibrium in the gene region, basing
on the analyzed SNPs; and (3) to analyze the importance ofADRB2 polymorphism for response to bronchodilator drugs in children diagnosed with bronchial asthma. We compared 113 asthmatic
children and 123 healthy subjects from the Polish population. Genotyping was performed by PCR-RFLP. We found an association
of the A allele of −1023A/GADRB2 polymorphism with asthma (P = 0.024). No significant associations with other SNPs were detected. Moderate linkage was found between Gln27Glu and −47C/T
polymorphisms in linkage disequilibrium analysis (D’ = 0.85,r
2 = 0.429, LOD = 31.97). No significant differences were found in haplotype frequencies in comparison to the control group,
implicating that they are not associated with susceptibility to asthma in the analyzed population. There was no significant
correlation between the analyzed SNPs of theADRB2 gene and the response to β2-agonists. This is the first report providing suggestive evidence for association of —1023A/GADRB2 polymorphism with an increased risk of asthma. The analyzed SNPs may not play a major role in response to β2-agonists in asthmatic children.

Keywordsassociation-asthma-β2-adrenergic receptor gene-β2-adrenoceptor gene-linkage disequilibrium-pharmacogenetics-polymorphism

Full text

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Introduction
Selective agonists of the ?2-adrenergic receptor
(also known as ?2-adrenoceptor) are crucial bron-
chodilators inasthma
chronic exposure to ?2-agonists leads to receptor
desensitization (tachyphylaxis) and drug toler-
ance,andtheefficacyofthelong-andshort-acting
?2-agonists and the clinical course of asthma de-
pend on genetic variability of the receptor .
The ?2-adrenergic receptor gene (ADRB2, also
known as ?2-AR) is located in chromosomal re-
gion 5q31-32, which has been reported to be
linked to asthma . ADRB2 is a non-intronic, rela-
tivelysmall(1.2-kb)genethatencodesaproteinof
413 amino acids. In the ADRB2 gene, numerous
treatment.However,
polymorphisms have been identified. The most
widely analyzed single-nucleotide polymorphisms
(SNPs)inthecodinggeneregionincludeArg16Gly
and Gln27Glu. The former is associated with an in-
creased repression of gene transcription and a de-
creased amount of receptors on the cell surface
(Liggett 2000). The Gly variant is associated with
severe asthma (Reihsaus et al. 1993), which has
been confirmed in a meta-analysis summarizing
the results of 28 association studies in asthma
(Contopoulos-Ioannidis et al. 2005). The influ-
ence of this SNP on tachyphylaxis after chronic
exposure to ?2-agonists observed both in vitro and
in vivo suggested that the Gly variant is more sus-
ceptible than the Arg variant to receptor desensiti-
zation (Martinez et al. 1997; Tan et al. 1997). For
Gln27Glupolymorphismithasbeendemonstrated
J Appl Genet 50(3), pp. 275–281
Original article
Role of ADRB2 gene polymorphism in asthma and response to
?2-agonists in Polish children
A. Szczepankiewicz1,2, A. Brêborowicz1, P. Sobkowiak1, L. Kramer3, A. Popiel1
1Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical Sciences, Poland
2Laboratory of Psychiatric Genetics, Department of Psychiatry, Poznan University of Medical Sciences, Poland
3Department of Computer Science and Statistics, Poznan University of Medical Sciences, Poland
Abstract. The aims of this study were: (1) to find associations of asthma with single-nucleotide polymorphisms
(SNPs)withintheADRB2gene:Arg16Gly,Gln27Glu,–1023G/A,–367T/C,–47C/T;(2)todefinelinkagedis-
equilibrium in the gene region, basing on the analyzed SNPs; and (3) to analyze the importance of ADRB2 poly-
morphism for response to bronchodilator drugs in children diagnosed with bronchial asthma. We compared 113
asthmatic children and 123 healthy subjects from the Polish population. Genotyping was performed by
PCR-RFLP. We found an association of the A allele of –1023A/G ADRB2 polymorphism with asthma
(P = 0.024). No significant associations with other SNPs were detected. Moderate linkage was found between
Gln27Glu and –47C/T polymorphisms in linkage disequilibrium analysis (D’ = 0.85, r2= 0.429, LOD = 31.97).
