Genetics

Association of Hypertension Drug Target Genes With Blood

Pressure and Hypertension in 86 588 Individuals
Andrew D. Johnson, Christopher Newton-Cheh, Daniel I. Chasman, Georg B. Ehret, Toby Johnson,
Lynda Rose, Kenneth Rice, Germaine C. Verwoert, Lenore J. Launer, Vilmundur Gudnason,
Martin G. Larson, Aravinda Chakravarti, Bruce M. Psaty, Mark Caulfield, Cornelia M. van Duijn,
Paul M. Ridker, Patricia B. Munroe, Daniel Levy, on Behalf of the Cohorts for Heart and Aging Research
in Genomic Epidemiology Consortium, Global BPgen Consortium, and Women’s Genome Health Study

Abstract—We previously conducted genome-wide association meta-analysis of systolic blood pressure, diastolic blood
pressure, and hypertension in 29 136 people from 6 cohort studies in the Cohorts for Heart and Aging Research in
Genomic Epidemiology Consortium. Here we examine associations of these traits with 30 gene regions encoding known
antihypertensive drug targets. We find nominal evidence of association of ADRB1, ADRB2, AGT, CACNA1A,
CACNA1C, and SLC12A3 polymorphisms with 1 or more BP traits in the Cohorts for Heart and Aging Research in
Genomic Epidemiology genome-wide association meta-analysis. We attempted replication of the top meta-analysis
single nucleotide polymorphisms for these genes in the Global BPgen Consortium (n⫽34 433) and the Women’s
Genome Health Study (n⫽23 019) and found significant results for rs1801253 in ADRB1 (Arg389Gly), with the Gly
allele associated with a lower mean systolic blood pressure (␤: 0.57 mm Hg; SE: 0.09 mm Hg; meta-analysis:
P⫽4.7⫻10⫺10), diastolic blood pressure (␤: 0.36 mm Hg; SE: 0.06 mm Hg; meta-analysis: P⫽9.5⫻10⫺10), and
prevalence of hypertension (␤: 0.06 mm Hg; SE: 0.02 mm Hg; meta-analysis: P⫽3.3⫻10⫺4). Variation in AGT
(rs2004776) was associated with systolic blood pressure (␤: 0.42 mm Hg; SE: 0.09 mm Hg; meta-analysis:
P⫽3.8⫻10⫺6), as well as diastolic blood pressure (P⫽5.0⫻10⫺8) and hypertension (P⫽3.7⫻10⫺7). A poly-
morphism in ACE (rs4305) showed modest replication of association with increased hypertension (␤: 0.06 mm Hg;
SE: 0.01 mm Hg; meta-analysis: P⫽3.0⫻10⫺5). Two loci, ADRB1 and AGT, contain single nucleotide
polymorphisms that reached a genome-wide significance threshold in meta-analysis for the first time. Our findings
suggest that these genes warrant further studies of their genetic effects on blood pressure, including pharmaco-
genetic interactions. (Hypertension. 2011;57:903-910.) ● Online Data Supplement
Key Words: drug target 䡲 genome-wide 䡲 SNP 䡲 hypertension 䡲 blood pressure

E levated blood pressure (BP) is a critical risk factor for

cardiovascular diseases,1 and BP control in hypertensive
individuals is an effective intervention for reducing cardio-
vascular disease risk. Hundreds of compounds representing
multiple drug classes have been approved for treatment of
hypertension. Achieving BP control in patients often requires
multiple medications and trial-and-error switching of drug
classes to achieve control. This suggests that interindividual

