Group Title: Journal of Translational Medicine 2008, 6:11
Title: Adrenergic gene polymorphisms and cardiovascular risk in the NHLBI-sponsored Women's Ischemia Syndrome Evaluation
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Title: Adrenergic gene polymorphisms and cardiovascular risk in the NHLBI-sponsored Women's Ischemia Syndrome Evaluation
Series Title: Journal of Translational Medicine 2008, 6:11
Physical Description: Archival
Creator: Pacanowski MA
Zineh I
Li H
Johnson BD
Cooper-DeHoff RM
Bittner V
McNamara DM
Sharaf BL
Merz CNB
Pepine CJ
Johnson JA
Publication Date: 39517
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Bibliographic ID: UF00100182
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Adrenergic gene polymorphisms and cardiovascular risk in the
NHLBI-sponsored Women's Ischemia Syndrome Evaluation
Michael A Pacanowski1, Issam Zineh*1, Haihong Li2, B Delia Johnson3,
Rhonda M Cooper-DeHoff4, Vera Bittner5, Dennis M McNamara6,
Barry L Sharaf7, C Noel Bairey Merz8, Carl J Pepine4 and Julie A Johnson'

Address: 'Department of Pharmacy Practice and Center for Pharmacogenomics, University of Florida College of Pharmacy, Gainesville, FL, USA,
2Department of Epidemiology and Health Policy Research, University of Florida College of Medicine, Gainesville, FL, USA, 3Department of
Epidemiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA, 4Department of Medicine, University of Florida
College of Medicine, Gainesville, FL, USA, 5Department of Medicine, University of Alabama Birmingham, Birmingham, AL, USA, 6Department of
Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 7Department of Medicine, Rhode Island Hospital, Providence, RI,
USA and 8Department of Medicine and Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
Email: Michael A Pacanowski; Issam Zineh*; Haihong Li; B
Delia Johnson; Rhonda M Cooper-DeHoff;
Vera Bittner; Dennis M McNamara; Barry L Sharaf; C
Noel Bairey Merz; Carl J Pepine; Julie A Johnson
* Corresponding author

Published: 10 March 2008 Received: 18 October 2007
journal of Translational Medicine 2008, 6:11 doi: 10. 1186/1479-5876-6- 1 Accepted: 10 March 2008
This article is available from: I
2008 Pacanowski et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Adrenergic gene polymorphisms are associated with cardiovascular and metabolic
phenotypes. We investigated the influence of adrenergic gene polymorphisms on cardiovascular risk in
women with suspected myocardial ischemia.
Methods: We genotyped 628 women referred for coronary angiography for eight polymorphisms in the
(IA-, PI-, P2- and 33-adrenergic receptors (ADRAIA, ADRBI, ADRB2, ADRB3, respectively), and their
signaling proteins, G-protein P 3 subunit (GNB3) and G-protein a subunit (GNAS). We compared the
incidence of death, myocardial infarction, stroke, or heart failure between genotype groups in all women
and women without obstructive coronary stenoses.
Results: After a median of 5.8 years of follow-up, I 15 women had an event. Patients with the ADRBI
Gly389 polymorphism were at higher risk for the composite outcome due to higher rates of myocardial
infarction (adjusted hazard ratio [HR] 3.63, 95% confidence interval [95%CI] 1.17-1 1.28; Gly/Gly vs. Arg/
Arg HR 4.14, 95%CI 0.88-19.6). The risk associated with ADRBI Gly389 was limited to those without
obstructive CAD (n = 400, Pinteraction = 0.03), albeit marginally significant in this subset (HR 1.71, 95%CI
0.91-3.19). Additionally, women without obstructive CAD carrying the ADRB3 Arg64 variant were at
higher risk for the composite endpoint (HR 2.10, 95%CI 1.05-4.24) due to subtle increases in risk for all
of the individual endpoints. No genetic associations were present in women with obstructive CAD.
Conclusion: In this exploratory analysis, common coding polymorphisms in the PI- and P3-adrenergic
receptors increased cardiovascular risk in women referred for diagnostic angiography, and could improve
risk assessment, particularly for women without evidence of obstructive CAD.
Trial Registration: NCT00000554.