No significant differences were found in haplotype frequencies in comparison to the control group, implicating
thattheyarenotassociatedwithsusceptibilitytoasthmaintheanalyzedpopulation.Therewasnosignificantcor-
relation between the analyzed SNPs of the ADRB2 gene and the response to ?2-agonists. This is the first report
providing suggestive evidence for association of –1023A/G ADRB2 polymorphism with an increased risk of
asthma. The analyzed SNPs may not play a major role in response to ?2-agonists in asthmatic children.
Keywords: association, asthma, ?2-adrenergic receptor gene, ?2-adrenoceptor gene, linkage disequilibrium,
pharmacogenetics, polymorphism.
Received: October 13, 2008. Accepted: December 1, 2008.
Correspondence: A. Szczepankiewicz, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan Uni-
versity of Medical Sciences, Szpitalna 27/33, 60–572 Poznan, Poland; e-mail: alszczep@amp.edu.pl

Page 2

that the Glu variant is associated with a 4-fold de-
crease in airway hyperreactivity in response to
methacholine (Postma et al. 1995) and may be a
factor protecting against receptor desensitization
(Greenetal.1994).Incontrast,amorerecentinvi-
tro study on human airway smooth muscle cells
showedanincreasedshort-andlong-termdesensi-
tization of cells with at least one Glu allele after
treating them with isoproterenol (Moore et al.
2000). Frequencies of both SNPs are similar in
asthmatic patients and healthy subjects (Dewar
etal.1998),sotheSNPsareunlikelytobedirectly
involved in asthma pathogenesis. They may, how-
ever, affect symptom severity and treatment re-
sponse.
Common SNPs in the promoter region of the
ADRB2 gene include –1023A/G, –367C/T and
–47C/T. The first one is located 2 bp upstream
from the sequence binding NF-IL6 transcription
factor (nuclear factor for IL-6) (Scott et al. 1999)
and therefore may influence gene expression. The
others were found to affect gene expression at the
transcription level (–367C/T) and translation level
(–47C/T).
Theaimsofthisstudywere:(1)tofindassocia-
tions of asthma with SNPs within the ADRB2
gene: Arg16Gly, Gln27Glu, –1023 G/A, –367
T/C,–47C/T;(2)todefinelinkagedisequilibrium
in the gene region, basing on the analyzed SNPs;
and (3) to analyze the importance of ADRB2 poly-
morphism for response to bronchodilator drugs in
children diagnosed with bronchial asthma. This is
the first such study done in the Polish pediatric
population.
Materials and methods
Patients
The study was performed on 113 Polish asthmatic
patients of Caucasian origin aged 6–18 years
(68 boys with a mean age of 11.8 years, SD = 2.9;
45 girls with a mean age of 12.4 years, SD = 3.8).
Patients were recruited from inpatients from the
Wielkopolska region, considered as ethnically ho-
mogenous(Cavalli-Sforza1994),andweretreated
for asthma in the Department of Pediatric
Pulmonology, Allergy, and Clinical Immunology
of the Poznan University of Medical Sciences.
Asthma diagnosis was made according to GINA
recommendations, based on clinical asthma symp-
tomsandlungfunctiontest(bronchodilatorrespon-
siveness, exercise-induced hyperresponsiveness).
We analyzed separately a subgroup of children
withsevereasthma(n=54).Severeasthmawasde-
fined as follows: symptoms requiring daily ther-
apy with high-dose inhaled corticosteroids (> 800
budesonide or > 500 fluticasone), despite regular
treatment with long-acting ?2-agonists and/or
leukotriene antagonist and/or theophylline (slow
releasing), and 1 or more emergency care visit or
oral steroid burst per year.