Received June 28, 2010; first decision July 15, 2010; revision accepted February 22, 2011.
From the National Heart, Lung, and Blood Institute’s The Framingham Heart Study (A.D.J., M.G.L., D.L.), Framingham, MA; Center for Population
Studies (A.D.J., D.L.), National Heart, Lung, and Blood Institute, Bethesda, MD; Center for Human Genetic Research (C.N.-C.), Cardiovascular Research
Center, Massachusetts General Hospital, Boston, MA; Program in Medical and Population Genetics (C.N.-C.), Broad Institute of Harvard and MIT,
Cambridge, MA; Division of Preventive Medicine (D.I.C., L.R., P.M.R.), Brigham and Women’s Hospital, Boston, MA; Harvard Medical School (D.I.C.,
L.R., P.M.R.), Boston, MA; Center for Complex Disease Genomics (G.B.E., A.C.), McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins
University School of Medicine, Baltimore, MD; Institute of Social and Preventive Medicine (IUMSP) (G.B.E.), Centre Hospitalier Universitaire Vaudois
and University of Lausanne, Lausanne, Switzerland; Cardiology Center (G.B.E.), Department of Medicine, Geneva University Hospital, Geneva,
Switzerland; Clinical Pharmacology and The Genome Centre (T.J., M.C., P.B.M.), William Harvey Research Institute, Barts and The London School of
Medicine and Dentistry, Queen Mary University of London, London, UK; Department of Biostatistics (K.R.), University of Washington, Seattle, WA;
Department of Epidemiology (G.C.V., C.M.v.D.), Erasmus Medical Center, Rotterdam, The Netherlands; Department of Internal Medicine (G.C.V.),
Erasmus Medical Center, Rotterdam, The Netherlands; Netherlands Centre for Healthy Aging (G.C.V.), Leiden University Medical Centre, Department
of Molecular Epidemiology, Leiden, The Netherlands; Laboratory of Epidemiology, Demography, Biometry (L.J.L.), National Institute on Aging,
National Institutes of Health, Bethesda, MD; Icelandic Heart Association (V.G.), Kopavogur, Iceland; Faculty of Medicine, University of Iceland (V.G.),
Reykjavik, Iceland; Department of Mathematics (M.G.L.), Boston University, Boston, MA; Cardiovascular Health Research Unit (B.M.P.), Department
of Medicine, University of Washington, Seattle, WA; Group Health Research Institute (B.M.P.), Group Health Cooperative, Seattle, WA; Centre of
Medical Systems Biology (C.M.v.D.), Erasmus Medical Center, Rotterdam, The Netherlands; Division of Cardiology (P.M.R.), Brigham and Women’s
Hospital, Boston, MA.
Correspondence to Daniel Levy, Framingham Heart Study and Center for Population Studies, National Heart, Lung, and Blood Institute, 73 Mt Wayte
Ave, Suite #2, Framingham, MA 01702. E-mail levyd@nhlbi.nih.gov
© 2011 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.110.158667

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903 at University of Arizona on April 19, 2015
904 Hypertension May 2011

Table 1. Results for SNPs Associated With SBP in CHARGE

CHARGE (5 Cohorts, n⫽29 136) GBPG (17 Cohorts, n⫽34 433)

Gene SNP Minor Allele ␤ SE P* MAF ␤ SE P* MAF

ⴚ4
ADRB1 rs1801253 G ⴚ0.60 0.17 7.3ⴛ10 27.3% ⴚ0.31 0.15 0.048 31.7%
I
2ⴝ 0% I
2ⴝ 13%

AGT rs2004776 T 0.58 0.17 9.4ⴛ10ⴚ4 23.8% 0.09 0.15 0.58 22.8%
I
2ⴝ 27% I
2ⴝ 6%

CACNA1A rs1985579 A ⫺0.70 0.16 2.6⫻10⫺5 36.4% ⫺0.09 0.14 0.53 39.8%
I
2⫽ 0% I
2⫽ 0%

ADRB2 rs6580586 C 0.76 0.23 1.6⫻10⫺3 11.0% 0.09 0.20 0.66 8.3%
I
2⫽ 27% I
2⫽ 21%

CACNA1C rs2239101 C ⫺0.92 0.27 9.6⫻10⫺4 12.8% 0.09 0.22 0.70 12.5%
I
2⫽ 52% I
2⫽ 55%

SCNN1A rs11064160 C ⫺0.90 0.33 7.8⫻10⫺3 12.0% 0.55 0.29 0.07 12.5%
I
2⫽ 0% I
2⫽ 98%

␭ values for SBP are as follows: CHARGE (␭⫽1.06), GBPG (␭⫽1.08), and WGHS (␭⫽1.06). ␤ reflects the unit change in millimeters of mercury in SBP/DBP or
in log odds of hypertension per allele dose. SE indicates SEM. Replicated SNPs are in bold. SBP indicates systolic blood pressure; DBP, diastolic blood pressure;
CHARGE, Cohorts for Heart and Aging Research in Genomic Epidemiology; GWAS, genome-wide association study; SNP, single nucleotide polymorphism; GBPG, Global
BPgen Consortium; WGHS, Women’s Genome Health Study; MAF, minor allele frequency.
*P values have been taken from GWAS results corrected by genomic control.9

differences in BP and in response to treatment may be ␤-blockers, angiotensin-receptor blockers, calcium-channel