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Journal of Translational Medicine 2008, 6:11

Coronary artery disease (CAD) is the leading cause of
morbidity and mortality among women in the United
States [ 1 More than half of women presenting with chest
pain or suspected myocardial ischemia do not have angi-
ographic evidence of stenosis [2]. Despite the absence of
obstructive lesions, many of these women have been
shown to have myocardial ischemia due to microvascular
disease [3,4] and are at high risk for cardiovascular events
[5,6]. Diagnosing CAD and assessing cardiovascular risk
among women continues to be clinically challenging and
represents a major public health concern. Therefore, alter-
native methods to estimate cardiovascular risk in women
are necessary to reduce the burden of cardiovascular dis-

Cardiovascular disease has been observed in families, and
a genetic predisposition has long been appreciated [7].
The literature is replete with studies have demonstrated
the potential prognostic value of genetic polymorphisms
[8], even in patients with established cardiovascular dis-
ease [9-11]. Studies have also demonstrated a sex-specific
associations between genetic variants and cardiovascular
disease phenotypes such as myocardial infarction and
ischemic heart disease [12]. However, the potential
genetic mechanisms remain incompletely explored.

Genetic polymorphisms in the adrenergic system have
been linked to various cardiovascular and metabolic dis-
orders, such as hypertension, heart failure, and diabetes
[13] Namely, the genes that encode the P1-, P2-, and a-
adrenergic receptors are important in myocardial and vas-
cular function, the P3-adrenergic receptors are involved in
thermogenesis and lipolysis, and the subunits of their cog-
nate G proteins all have documented associations with
cardiovascular or metabolic phenotypes. We investigated
the association of these genes with cardiovascular out-
comes in women with clinical indications for a cardiac
angiography who participated in the National Heart,
Lung, and Blood Institute (NHLBI)-sponsored Women's
Ischemia Syndrome Evaluation (WISE) study.

Study population and procedures
We studied 628 women enrolled in the NHLBI-sponsored
WISE study who consented to genetic analyses and had
complete clinical data. The WISE protocol has been previ-
ously described [14]. Briefly, the WISE was a multicenter
prospective cohort study of 936 women that was designed
to evaluate diagnostic techniques, disease mechanisms,
and the prognosis of ischemic heart disease in women,
particularly those without coronary artery stenosis. The
WISE population consisted of women over the age of 18
undergoing coronary angiography as clinically indicated
for the evaluation of chest pain or suspected myocardial

ischemia. The baseline evaluation included collection of
demographic data and a detailed medical history, as well
as a symptom and psychosocial evaluation, physical
examination, and blood sampling. Quantitative angiogra-
phy was performed at a core laboratory by investigators
blinded to all other subject data. Follow-up data were col-
lected by telephone or mail contact six weeks after angiog-
raphy, then yearly. Women were followed for death from
any cause and hospitalization for nonfatal myocardial inf-
arction (MI), heart failure, or stroke. Death certificates
were obtained for verification and, where possible, other
events were verified against the medical record. Nonfatal
events were adjudicated at one center and shown to be
98.2% concordant with data gathered through standard
follow-up procedures. The WISE protocol was approved
by the institutional review boards of all participating sites,
and all study participants gave written informed consent
before undergoing evaluation and sample collection for
genetic analyses.

Selection of polymorphisms and genotyping methods
Genomic DNA was isolated from whole blood using a
commercially available kit (Puregene; Gentra Systems,
Minneapolis, MN). Genotypes were determined for the
following 8 single nucleotide polymorphisms (SNPs)
with known or putative functional consequences in 6
adrenergic system genes: P,-adrenergic receptor (ADRB1;
Ser49Gly and Arg389Gly), B2-adrenergic receptor
(ADRB2; Argl6Gly and Gln27Glu), P3-adrenergic recep-
tor (ADRB3; Trp64Arg), a,,-adrenergic receptor
(ADRA1A; Arg347Cys), stimulatory G-protein a subunit
(GNAS; 393 T>C), and G-protein 33 subunit (GNB3; 825
C>T). Genotyping was performed in duplicate using opti-
mized polymerase chain reaction protocols and either sin-
gle-primer extension (SNP-IT; Orchid Biosciences,
Princeton, NJ) or luciferase-based assays with the Pyrose-
quencing PSQ HS 96 system (Biotage AB, Uppsala, Swe-

Data analysis
Baseline characteristics were compared between genotype
groups using X2 tests for categorical data and t-tests, anal-
ysis of variance, or a nonparametric equivalent for contin-
uous data. Departures from Hardy-Weinberg equilibrium
were tested by X2 or Fisher's exact tests. The primary out-
come was a composite of death from any cause, or hospi-
talization for nonfatal MI, heart failure, or stroke. The
effect of each of the eight SNPs on the primary outcome
was evaluated using Kaplan-Meier analysis and pooled
log-rank tests adjusted for race. Hazard ratios (HR) and
95% confidence intervals (95%CI) were estimated using
Cox proportional hazards regression. The regression
model included age and race as forced covariates, in addi-
tion to clinical variables that were significant predictors of
the outcome in univariate analysis (P < 0.1) that remained

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Journal of Translational Medicine 2008, 6:11

significant in multivariate analysis (P < 0.05). Given the
heterogeneous nature of the primary outcome, significant
associations were followed by exploratory analysis of the
individual outcomes. All analyses compared heterozy-
gotes and variant homozygotes to common allele
homozygotes, except SNPs with a minor allele frequency
< 0.1 were treated as dominant in the interest of ensuring
adequate power. Furthermore, where more than 1 SNP
was typed in a single gene (i.e. ADRB1 and ADRB2), hap-
lotypes were inferred using PHASE version 2.1 [15 coded
based on the number of copies (0, 1, or 2), and individu-
ally entered into the regression with 0 copies as the refer-
ent category. To probe for modification of genotype
effects by race and CAD severity, interaction terms were
tested and stratified analysis was performed.