Clinical diagnosis of atopy depended on cur-
rentorpastsymptomsofatopicdermatitis,allergic
rhinoconjunctivitis (seasonal or perennial) or food
allergy,asdescribedpreviously(Szczepankiewicz
et al. 2007).
Response to ?2-agonists was evaluated in the
bronchodilator test (obstruction reversibility) with
salbutamol MDI (200 mcg) applied through
Volumatic holding chamber. Before drug applica-
tion and 15 min after, spirometry was performed.
A ? 15% increase in FEV1 was diagnostic.
Spirometric measures (flow-volume curve) were
performed on LungTest 1000 apparatus according
to ATS guidelines. Response to salbutamol, a
short-acting-?2-agonist (SABA), was analyzed
twice: during chronic treatment with salmeterol, a
long-acting-?2-agonist (LABA), and at least
4weeksaftersalmeterolwithdrawal(wash-outpe-
riod). The percentage inhibition of salbuta-
mol-induced change in FEV1% between both
trialswasanalyzedasthedegreeoftachyphylaxis.
Control group
The control group consisted of 123 healthy sub-
jectsofCaucasianorigin(59boyswithameanage
of10.0years,SD=2.2;64girlswithameanageof
9.6 years, SD = 1.8). Control subjects were re-
cruited from the same Wielkopolska region, from
a group of carefully chosen volunteers without
asthma and allergy symptoms. Any allergic dis-
easesorasthmawereexcludedonthebasisofclin-
ical examination, spirometry, and exhaled NO
measurement.
All participants as well as their parents gave
written informed consent. The local ethics com-
mittee accepted the project. The study was per-
formed in compliance with the Code of Ethics of
the World Medical Association (Declaration of
Helsinki).
Genotyping
DNA
EDTA-anticoagulated whole blood by using the
salting-out method (Miller et al. 1988). The
ADRB2 polymorphisms were analyzed by the
wasextractedfrom 10 mLof
276 A. Szczepankiewicz et al.

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PCR-RFLP method. The conditions of PCR and
sequences of the primers for the following SNPs
were used as described previously: Arg16Gly and
Gln27Glu (Martinez et al. 1997), –367C/T
(Lipworth et al. 2002). For –47C/T and –1023A/G
polymorphisms, primers were designed according
to thePrimer3software
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_
www.cgi.ForArg16Glyand–1023A/Gtheforward
primer was modified (mismatch primer) to create a
recognitionsiteforappropriaterestrictionenzymein
the substitution site with the use of the SNP cutter
available at: http://sky.bsd.uchicago.edu/SNP_cut-
ter.htm. The PCR-RFLP conditions are presented in
Table 1.
available at:
The uncut PCR products for Gln27Glu,
–367C/T and –1023A/G polymorphisms were
re-digested independently to confirm the obtained
genotypes. The control of the whole PCR-RFLP
analysis was also performed (25% of duplicates –
randomly chosen DNA samples from both groups
and blind-tested). The genotyping was performed
without knowing the clinical outcome of the pa-
tient.
Statistical analysis
The Pearson chi-square (?2) test and Fisher exact
test were used to analyze differences in the
genotypic and allelic (respectively) distribution
between the groups of patients and control sub-
jects. Calculationswere
STATISTICAversion7.1software.Datasetscon-
taining < 5 observations per subgroup were sub-
jected to the Fisher-Freeman-Halton exact test
with the use of StatXact 8 software (Cytel, Cam-
bridge, USA). Odds ratios were calculated using a
demo of InStat 3 program (GraphPad Software, La
Jolla,USA).ConcordancewiththeHardy-Weinberg
lawwasverifiedusingUtilityProgramsforAnalysis
performedwith
of Genetic Linkage (© 1988 J. Ott). We also
performedlinkagedisequilibriumanalysisofthean-
alyzed SNPs of the ADRB2 gene by using free on-
line software Haploview version 3.2 from the
website:
http://www.broad.mit.edu/mpg/haplview/index.php
(Barrettetal.2005).Inpharmacogeneticanalysisthe
response to treatment with ?2-agonists, expressed as
the mean values of spirometric measures (FEV1%,
FVC%, PEF), was compared in patients with differ-
entallelesofADRB2polymorphisms.We also ana-
lyzed bronchodilator response to ?2-agonists in
patients stratified by genotype, using one-way
analysisofvariance(ANOVA)ifthedatadistribu-
tion was normal (Shapiro-Wilk test) and after
checking variance homogeneity (Levene test).