influenced by genetic variation or environmental or other
nongenetic factors.
We recently completed a genome-wide association study
(GWAS) and meta-analyses of systolic BP (SBP), diastolic
BP (DBP), and hypertension in 29 136 individuals from 6
population-based cohorts of European ancestry in the Cohorts
for Heart and Aging Research in Genomic Epidemiology
(CHARGE) Consortium, identifying and replicating novel BP
loci at genome-wide significance levels.2 Although GWASs
have been successful in identifying new genes with common
variants that exhibit small effects on BP, standard methods of
analysis ignore all a priori information about specific genes.
The strict requirements for controlling the occurrence of
false-positives in such an “unbiased” approach lead to severe
multiple testing corrections, whereby true-positive associa-
tions will be missed, particularly when replication resources
are limited.
Examining subsets of GWAS associations based on a priori
hypotheses is one way to identify genes of interest for further
investigation3 while paying a smaller penalty for multiple
testing. Evidence from lipid GWASs and candidate gene
studies indicates that some polymorphisms in drug target
genes (eg, HMGCR and APOE) are associated with main
effects on lipids, as well as effects on drug response.4,5 We
hypothesized that GWAS approaches have missed some true
BP association signals in antihypertensive drug target genes.
We identified 30 drug target genes, including the targets of
␣-blockers, angiotensin-converting enzyme (ACE) inhibitors,
blockers, diuretics, and vasodilators, and analyzed single
nucleotide polymorphisms (SNPs) in these gene regions for
association with BP and hypertension.
Methods
Description of Cohorts, Participants, Genotypes,
and Phenotypes
The CHARGE consortium cohorts, their genotyping, SNP imputa-
tion,6 and BP and hypertension GWASs have been described
previously.2 Participants underwent standardized resting seated BP
readings (means of 2 repeated measures used in analysis) and had
GWAS results available (n⫽29 136). BP readings from the first
examination attended were used. Hypertension was defined as SBP
ⱖ140 mm Hg, DBP ⱖ90 mm Hg, or drug treatment for hypertension
at BP assessment.
The Global BPgen Consortium (GBPG) included 17 cohorts of
European ancestry with either population-based designs or controls
drawn from case-control designs.7 In most participants, BP analysis
was based on the mean of 2 resting sitting measurements.7
The Women’s Genome Health Study population sample with BP
and hypertension data consisted of 23 019 female health profession-
als of European descent ⱖ45 years of age at enrollment, free of
cardiovascular disease or other major chronic illnesses, with GWAS
and genotyping described previously.8 BP was determined by self-
report in ranges (see the online Data Supplement, available at
http://hyper.ahajournals.org), with the midpoint of these ranges used
in analyses, and hypertension defined as above.
For individuals in CHARGE who were taking antihypertensive
medication, we added 10/5 mm Hg to the observed SBP and DBP;
for those in GBPG we added 15/10 mm Hg. Association results for
different treatment adjustments were highly correlated in CHARGE
(Table S1, available in the online Data Supplement at http://

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 Johnson et al Associations in BP Drug Target Genes 905

Table 1. Continued SNPs creating a protein change or (based on HapMap populations) in