Logic regression under the Cox proportional hazards
model, implemented as a package for R statistical soft-
ware, was used to define epistatic interactions as previ-
ously described [16-18]. Logic regression is a powerful
adaptive regression method that tests multiple variable
combinations (leaves) using logical, Boolean operators
(and, or) and stochastic modeling to define the best fitting
model (tree) for a binary outcome. As an example, this
method yields a model that is interpreted as follows: (SNP
A carrier AND SNP B variant homozygote) OR ((SNP C
carrier OR SNP D carrier) AND SNP E carrier). Cross-vali-
dation was performed to identify the best scoring model
containing 1 to 8 predictors, and a single logic regression
tree was constructed. Default settings were used with
25,000 iterations. All SNPs were eligible for inclusion in
the model. Additional models were fit for women without
obstructive CAD.

An additional analysis was performed to determine if
genetic markers retained predictive value along with clin-
ical data and inflammatory biomarkers for women with
available data (n = 559, 89%). This model added global
inflammatory status, as defined by the number of inflam-
matory biomarkers in the upper quartile (C-reactive pro-
tein, interleukin-6, and serum amyloid A) [19]. The
contribution of genetic variables to the overall risk assess-
ment beyond clinical and inflammatory variables among
women without obstructive CAD was determined by com-
paring the global -2 log-likelihood of the models contain-
ing clinical and inflammatory biomarker data, with and
without significant genetic variables.

Statistical analyses were performed using SAS version 9.1
(SAS Institute, Inc., Cary, NC) and R (R Foundation,
Vienna, Austria). The significance threshold for all analy-
ses was set at a = 0.05. At minor allele frequencies greater
than 0.1, this study had 80% power to detect a relative
hazard of approximately 1.81 assuming a standard devia-

tion of 0.5 and a dominant model. Power calculations
were performed using PASS (NCSS, Kaysville, UT).

Complete clinical and genetic data were available for 628
women. Baseline characteristics are presented in Table 1.
The majority of women were non-Hispanic whites (83%).
The mean age was 58 12 years and 32% of the women
were over the age of 65. Obstructive lesions on angiogra-
phy were present in 228 (36%) patients. Hardy-Weinberg
equilibrium was satisfied for all loci. Genotype and allele
frequencies differed significantly by race at all loci (Table
2). Notable differences in baseline characteristics by gen-
otype were as follows: ADRA1A Cys347 homozygotes had
a lower prevalence of dyslipidemia (Arg/Arg 53%, Arg/Cys
50%, Cys/Cys 40%, P = 0.04) and higher diastolic blood
pressure (Arg/Arg 75 10 mmHg Arg/Cys 77 11 mmHg,
Cys/Cys 79 + 11 mmHg, P = 0.02), GNAS 393C homozy-
gotes had a lower hypertension prevalence (T/T 65% T/C
58% C/C 52%, P = 0.05) and systolic BP (139 23
mmHg, 137 21 mmHg, 133 + 19 mmHg, P = 0.02), and
GNB3 825T homozygotes had a higher prevalence of dia-
betes (C/C 21%, C/T 20%, T/T 40%, P = 0.0002).

The median duration of follow-up was 5.8 years (inter-
quartile range 3.6-8.1 years), over which time 115
women (18.3%) experienced a primary event. The event
rate was higher in women with (versus without) obstruc-
tive CAD (28.9% vs. 12.3%). Clinical correlates with
event risk identified in univariate analysis included base-
line systolic BP, age, black race, ever-smoking, diabetes
history, hypertension history, dyslipidemia history, BMI,
and obstructive CAD. Hypertension and dyslipidemia his-
tory did not retain significance in the multivariate model
and were eliminated. Additionally, the risk for the com-
posite outcome was higher among patients with 2 or more
inflammatory markers in the highest quartile, as previ-
ously reported [19]. All of these factors were predictive in
patients without obstructive CAD, whereas only diabetes,
ever-smoking, and inflammatory biomarkers were associ-
ated with a greater risk for the primary outcome in women
with obstructive CAD (data not shown).