Meanswerecompared
Newman-Keuls test. If the data did not meet the
criteria mentioned above, then the nonparametric
Kruskal-Wallis test was performed.
For the analyzed SNPs, we calculated the fol-
lowing power values: 19.5% for allele A and 27%
for allele G (Arg16Gly); 6% for alleles C and G
(Gln27Glu); 7% for allele C and 8.5% for allele T
(–47C/T); 10.6% for allele C and 13.9% for allele
T (–367C/T); 28.6% for allele A and 39.7% for al-
lele G (–1023A/G).
by thepost-hoc
Results
Genotype distributions for all the analyzed SNPs
in theADRB2gene
Hardy-Weinberg equilibrium in both the studied
patients and control subjects (P > 0.05), except
Gln27Glu polymorphism in the control group
(P = 0.015) and –47C/T polymorphism in patients
(P = 0.019). We observed a significant difference
in allele frequencies for –1023A/G polymor-
were mostly in
ADRB2 polymorphism in asthmatic children277
Table 1. Description of PCR-RFLP reaction conditions for the studied single-nucleotide polymorphisms (SNPs)
SNPPrimers PCR
product
Tm Restriction
enzyme
Allele sizes (bp) Source
Arg16Gly
F 5'-GCCTTCTTGCTGGCACCCCAT-3'
R 5'-CAGACGCTCGAACTTGGCCATG-3'
F 5'-GCCTTCTTGCTGGCACCCCAT-3'
R 5'-CAGACGCTCGAACTTGGCCATG-3'
F 5'-CCTCTGCCTCGAGACCTCAAGCC-3'
R 5'-CCGTCTGCAGACGCTCGAAC-3'
F 5'-TGGCCGAAAGTTCCCGTAC-3'
R 5'-TTGGGTGCCAGCAAGAAGG-3'
F 5'-CACAACTTTCTCTCTCTGTCCCAA-3'
R 5'-CAGGAGGTGACTTCAACAGCAGC-3'
168 bp59°CNcoI
A-146, 22
G-128, 22, 18
G-168
C-105, 63
C-758
T-566, 192
C - 186, 89, 60
T-186, 149
A-144, 22
G-166
Martinez
1997
Gln27Glu
168 bp 59°CBesXI
Martinez
1997
–367C/T
740 bp64°C Bsu36I
Lipworth
2002
–47C/T
336 bp56°C MspA1I
own design
–1023A/G
166 bp59°C AluI
own design

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phism,withalleleAsignificantlymorefrequentin
patients than in the control subjects (Table 2). We
did not find any significant differences in geno-
type distribution and allele frequencies for the
other 4 polymorphisms studied (Table 2).
In subgroup analysis, we found an association
of heterozygous genotype of Gln27Glu polymor-
phism with severe asthma (P = 0.019). However,
dueto theobserved
Hardy-Weinberg equilibrium in this subgroup,
this SNP was excluded from interpretation (data
not shown).
In linkage disequilibrium analysis, we ob-
served a moderate linkage between 2 of the SNPs
analyzed, Gln27Glu and –47C/T (D’ = 0.85,
r2= 0.429, LOD = 31.97). On the basis of moder-
ately high D’ value, it was possible to define
haplotype block for them. However, no statistical
differences in frequencies of particular haplotypes
were observed when we compared asthmatic pa-
tients to the control subjects (data not shown). The
linkage values for other SNPs were lower, so
haplotypes could not be defined.