linkage disequilibrium (LD) with protein-changing variants or SNPs
WGHS (1 Cohort, n⫽23 019) Meta-Analysis (n⫽86 588) with previous associations with SBP, DBP, or hypertension.
␤ SE P* MAF Trait ␤ SE P
ⴚ7
Results
ⴚ0.82 0.16 7.3ⴛ10 27.0% SBP ⴚ0.57 0.09 4.7ⴛ10ⴚ10 For 30 regions that encode antihypertensive drug targets, the
DBP ⴚ0.36 0.06 9.5ⴛ10ⴚ10 single strongest SNP associations for SBP, DBP, and hyper-
HTN ⴚ0.06 0.02 3.3ⴛ10ⴚ4 tension in/near each drug target gene for the initial CHARGE
0.69 0.16 2.8ⴛ10ⴚ5 24.1% SBP 0.42 0.09 3.8ⴛ10ⴚ6 analysis are in Table S2, along with the number of SNPs
DBP 0.32 0.06 5.0ⴛ10ⴚ8 tested within each gene region. The most significant SNP
HTN 0.08 0.02 3.7ⴛ10ⴚ7 association among the drug target genes tested in CHARGE
⫺0.21 0.15 0.16 34.5% SBP ⫺0.29 0.08 4.4⫻10⫺4
was rs1985579 in CACNA1A with SBP (P⫽2.6⫻10⫺5).
Using resampling to account for multiple SNPs per locus, 2
DBP ⫺0.14 0.05 8.7⫻10⫺3
significant SNP associations were identified for SBP (in
HTN ⫺0.04 0.01 2.5⫻10⫺3
ADRB1 and CACNA1A), 4 for DBP (in ADRB1, AGT,
0.03 0.23 0.89 10.1% SBP 0.27 0.13 0.033 CACNA1A, and SLC12A3), and 4 for hypertension (in
DBP 0.21 0.08 8.5⫻10⫺3 ADRB2, AGT, CACNA1C, and CACNA1H). At a less restric-
HTN 0.04 0.02 0.10 tive cutoff of P⬍1/(the number of SNPs tested in/near a
0.15 0.25 0.55 11.5% SBP ⫺0.16 0.14 0.24 gene), 11 additional SNPs in 9 genes were selected (ACE,
DBP ⫺0.14 0.09 0.13 ADRB2, AGT, CA1, CACNA1C, MME, REN, SCNN1A, and
HTN ⫺0.01 0.02 0.65 SLC9A1), for a total of 19 SNPs in 13 genes selected for
⫺0.39 0.38 0.32 10.8% SBP ⫺0.15 0.19 0.41
replication. Two genes (CACNA1H and MME) were dropped
from replication because their most associated SNPs had poor
DBP ⫺0.12 0.12 0.30
imputation in ⱖ2 groups, and attempts to find satisfactory
HTN ⫺0.01 0.03 0.85
surrogate SNPs in LD were unsuccessful.
For SBP we replicated associations for variants in ADRB1
and AGT (Table 1). In ADRB1 the minor allele of rs1801253
(nonsynonymous Arg389Gly) was associated with decreased
SBP (replication: P⫽7.3⫻10⫺7; meta-analysis: ␤ (units
as mm Hg) ⫺0.57, SE 0.09, P⫽4.7⫻10⫺10), and in AGT the
hyper.ahajournals.org). Individual studies obtained approval from minor allele of rs2004776 was associated with increased SBP
their institutional review boards for consent procedures, examina- (replication: P⫽2.8⫻10⫺5).
tion, data security, and DNA collection and use in genetic research. We also replicated associations for ADRB1 and AGT with
All of the cohorts in the current study conducted imputation using a DBP (Table 2). The minor allele of rs1801253 in ADRB1 was
HapMap reference panel of samples from Centre d’Etude du Poly-
morphisme Humain Utah residents with ancestry from northern and associated with decreased DBP (replication: P⫽2.5⫻10⫺7;
western Europe (CEU). meta-analysis: ␤ ⫺0.36, SE 0.06, P⫽9.5⫻10⫺10), and in
AGT the minor allele of rs11122587 was associated with
Discovery in CHARGE and Replication in increased DBP (replication: P⫽2.4⫻10⫺5).
GBPG/Women’s Genome Health Study We sought replication for 6 SNPs where hypertension was the
Within each cohort, regression models for BP phenotypes were fit primary trait (Table 3). The minor allele of an intron 5 SNP
adjusting for sex, age, age squared, and body mass index. Genomic (rs4305) in ACE replicated with all of the cohort associations
control (␭) parameter values9 were calculated and applied to account
for within-study heterogeneity. Meta-analyses of the SNP-trait asso-
with increased odds of hypertension (replication: P⫽7.5⫻10⫺3;
ciation estimates were inverse-variance weighted and reflect the meta-analysis: ␤ 0.06, SE 0.01, P⫽3.0⫻10⫺5). In secondary
combination of additive model analyses from the cohorts.2 analysis, this SNP also showed association with increased levels
We identified a priori 30 candidate genes that code for proteins of SBP (P⫽4.6⫻10⫺4) and DBP (P⫽6.0⫻10⫺5).
that are direct targets of antihypertensive drugs based on general In secondary analyses, the AGT SNP selected for SBP,
knowledge and DrugBank (www.drugbank.ca), a database of human
drug target genes.10 We analyzed all of the CHARGE BP/hyperten-
rs2004776, reached a low P value for DBP (meta-analysis:
sion associations within 60 kb of each target gene and applied a P⫽5.0⫻10⫺8). ADRB1 and AGT SNPs were also associated
resampling-based test (Reference 11, see Figure S1, available in the with hypertension, in the same direction as expected based on
online Data Supplement at http://hyper.ahajournals.org). To aug- their BP associations (ADRB1: rs1801253 P⫽3.3⫻10⫺4;
ment SNPs for replication, we additionally selected SNPs at a AGT: rs2004776 P⫽3.7⫻10⫺7).
P⬍1/(the number of SNPs tested). SNPs selected for replication
are in bold in Table S2 (available in the online Data Supplement Heterogeneity analyses for ADRB1, AGT, or ACE in the
at http://hyper.ahajournals.org). multistudy cohorts (CHARGE and GBPG) or within the full
For selected gene regions, we examined the most significant meta-analysis found no evidence for heterogeneity (all
CHARGE SNP-trait association for the same trait in GBPG and I2⬍0.50). In additional analyses for ADRB1 and AGT, con-
Women’s Genome Health Study (WGHS). Replication was defined ditioning on the top variant for SBP or DBP, we found no
a priori as allelic association in the same direction as in CHARGE
(thresholds: SBP, P⬍8.3⫻10⫺3; DBP, P⬍7.1⫻10⫺3; hypertension, additional SNPs that contributed to these phenotypes after
P⬍8.3⫻10⫺3). We also conducted meta-analysis to provide esti- multiple test correction (see the online Data Supplement).
mates comparable to GWAS thresholds. We used SNAP12 to identify Summary results for the 3 replicated genes and 4 promising,
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906 Hypertension May 2011