Associations between the adrenergic SNPs and the pri-
mary outcome are depicted in Table 3. Patients with the
ADRB1 Gly389 allele had a significantly higher incidence
of the primary outcome (log-rank P = 0.047; Table 3).
When restricted to whites only, the risk in ADRB1 Gly389
homozygotes was elevated but marginally significant
(Table 3). The primary outcome association was driven by
higher rates of MI among patients with the variant allele
and death and heart failure trended in the same direction,
while stroke risk was neutral (Table 4). For ADRB 1, 3 com-
mon haplotypes were defined; the Gly49-Gly389 haplo-
type was not observed. Consistent with the SNP

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Table I: Baseline characteristics

> 50% stenosis (n = 228)

White/Caucasian (%)
Age (years)*
Past medical history (%)
Diabetes mellitus*
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Body mass index (kg/m2)
Waist circumference (in)
Cigarette smoking (%)
Menopausal status (%)*
Family history of premature CAD (%)
Current medication use (%)
History of HRT (%)*
CRP (n = 547; median, IQR)
SAA (n = 519; median, IQR)*
IL-6 (n = 547; median, IQR)

62 12

138 22
77 II
29.1 6.4
36 8



0.37, 0.17-0.97
0.66, 0.36-1.15
3.37, 1.85-6.87


Abbreviations: ACE, angiotensin-converting enzyme; CAD, coronary artery disease; HRT, hormone replacement therapy; IQR, interquartile range;
CRP, C-reactive protein; SAA, serum amyloid A; IL-6, interleukin-6
Values presented as means+SD unless otherwise specified
*P < 0.05 for comparison between > 50% stenosis and < 50% stenosis; for CRP, SAA, and IL-6 based on Wilcoxon rank-sum test
tHistory of high cholesterol or use of lipid-lowering medication

associations, patients with 2 copies of the Ser49-Gly389
haplotype were at increased risk for the composite out-
come (frequency 0.30; HR 2.00, 95%CI 1.08-3.71), and
similar trends were noted for the individual endpoints as
above. No other significant SNP associations were identi-
fied in the overall population (Table 3). For ADRB2, 3
common haplotypes were defined; the Argi 6-Gly27 allele
was present on only 9 chromosomes in whites. ADRB2
haplotypes were not associated with the primary outcome
(data not shown).

The presence or absence of CAD appeared to modify gen-
otype risks for ADRB1 Arg389Gly (Pinteraction= 0.03; Figure
1) and ADRB3 Trp64Arg (Pinteraction= 0.1; Figure 2). None
of the SNPs were associated with the primary outcome in
patients with obstructive CAD (data not shown). How-
ever, in women without obstructive CAD, the association
between ADRB1 Arg389Gly polymorphism and the pri-
mary outcome remained significant (log-rank P = 0.03)
but was attenuated when adjusted for clinical covariates
(Arg/Gly vs. Arg/Arg HR 1.56, 95%CI 0.82-2.97; Gly/Gly
vs. Arg/Arg HR 1.95, 95%CI 0.82-2.97). Gly389 carriers

were at increased risk in both racial groups (data not
shown). Again, this was related to increased MI risk
among Gly389 carriers, in addition to an increase in heart
failure risk (Table 4). Haplotype associations were pre-
dominantly driven by patients with 2 copies of the Ser49-
Gly389 haplotype (HR 1.74, 95%CI 0.52-1.50), but not
statistically significant. Also in the subgroup of women
without obstructive CAD, a trend toward higher risk was
apparent in patients carrying the Arg64 allele of ADRB3
(log-rank P = 0.074). The association strengthened after
adjustment for clinical covariates (Trp/Arg+Arg/Arg vs.
Trp/Trp HR 2.10, 95%CI 1.05-4.24). The risk among Arg
64 carriers remained significant when restricted to whites
(Trp/Arg+Arg/Arg vs. Trp/Trp HR 2.49, 95%CI
1.11-5.60). ADRB3 genotype was associated with subtle
trends toward higher rates of all events (Table 4).

Logic regression models with 1 to 8 predictors were eval-
uated. Figure 3 illustrates the test-set deviance for different
size models in the overall population. Models containing
more than 1 predictor did not improve test-set deviance,
and the best fitting model for the overall population and

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< 50% stenosis (n = 400)

56 II

136 21
77 II
29.8 6.8
36 7



0.36, 0.16-0.84
0.55, 0.31-0.94
2.79, 1.72-4.61

Journal of Translational Medicine 2008, 6:11

Journal of Translational Medicine 2008, 6:11

Table 2: Genotype and allele frequencies by race*










393 T > C

825 C > T

Abbreviations: MAF, minor allele frequency
*all P < 0.05 for genotype frequency by race

the subset of patients with obstructive CAD (not includ-
ing other clinical predictors) contained only the ADRB1
Arg389 Gly polymorphism.