Association between the ADRB2 gene SNPs
and response to ?2-agonists was analyzed in 3
groups of data: (1) response of the receptors
chronically exposed to LABA (salmeterol), in the
deviationfrom
group of patients treated with salmeterol after
application of salbutamol (SABA) (n = 96); (2) re-
sponse of the unexposed receptors after applica-
tionofsalbutamol,inthegroupofpatientswithout
concurrent treatment with salmeterol (n = 48); (3)
degree of receptor desensitization (tachyphylaxis)
after application of salbutamol, in the group of pa-
tientsconcurrentlytreatedwithsalmeterolincom-
parison to the same group in the wash-out period
(no treatment with LABA for at least 4 weeks)
(n = 34). In the first 2 settings, mean values of
bronchodilator response after salbutamol inhala-
tion were not significantly different between pa-
tients stratified by genotypes (data not shown).
Similarly, no significant differences between pa-
tients stratified by genotype of particular ADRB2
polymorphisms were found when the degree of
tachyphylaxis was assessed (data not shown).
Significance level was not corrected for multi-
ple testing.
Discussion
The main finding of this study is an association of
–1023A/Gpolymorphismwithasthma,whichhas
278A. Szczepankiewicz et al.
Table 2. Genotype distributions and allele frequencies of five
ADRB2genepolymorphismsinasthmaticpatientsandthecontrol
group (figures in parentheses indicate percentages)
Polymorphism Asthma
(N = 113)
Control
(N = 123)
P value
Arg16Gly
genotypes
A/A
A/G
G/G
A
G
G/G
G/C
C/C
G
C
CC
CT
TT
C
T
CC
CT
TT
C
T
GG
GA
AA
G
A
16 (14.16)
48 (42.48)
49 (43.36)
80 (35.40)
146 (64.60)
24 (21.24)
58 (51.33)
31 (27.43)
106 (46.90)
120 (53.10)
27 (23.89)
22 (19.47)
64 (56.64)
94 (41.60)
132 (58.40)
21 (18.58)
41 (36.28)
51 (45.13)
83 (36.73)
143 (63.27)
32 (28.32)
59 (52.21)
22 (19.47)
123 (54.42)
103 (45.58)
26 (21.49)
54 (44.63)
41 (33.88)
106 (43.80)
136 (56.20)
36 (29.27)
48 (39.02)
39 (31.71)
120 (48.79)
126 (51.21)
19 (15.57)
51 (41.80)
52 (42.62)
89 (36.48)
155 (63.52)
17 (13.82)
49 (39.84)
57 (46.34)
83 (33.74)
163 (66.26)
50 (40.50)
59 (47.97)
14 (11.38)
159 (64.63)
87 (35.37)
0.204
alleles
0.072
Glu27Gln
genotypes
0.146
alleles
0.123
–367C/T
genotypes
0.550
alleles
0.258
–47C/T
genotypes
0.593
alleles
0.501
–1023A/G
genotypes
0.070
alleles
0.024*
* indicates significance: ÷2= 5.314, df = 2

Page 5

not been observed in any other study. The second
main finding is a lack of relationship between
bronchodilatorresponse
ADRB2 receptor genotypes for all studied SNPs.
to
?2-agonists and
Association analysis
The extensive studies on Arg16Gly and Gln27Glu
polymorphisms were based on their localization
within(codon16)orinthevicinity(codon27)of2
tripeptides(N-X-S/T)
N-glycosylation involved in receptor expression
on the cell surface (Rands et al. 1990) and in bind-
ing to Gsproteins (Boege et al. 1988). Functional-
ity of Arg16Gly polymorphism was determined in
vitro by Green et al. (1994), who demonstrated
that the Gly allele was associated with more rapid
receptor desensitization
?2-agonists, in comparison to the Arg allele. In the
case of Gln27Glu polymorphism, Green et al.