Table 2. Results for SNPs Associated With DBP in CHARGE

CHARGE (5 Cohorts, n⫽29 136) GBPG (17 Cohorts, n⫽34 433)

Gene SNP Minor Allele ␤ SE P* MAF ␤ SE P* MAF

ⴚ4
ADRB1 rs1801253 G ⴚ0.35 0.10 7.5ⴛ10 27.3% ⴚ0.19 0.10 0.08 31.7%
I
2ⴝ 0% I
2ⴝ 0%

AGT rs11122587 G 0.42 0.11 1.1ⴛ10ⴚ4 20.9% 0.11 0.10 0.29 24.2%
I
2ⴝ 0% I
2ⴝ 0%

SLC12A3 rs2399594 G ⫺0.32 0.09 6.4⫻10⫺4 38.5% ⫺0.15 0.09 0.10 42.1%
I
2⫽ 21% I
2⫽ 24%

SCNN1A rs4149570 A ⫺0.25 0.09 7.5⫻10⫺3 39.6% ⫺0.08 0.09 0.37 30.8%
I
2⫽ 0% I
2⫽ 0%

CACNA1A rs1985579 A ⫺0.37 0.10 1.5⫻10⫺4 36.4% ⫺0.02 0.09 0.82 39.8%
I
2⫽ 0% I
2⫽ 0%

REN rs12089381 C 0.75 0.25 3.7⫻10⫺3 4.5% ⫺0.08 0.24 0.75 2.5%
I
2⫽ 41% I
2⫽ 0%

CA1 rs13278559 T ⫺0.51 0.16 2.5⫻10⫺3 10.0% 0.15 0.14 0.31 9.2%
I
2⫽ 0% I
2⫽ 9%

␭ values for DBP are as follows: CHARGE (␭⫽1.05), GBPG (␭⫽1.07), and WGHS (␭⫽1.06). ␤ reflects the unit change in millimeters of mercury in SBP/DBP or
in log odds of hypertension per allele dose. SE indicates SEM. Replicated SNPs are in bold. SBP indicates systolic blood pressure; DBP, diastolic blood pressure;
CHARGE, Cohorts for Heart and Aging Research in Genomic Epidemiology; GWAS, genome-wide association study; SNP, single nucleotide polymorphism; GBPG, Global
BPgen Consortium; WGHS, Women’s Genome Health Study; MAF, minor allele frequency.
*P values have been taken from GWAS results corrected by the genomic control approach.9

nonreplicated genes (ADRB2, CACNA1C, CACNA1A, and
SLC12A3) compared with previous results and meta-analyses
from the literature are presented in Tables S3 and S4,
respectively (available in the online Data Supplement at
http://hyper.ahajournals.org).
Discussion
Within one of the largest genetic studies of BP traits to date,
we examined evidence for associations in gene regions
encoding protein targets of antihypertensive medications. We
conducted a discovery scan in ⬎29 000 individuals from the
CHARGE consortium, with validation of significant results in
⬎57 000 individuals from GBPG and WGHS. Of note, in
previously published GWAS meta-analysis reports from
CHARGE and GBPG,2,7 none of the SNPs that we tested
reached genome-wide significance (P⬍5.0⫻10⫺8). Associa-
tions at 3 loci in our study (ADRB1, AGT, and ACE)
successfully replicated in independent populations.
The ␤-adrenergic receptors (ADRB1 and ADRB2) are
targets of a variety of endogenous and pharmacological
agonists and antagonists, including epinephrine, norepineph-
rine, and ␤-blocker drugs, and they mediate important car-
diovascular responses, including cardiac contractility and
heart rate. A nonsynonymous variant of ADRB1 (rs1801253,
Arg389Gly) was reported to alter BP response to ␤-blocker
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therapy in multiple studies (eg, References 13 and 14) and
was also reported to affect outcomes after treatment.15–17
However, tests of this variant with baseline BP have generally
been conducted in modestly sized samples not drawn from
general population cohorts, with conflicting reports about
association of the Arg389 allele with increased BP (Table
S3).14,16,18 –23 In our survey of 142 SNPs in/near ADRB1, we
found the strongest association at rs1801253 with the Gly389
allele being associated with decreased SBP (P⫽4.7⫻10⫺10)
and DBP (P⫽9.5⫻10⫺10). Our study is consistent with
several studies (Table S3) indicating that there is a small
reduction in BP associated with Gly389.16,19,21–23 This result
is also consistent with experimental observations that Gly389
acts functionally to reduce basal and agonist-stimulated
receptor responses.24 –26
The renin-angiotensin system plays critical roles in BP
regulation, is targeted by multiple drug classes, and has been
the subject of previous genetic studies for candidate genes
(eg, AGT and ACE). Among 3 AGT SNPs (rs2004776,
rs12046196, and rs11122587) associated with BP in
CHARGE, rs2004776, in intron 1 (between the AGT 5⬘UTR
and exon 1), showed the strongest validation in an indepen-
dent European ancestry cohort (meta-analysis P⫽5.0⫻10⫺8).
Of note, rs2004776 is in partial LD (r2⫽0.49, HapMap CEU)
with a Met235Thr (rs699) that has been widely studied for
 Johnson et al Associations in BP Drug Target Genes 907