Including inflammatory biomarkers in the model reduced
the sample size by 11%. Consequently, while the
increased risk for the composite outcome associated with
the Gly389 persisted after adjustment for inflammatory
biomarkers, the estimate lost significance (Arg/Gly vs.
Arg/Arg HR 0.96, 95%CI 0.63-1.47; Gly/Gly vs. Arg/Arg
HR 1.83, 95%CI 0.95-3.52). The association with MI was
similarly borderline in the overall population (Arg/Gly vs.
Arg/Arg HR 3.12, 95%CI 0.94-10.4; Gly/Gly vs. Arg/Arg
HR 3.37, 95%CI 0.56-20.4) and when restricted to
women without obstructive CAD (Arg/Gly vs. Arg/Arg HR
4.83, 95%CI 0.84-20.32; Gly/Gly vs. Arg/Arg not calcu-
lated because no events in Gly/Gly patients). However,
despite the sample size reduction, the risk associated with
carrying the ADRB3 Arg64 allele remained significant in
women without obstructive CAD (Trp/Arg+Arg/Arg vs.

Trp/Trp HR 2.37, 95%CI 1.08-5.20). For patients without
obstructive CAD, incorporating both the ADRB1 codon
389 and ADRB3 codon 64 genotypes significantly
improved the global -2 log-likelihood of the models
based on clinical variables (P < 0.001 for change) and
inflammatory biomarker data (P < 0.001 for change;
Table 5).

In this study, we examined the joint effect of traditional
risk factors, inflammatory mediators, and candidate SNPs
in several genes from the adrenergic pathway on cardio-
vascular risk in women undergoing clinical evaluation for
suspected ischemia. Women with symptoms of ischemia
prompting angiography have been shown to have micro-
vascular CAD, which is associated with a prognosis that is
similar to that of patients with obstructive CAD [4,6]. We
identified associations between incident cardiovascular
events and the P3-adrenergic receptor Arg389Gly and the
33-adrenergic receptor Trp64Arg polymorphisms. The

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White, n (%)

399 (76)
121 (23)
265 (51)
215 (41)
41 (8)
175 (33)
263 (50)
193 (37)
253 (48)
439 (84)
163 (31)
270 (52)
91 (17)
139 (27)
254 (49)
130 (25)
280 (53)

Black, n (%)

49 (47)
49 (47)
37 (36)
54 (52)
13 (12)
18 (17)
56 (54)
30 (29)
29 (28)
I (I)
81 (79)
21 (20)
I (I)
6 (6)
43 (41)
55 (53)
60 (58)
42 (40)
2 (2)
7 (7)
30 (29)
67 (64)

Table 3: Incidence and relative hazard of primary outcome by genotype

Gene Genotype No. Events Primary All Patients
(%) Outcome Adjusted HR
Incidence* (95%CI)t n = 628

Ser49Gly Ser/Ser
Arg389Gly Arg/Arg
Glyl6Arg Gly/Gly
Gln27Glu Gin/Gin



393 T > C

825 C > T

Trp/Arg +




3 (33)
55 (20)

34 (18)
24 (21)
52 (20)

93 (18)
21 (20)

31 (18)

36 (18)
58 (20)
21 (16)

26 (26)

1.00 (reference)
0.88 (0.57-1.37)
1.02 (0.31-3.36)
1.00 (reference)
1.15 (0.77-1.71)
1.94 (1.05-3.59)

1.00 (reference)
0.93 (0.60-1.43)
1.14 (0.67-1.93)
1.00 (reference)
0.88 (0.59-1.30)
0.99 (0.53-1.84)

4.0 1.00 (reference)
4.8 1.32 (0.81-2.14)

1.00 (reference)
1.03 (0.66-1.62)
0.68 (0.37-1.18)

1.00 (reference)
1.10 (0.71-1.69)
1.31 (0.74-2.31)

1.00 (reference)
1.06 (0.69-1.64)
1.51 (0.82-2.77)

P Whites
Adjusted HR
(95%CI)t n = 524

1.00 (reference)
0.58 0.89 (0.51-1.53)
0.98 ...
1.00 (reference)
0.49 1.09 (0.69-1.72)
0.03 1.82 (0.92-3.61)

1.00 (reference)
0.73 0.90 (0.56-1.46)
0.63 0.81 (0.42-1.57)

0.51 1.06 (0.67-1.71)
0.98 1.13 (0.59-2.15)

1.00 (reference)
0.26 1.19 (0.67-2.14)

1.00 (reference)
0.88 0.97 (0.60-1.57)
0.16 0.83 (0.43-1.58)