(1994) observed that the Glu variant underlies
downregulation to a lesser degree than Gln in cul-
tures of human primary airway smooth muscle
cells after chronic exposure to ?2-agonists. That
suggestedaprotectiveroleofthisvariantinregard
to receptor desensitization. In our analysis, we did
not observe any association of Arg16Gly and
Gln27Glu polymorphisms with asthma, although
it wasearlierconfirmed
(Contopoulos-Ioannidis, Manoli et al. 2005;
Migita et al. 2004).
Promoter polymorphism –1023A/G was not
analyzedpreviouslyinrelationtoasthmasuscepti-
bility or with bronchodilator response. In our
study, we observed that allele A was associated
with 1.5-fold increased risk of asthma in compari-
son to allele G (OR = 1.53, 95%CI = 1.057–2.216).
It is difficult to verify our results, as there are no
previous association studies with asthma in other
cohorts. Its location in the promoter region adja-
cent to the NF-IL-6 transcription factor binding
site may affect gene expression (Scott et al. 1999).
However, the true importance of this substitution
has not been analyzed in experimental studies.
The other promoter polymorphism, –367C/T,
islocatedwithinthesequencethatformsaconsen-
sussequencerecognizedbytheAP-2transcription
factor, which affects gene transcription and ex-
pression (Scott, Swan et al. 1999). This was con-
firmed by Westland et al. (2004), indicating a
decreased affinity to the transcription factor com-
plex in the case of allele C. In our study, no rela-
tionship was found with asthma. No previous
association studies in asthma have been reported
being consensus for
inresponse to
inmeta-analyses
for this SNP, so we cannot compare our results.
The third promoter polymorphism, –47C/T, is lo-
cated within thesequence
?2-adrenoreceptor upstream peptide, responsible
for regulation of receptor translation with allele C
(encoding Arg), correlated with decreased recep-
tor expression. In this study, no association was
observed with asthma; however, studies on larger
populations seem to be necessary to verify our re-
sults.
encodingfor
Pharmacogenetic analysis
The present study is the first pharmacogenetic
analysis of the ADRB2 gene performed in the Pol-
ish population of asthmatic children. Our negative
results are concordant with a recent study by
Bleecker et al. (2006), as they also did not observe
significant differences in response to salmeterol
when analyzing patients with different ADRB2
polymorphisms. Similarly to their study, we also
analyzed patients treated simultaneously with in-
haled glucocorticoids and observed improvement
after salmeterol treatment.
Numerous clinical studies of Arg16Gly poly-
morphism in association with bronchodilator re-
sponse showed inconsistent results: both positive
(Kukreti et al. 2005; Telleria et al. 2006; Wechsler
et al. 2006) and negative (Bleecker et al. 2006;
Joos et al. 2001; Martinez et al. 1997; Tsai et al.
2006). In a meta-analysis by Lee et al. (2004),
summarizing the results of 6 randomized studies,
they foundthatArg16
bronchoprotectivesubsensitivity
(formoterol and salmeterol) in patients treated
with glucocorticoids. In the case of Gln27Glu
polymorphism, the results from clinical studies
were inconsistent again, showing either an associ-
ation of the Gln27 allele with tachyphylaxis
(Telleria et al. 2006) or a lack of relationship of
this SNP with response to ?2-agonists (Martinez
et al. 1997). The latter negative finding is consis-
tent with our results.
In experimental studies, –367C/T polymor-
phism did not influence the degree of receptor de-
sensitizationafter exposure
(Westland et al. 2004). Therefore, it seems un-
likely to be involved in the response to those
drugs, which was confirmed by our study. In the
case of –47C/T polymorphism, a significant asso-
ciationofpoorbronchodilatorresponsewithallele
T was observed in a SAGE (Study of Afri-
can-Americans, Asthma, Genes and Environ-
ments)report(Tsaietal.2006).Thoseresultswere
predisposes
to
to
LABA
to
?2-agonists
ADRB2 polymorphism in asthmatic children 279