Table 2. Continued minor allele of rs4343 with increased activity in Han Chinese
(P⫽3.0⫻10⫺25).34 The nonsynonymous SNP rs4343 is in
WGHS (1 Cohort, n⫽23 019) Meta-Analysis (n⫽86 588)
moderate-high LD in Asian and European populations with
␤ SE P* MAF Trait ␤ SE P our replicated BP SNP, rs4305 (HapMap CEU r2⫽0.48;
ⴚ0.54 0.10 2.5ⴛ10 ⴚ7
27.0% DBP ⴚ0.36 0.06 9.5ⴛ10ⴚ10 JPT/CHB r2⫽0.80), suggesting a potential common link
SBP ⴚ0.57 0.09 4.7ⴛ10ⴚ10 between the studies. We examined a sample of 944 unrelated
individuals in The Framingham Heart Study and found
HTN ⴚ0.06 0.02 3.3ⴛ10ⴚ4
rs4350-I/D is also in modest LD (r2⫽0.55).
0.48 0.11 2.4ⴛ10ⴚ5 21.4% DBP 0.32 0.06 1.2ⴛ10ⴚ7
The effects of variation in ADRB1, AGT, and ACE on BP
SBP 0.45 0.09 2.3ⴛ10ⴚ6 variation and treatment response were the subject of previous
HTN 0.08 0.02 2.5ⴛ10ⴚ6 research (Table S3). Our large study validates their role in BP
0.01 0.09 0.93 38.8% DBP ⫺0.16 0.05 2.7⫻10⫺3 genetics. The effect sizes of the ADRB1 and AGT variants are
SBP ⫺0.15 0.08 0.07 on par with variants identified in GWAS for BP, lipids, and
HTN ⫺0.001 0.01 0.93 similar traits, accounting for only ⬇0.25% to 0.50% of the
variation in BP.2,7,35 These results demonstrate that surveying
⫺0.09 0.09 0.37 39.4% DBP ⫺0.14 0.05 8.2⫻10⫺3
previous biological candidates in large genetic studies may be
SBP ⫺0.18 0.08 0.026
a useful approach to identify and replicate additional loci.
HTN ⫺0.02 0.01 0.15 This observation is consistent with 2 recent surveys of
⫺0.04 0.10 0.65 34.5% DBP ⫺0.14 0.05 8.7⫻10⫺3 published GWASs that indicate some a priori candidates
SBP ⫺0.30 0.08 4.4⫻10⫺4 show true associations.36,37 Candidate genes for variation in
HTN ⫺0.04 0.01 2.5⫻10⫺3 lipid levels have also been validated in GWASs and shown to
⫺0.12 0.27 0.66 4.0% DBP 0.19 0.15 0.20 have treatment effects.4,5,35
SBP 0.19 0.23 0.41
The loci and variants identified here may also influence
treatment response, a hypothesis that we did not assess. Our
HTN 0.01 0.04 0.76
study is limited in that treated and untreated individuals are
0.02 0.15 0.89 9.8% DBP ⫺0.08 0.09 0.36 included, with variable ascertainment of treatment across
SBP ⫺0.19 0.13 0.17 cohorts. We applied differing treatment adjustments in dif-
HTN ⫺0.01 0.02 0.55 ferent cohorts to impute expected baseline effects. Further
analysis in CHARGE indicates that ⫹15/10 and ⫹10/5
adjustments generate similar results for BP associations, so
this is unlikely to have greatly affected replication (Table S1
and Figures S2 and S3, available in the online Data Supple-
ment at http://hyper.ahajournals.org). Because treatment of
participants in our GWAS was nonrandomized, it is difficult
association with hypertension.27 Previous analyses suggest a to assess gene-by-treatment interactions without confound-
role for Met235Thr27,28 or other variants affecting AGT ing. Prospective studies, or more sophisticated cross-sectional
function29,30 in hypertension, with multiple variants being analyses, will be required to determine whether gene variants
supported in meta-analyses (Table S3).27,29 In CHARGE, the identified from large GWASs influence treatment outcomes;
minor allele of Met235Thr showed nominal association with our results indicate that these could be worthwhile pursuits.
hypertension (P⫽0.0011), SBP (P⫽0.012), and DBP Another potential limitation of the study is reliance in WGHS
(P⫽0.0042). These results suggest that Met235Thr may not on self-reported BP values. However, past studies indicate
be the most relevant AGT variant and that there may be other that self-report is reasonably reliable in assessment of BP and
functional alleles to be discovered and characterized. hypertension (eg, References 38 and 39). Furthermore, we
Another important gene within the renin-angiotensin sys- independently found high replication rates for BP GWAS loci
tem, ACE, showed significant positive association with hy- in the WGHS.40
pertension (rs4305; P⫽3.0⫻10⫺5), and with SBP
(P⫽4.6⫻10⫺4) and DBP (P⫽6.0⫻10⫺5). ACE genotypes Perspectives
Our results indicate that candidate genes, with clinical and
have been studied for association with various traits, includ-
physiological relevance by virtue of their role as antihyper-
ing BP and hypertension, where results from a literature-
tensive drug targets, harbor true BP-associated variants. Such
based meta-analysis indicate no significant effect.31 A large
loci, not identified in previous large GWAS meta-analyses
study of antihypertensive drug response also indicates no
but detected in our drug target gene approach, account for a
genotype-treatment effect.32 However; most previous studies
portion of the unexplained proportion of BP variance. These
focused only on the well-known intronic I/D polymorphism results suggest that revisiting GWAS scans from the perspec-
(Table S3) and lacked detailed information on the genetic tive of biological and clinical knowledge may be useful for
architecture of ACE. The importance of this point is empha- discovery and validation of new genetic associations.
sized by a recent resequencing study of ACE in blacks that
found novel functional variation associated with myocardial Acknowledgments
infarction in hypertensives.33 Furthermore, a recent GWAS We thank the staff and participants of the Age, Gene/Environment
for ACE enzyme activity found strong association of the Susceptibility Reykjavik Study, Atherosclerosis Risk in Communities