1.00 (reference)
0.67 1.26 (0.73-2.17)
0.36 1.60 (0.85-3.01)

1.00 (reference)
0.78 1.26 (0.73-2.17)
0.19 1.60 (0.85-3.01)

P Blacks Adjusted HR
(95%CI)t n = 104

1.00 (reference)
1.12 (0.51-2.48)
1.95 (0.47-8.03)
1.00 (reference)
1.23 (0.56-2.83)
2.27 (0.53-9.79)

1.00 (reference)
1.09 (0.38-3.19)
2.61 (0.81-8.42)

0.51 (0.20-1.30)

1.00 (reference)
1.33 (0.51-3.50)

1.00 (reference)
1.05 (0.20-5.43)
0.46 (0.08-2.39)

1.00 (reference)
0.97 (0.39-1.93)

1.00 (reference)
0.87 (0.40-1.93)

Abbreviations: HR, hazard ratio; 95%CI, 95% confidence interval
* incidence per 100 patient-years
adjusted for age, race, presence of obstructive CAD, history of diabetes, ever-smoking, baseline systolic BP, BMI
analyzed using dominant model based on minor allele frequency < 0.1

association with ADRB3 was present only in women with-
out obstructive CAD and maintained significance in the
presence of other robust predictors of cardiovascular risk.

The Arg389Gly polymorphism in ADRB1 was associated
with nearly a two-fold increase in the risk for major cardi-
ovascular events. In vitro, the Arg389 allele demonstrates
higher basal and agonist-stimulated adenylyl cyclase
activity than the Gly389 variant, resulting in increased
sympathetic tone [20]. In human studies, the Gly389
allele was under-represented in Japanese acute MI patients
relative to controls, and similar findings in heart failure
suggested that this variant might be protective [21,22].
However, subsequent cohort studies did not identify any
genotype-related differences in adverse cardiovascular
outcomes [9,23]. To the contrary, we identified an excess
risk of cardiovascular events in patients with the Gly389

A recent investigation offers compelling mechanistic data
to support this unexpected finding of increased risk.
Akhter et al. [24] found that hearts from transgenic mice
over-expressing cardiac Gly389 showed significantly
poorer systolic and diastolic recovery after ischemia-reper-
fusion compared with Arg389 mice and non-transgenic
littermates. Functional data further showed that the
Arg389 receptor displayed enhanced phosphorylation,
leading to desensitization and increased anti-apoptotic
signaling. To the extent that women participating in WISE
were enrolled based on chest pain suggestive of ischemia,
diminished post-ischemic myocardial recovery associated
with the Gly389 allele could be a biologically plausible
explanation of our finding that the Gly389 allele
increased the risk for heart failure, more so among
patients without obstructive CAD. The role of the poly-
morphism in the pathogenesis of myocardial infarction is
less clear, although it is possible that less severe ischemic
episodes may actually result in myocardial damage in cer-
tain patients. Interestingly, the association was primarily

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Journal of Translational Medicine 2008, 6:11

Table 4: Individual endpoint risk by ADRBI and ADRB3 genotype


Myocardial Infarction

Heart Failure


No. Events Adjusted
(%) HR(95%CI)*
n =628

No. Events Adjusted No. Adjusted
(%) HR(95%CI)* Events (%) HR(95%CI)*
n =628 n =628

No. Events Adjusted
(%) HR(95%CI)*
n =628

All Patients
(n = 628)
ADRBI Arg389Gly



ADRB3 Trp64Argt

Trp/Arg + Arg/Arg

(n = 400)




Trp/Arg + Arg/Arg

Abbreviations: HR, hazard ratio; 95%CI, 95% confidence interval
* adjusted for age, race, presence of obstructive CAD, history of diabetes,
t analyzed using dominant model based on minor allele frequency < 0.1

seen in women without obstructive lesions, in whom col-
lateral development and ischemic preconditioning are
less likely to compensate. Thus, the outlined mechanisms
involving apoptotic signaling may be particularly relevant
in this subset of women, whereas other factors may be
pervasive in women with flow limiting stenosis.

Among women without obstructive CAD, we also identi-
fied an association between composite outcome and the
Trp64Arg variant in the P3-adrenergic receptor, which has
been associated with reduced agonist-stimulated adenylyl
cyclase activity in vitro [25]. Higashi, et al. [26] reported a
higher frequency of the variant among Japanese patients
with CAD, although this was not substantiated by subse-
quent investigations [27-29]. Considering the previously
reported associations between the Trp64Arg polymor-
phism and pro-atherogenic metabolic diseases such as
diabetes, we expected that the variant might be indirectly
associated with cardiovascular risk [30]. However, the

ever-smoking, baseline systolic BP, BMI

Trp64Arg polymorphism was not associated with BMI,
waist circumference, or diabetes in the WISE population
[31]. Moreover, the association with the primary outcome
was robust to adjustment for these factors.

Beyond metabolic functions, P3-adrenergic receptors reg-
ulate cardiac inotropy, angiogenesis, and endothelium-
dependent vasorelaxation in the coronary microvascula-
ture [32,33]. WISE data demonstrated that women with-
out obstructive CAD have evidence of ischemia using the
gold standard cardiac magnetic resonance spectroscopy
[4], and that this ischemia is associated with an adverse
prognosis [6]. Panting, et al. [3] further demonstrated that
subendocardial hypoperfusion may drive myocardial
ischemia in patients who have typical angina or abnormal
stress test results but no angiographic evidence of severe
coronary arterystenosis. RecentWISE data suggest that this
may be due to microvascular dysfunction. [34] Thus, com-
promised P3-adrenergic receptor signaling could ostensi-

Page 7 of 11
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22 (7.3)


7 (13.0)

50 (9.6)


8 (4.1)

8 (4.8)

4 (11.1)


4 (6.9)




1.9 1

4 (1.3)

13 (4.8)

3 (5.6)

16 (3.1)

4 (3.9)


8 (4.8)

2 (5.6)

7 (2.1)

3 (5.2)





II (3.6)

16 (6.0)

4 (7.4)

24 (4.6)

6 (5.8)

3 (1.5)

II (6.6)


II (3.2)

3 (5.2)





14 (4.6)

9 (3.4)

4 (7.4)

21 (4.0)

5 (4.8)

5 (2.6)

6 (3.6)

2 (5.6)


3 (5.2)





Journal of Translational Medicine 2008, 6:11

A vs. B P=0.015
C vs. D P=0.799
12 24 36 48 60

A: Non-obstructive CAD, Arg389 homozygote (n=196)

B: Non-obstructive CAD, Gly389 carrier (n=202)

C: Obstructive CAD, Arg389 homozygote (n=106)

D: Obstructive CAD, Gly389 carrier (n=121)

72 84 96

Time to Event (months)

Figure I
Kaplan-Meier plot for primary outcome by ADRB I coc

bly promote ischemia in the microvasculature. While the
association with clinical outcomes in WISE was modest,
our results suggest that this receptor may play an impor-
tant role in patients with ischemia in the absence of
obstructive CAD and warrant further investigation.

Logic regression is a novel and very powerful approach to
defining high-level gene-gene or gene-environment inter-
actions. As the SNPs included in this investigation


0 90

3 t

> 70

3 60 Log-rank
0 A v R P=n n75

Ion 389 genotype and CAD severity.

spanned a biological pathway, we examined whether the
genetic variations interacted with each other to modify
cardiovascular risk. Previously, this had been demon-
strated for renin-angiotensin system polymorphisms in
the Group Health Cooperative of Puget Sound [17]. We
modeled complex interactions between all of the SNPs,
although the best fitting model was actually based on the
single SNP in ADRB1 that was identified in the initial
analyses. These results highlight that single SNP associa-

-A: Non-obstructive CAD, Trp64 homozygote (n=341)
- B: Non-obstructive CAD, Arg64 carrier (n=58)

C: Obstructive CAD, Trp64 homozygote (n=179)

D: Obstructive CAD, Arg64 carrier (n=45)

0 12 24 36 48 60 72 84 96

Time to Event (months)

Figure 2
Kaplan-Meier plot for primary outcome by ADRB3 codon 64 genotype and CAD severity.

Page 8 of 11
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Journal of Translational Medicine 2008, 6:11

Model Size

Figure 3
Test-set deviance for different size logic regression models. Test-set deviances from cross-validation analysis are
shown for single logic regression trees containing I to 8 predictors. Lower test-scores represent the better-fitting model.

tions remain informative, although gene-gene interac-
tions within biological systems should not be ignored in
the setting of complex disease.

WISE was a prospective, multicenter cohort study with a
long follow-up period, although several limitations
deserve consideration. First, the population may not be
large enough to detect subtle genetic or epistatic influ-
ences on cardiovascular risk, particularly for the individ-
ual outcomes and patient subgroups. As an exploratory
investigation, analyses were not adjusted for multiple
comparisons, although statistical significance would not
have been met using the stringent Bonferonni correction.
Mechanistic or replication studies are therefore necessary
to make causal inferences for this patient population.
While independent replication is the most desirable
approach, the WISE cohort represents a unique popula-
tion of women with detailed angiography whose cardiac
disease is largely driven by microvascular defects. Thus,
the existing genetic databases (e.g. Framingham,
Women's Health Initiative, Wellcome Trust Case-Control
Consortium) may not adequately represent the women
enrolled in this investigation. Secondly, outcomes were
largely ascertained by patient report, although events were
adjudicated by the WISE Steering Committee when such
data were available. Thirdly, while potentially interesting,
genetic associations were not tested relative to B-blocker
therapy because data on new use and discontinuation
throughout the study period was not sufficient to justify

such comparisons. Fourthly, it is interesting that typical
risk factors such as hypertension and dyslipidemia were
not associated with outcomes. This may be a reflection of
active treatment, considering that the mean blood pres-
sure of hypertensive women at entry was 143/79. Lastly,
the results may not be generalizable beyond women with
ischemic symptoms.