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908 Hypertension May 2011

Table 3. Results for SNPs Associated With Hypertension in CHARGE

CHARGE (5 Cohorts, n⫽29 136) GBPG (17 Cohorts, n⫽34 433)

Gene SNP Minor Allele ␤ SE P* MAF ␤ SE P* MAF

ⴚ3 ⴚ3
ACE rs4305 A 0.06 0.02 9.3ⴛ10 44.7% 0.07 0.03 7.5ⴛ10 39.7%
I
2ⴝ 47% I
2ⴝ 0%

AGT rs12046196 A 0.14 0.04 5.6⫻10⫺4 9.1% ⫺0.01 0.05 0.80 7.5%
I
2⫽ 91% I
2⫽ 15%

CACNA1A rs1985579 A ⫺0.07 0.02 2.6⫻10⫺5 36.3% ⫺0.003 0.03 0.90 39.8%
I
2⫽ 86% I
2⫽ 11%

ADRB2 rs2082382 G ⫺0.07 0.02 7.0⫻10⫺4 41.1% 0.04 0.03 0.19 32.4%
I
2⫽ 89% I
2⫽ 0%

SLC9A1 rs484677 T 0.07 0.02 3.3⫻10⫺3 41.5% 0.04 0.03 0.24 38.3%
I
2⫽ 0% I
2⫽ 0%

CACNA1C rs16929470 T ⫺0.19 0.05 1.3⫻10⫺4 6.9% 0.05 0.07 0.46 8.3%
I
2⫽ 94% I
2⫽ 0%

␭ values for hypertension are as follows: CHARGE (␭⫽1.04), GBPG (␭⫽1.01), and WGHS (␭⫽1.07). ␤ reflects the unit change in millimeters of mercury in SBP/DBP
or in log odds of hypertension per allele dose. SE indicates SEM. Replicated SNP is in bold. SBP indicates systolic blood pressure; DBP, diastolic blood pressure;
CHARGE, Cohorts for Heart and Aging Research in Genomic Epidemiology; GWAS, genome-wide association study; SNP, single nucleotide polymorphism; GBPG, Global
BPgen Consortium; WGHS, Women’s Genome Health Study; MAF, minor allele frequency.
*P values have been taken from GWAS results corrected by the genomic control approach.9