This investigation demonstrated that SNPs in the genes
encoding the B and B3-adrenergic receptors may influ-
ence the risk for cardiovascular events among U.S. women
with suspected CAD. More importantly, the association in
the subset of women without obstructive lesions provides
mechanistic insight into the pathophysiology of myocar-
dial ischemia in this population and the relative impor-
tance of certain receptor subtypes in vascular function. If
replicated, these findings may have significant implica-
tions for assessing cardiovascular risk in women without
angiographic evidence of severe stenosis, a population for
whom risk stratification has been clinically challenging.
However, translating these findings to clinical practice
will require validation in larger populations, as well as
studies to determine the appropriate management strate-
gies for patients with different genetic characteristics.

Authors' contributions
MP was involved with analysis and interpretation of data,
and drafting of the manuscript. IZ was involved with the

Page 9 of 11
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-. .- '" _
. I---- 1 . "

Journal of Translational Medicine 2008, 6:11

Table 5: Multivariate Cox proportional hazards regression model for the primary outcome in women without obstructive CAD*


Clinical Model with Genetic Biomorkers (n = 396)
ADRBI Gly389 carrier
ADRB3 Arg64 carrier
Black race
Age (per decade)
History of diabetes
Baseline systolic BP (per 10 mmHg)
Body mass index (per 5 kg/m2)

Clinical Model with Inflammatory and Genetic Biomorkers (n = 355)
ADRBI Gly389 carrier
ADRB3 Arg64 carrier
Black race
Age (per decade)
History of diabetes
Baseline systolic BP (per 10 mmHg)
Body mass index (per 5 kg/m2)
I inflammatory biomarker in upper quartile*
2 or 3 inflammatory biomarkers in upper quartile

Abbreviations: HR, hazard ratio; 95%CI, 95% confidence interval*
* Hypertension and dyslipidemia eliminated from models after stepwise selection

conception and design of study or analysis and interpreta-
tion of data, and drafting of the manuscript. HL was
involved with analysis and interpretation of data, and
revising manuscript critically for important intellectual
content. RMCD, DJ, VB, DM, BS, and CNBM were
involved with interpretation of data and revising manu-
script critically for important intellectual content. CP was
involved with the conception and design of the study and
analysis and interpretation of data, manuscript or revising
it critically for important intellectual content JJ was
involved with the conception and design of the study and
analysis and interpretation of data, drafting of the manu-
script, manuscript or revising it critically for important
intellectual content. All authors read and approved the
final manuscript.

This work was supported by NIH Grants R03-HL65729, U01-HL64924,
K24-HL68834 and Orchid Biosciences, Inc. The WISE is supported by
NHLBI contracts NOI-HV68161, NOI-HV68162, NOI-HV68163, and N01-
HV68164, and grants UOI-HL64829, UOI-HL64914, UOI-HL65924, and
GCRC grant MOI-RR00425 from the National Center for Research
Resources, National Institutes of Health; grants from the Gustavus and
Louis Pfeiffer Research Foundation, Danville, New Jersey; the Women's
Guild of Cedars-Sinai Medical Center, Los Angeles, California; and the
Ladies Hospital Aid Society of Western Pennsylvania, Pittsburgh, Pennsyl-
vania. Dr. Pacanowski was an American Heart Association (Florida/Puerto
Rico affiliate) Postdoctoral Fellow at the time of this work. We thank Shan-
telle Noble for her technical support.

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HR (95%CI)

1.71 (0.91-
2.14 (1.06-
1.77 (0.90-
1.02 (0.99-
4.08 (2.01-
8.13 (3.55-
1.16 (0.99-
0.70 (0.54-

1.67 (0.85-
2.43 (1.11-
1.84 (0.92-
1.01 (0.97-
3.78 (1.78-
7.43 (3.13-
1.17 (0.98-
0.70 (0.53-
1.67 (0.75-
4.30 (2.02-


< 0.001
< 0.001

< 0.001
< 0.001
< 0.001

Journal of Translational Medicine 2008, 6:11

Journal of Translational Medicine 2008, 6:11

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