Study, Cardiovascular Health Study, Framingham Heart Study, GBPG,
Rotterdam Study, and WGHS for their important contributions. Analy-
ses from Framingham Heart Study reflect the efforts and resource
development from the Framingham Heart Study investigators partici-
pating in the SNP Health Association Resource Project. A full list of
principal Cardiovascular Health Study investigators and institutions can
be found at http://www.chs-nhlbi.org/pi.htm. Investigators of the Rot-
terdam Study thank Pascal Arp, Mila Jhamai, Michael Moorhouse,
Marijn Verkerk, and Sander Bervoets for help in creating the database
and Maxim Struchalin for his contributions to the imputations of
the data.
Sources of Funding
The Cardiovascular Health Study research reported in this article was
supported by contract numbers N01-HC-85079 through N01-HC-
85086, N01-HC-35129, N01 HC-15103, N01 HC-55222, N01-HC-
75150, N01-HC-45133, U01 HL080295, and R01 HL087652 from
the National Heart, Lung, and Blood Institute, with additional
contribution from the National Institute of Neurological Disorders
and Stroke. DNA handling and genotyping were supported in part by
National Center for Research Resources grant M01-RR00425 to the
Cedars-Sinai General Clinical Research Center Genotyping core and
National Institute of Diabetes and Digestive and Kidney Diseases
grant DK063491 to the Southern California Diabetes Endocrinology
Research Center.
The National Heart, Lung, and Blood Institute’s Framingham
Heart Study is a joint project of the National Institutes of Health and
Boston University School of Medicine and was supported by the
National Heart, Lung, and Blood Institute’s Framingham Heart
Study (contract N01-HC-25195) and its contract with Affymetrix,
Inc, for genotyping services (contract No. N02-HL-6-4278). A
portion of this research was conducted using the Linux Cluster for
Genetic Analysis (LinGA-II) funded by the Robert Dawson Evans
Endowment of the Boston University School of Medicine Depart-
ment of Medicine and Boston Medical Center.
The Rotterdam Study is supported by the Erasmus Medical Center
and Erasmus University Rotterdam; the Netherlands Organization
for Scientific Research; the Netherlands Organization for Health
Research and Development (ZonMw); the Research Institute for
Diseases in the Elderly; the Netherlands Heart Foundation; the
Ministry of Education, Culture, and Science; the Ministry of Health,
Welfare, and Sports; the European Commission; and the Municipal-
ity of Rotterdam. Support for genotyping was provided by the
Netherlands Organization for Scientific Research (NWO Groot
175.010.2005.011 and 911.03.012) and Research Institute for Dis-
eases in the Elderly (014.93.015; RIDE2). This study was supported
by the Netherlands Genomics Initiative/Netherlands Organization
for Scientific Research project 050-060-810 and the National
Genomics Initiative Centre for Medical Systems Biology and Neth-
erlands Centre for Healthy Aging.
C.N.-C. is supported by the Doris Duke Charitable Foundation, the
Burroughs Wellcome Fund, and the National Institutes of Health.
The Age, Gene/Environment Susceptibility Reykjavik Study is
funded by National Institutes of Health contract N01-AG-12100, the
National Institute on Aging Intramural Research Program, Hjar-
tavernd (the Icelandic Heart Association), and the Althingi (the
Icelandic Parliament). The Atherosclerosis Risk in Communities
Study is carried out as a collaborative study supported by National
Heart, Lung, and Blood Institute contracts N01-HC-55015, N01-HC-
55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-
55021, and N01-HC-55022, and grants R01HL087641,
R01HL59367, R37HL051021, R01HL086694, and U10HL054512;
National Human Genome Research Institute contract U01HG004402;
and National Institutes of Health contract HHSN268200625226C.
Infrastructure was partly supported by grant UL1RR025005, a compo-
nent of the National Institutes of Health and National Institutes of Health
Roadmap for Medical Research.

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 Johnson et al Associations in BP Drug Target Genes 909

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SBP 0.29 0.08 4.6ⴛ10ⴚ4 Najjar SS, Zhao JH, Heath SC, Eyheramendy S, Papadakis K, Voight BF,
DBP 0.21 0.05 6.0ⴛ10ⴚ5 Scott LJ, Zhang F, Farrall M, Tanaka T, Wallace C, Chambers JC, Khaw
KT, Nilsson P, van der Harst P, Polidoro S, Grobbee DE, Onland-Moret
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Samani NJ, Webster J, Zeggini E, Beckmann JS, Bergmann S, Lim N,
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SBP ⫺0.30 0.08 4.4⫻10⫺4 L, Orho-Melander M, Allione A, Di Gregorio A, Guarrera S, Panico S,
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⫺0.05 0.02 0.04 43.9% HTN ⫺0.04 0.01 5.0⫻10⫺3 castle LL, Collins FS, Jackson AU, Mohlke KL, Stringham HM, Valle
TT, Willer CJ, Bergman RN, Morken MA, Döring A, Gieger C, Illig T,
SBP ⫺0.02 0.08 0.84
Meitinger T, Org E, Pfeufer A, Wichmann HE, Kathiresan S, Marrugat J,
DBP ⫺0.04 0.05 0.43 O’Donnell CJ, Schwartz SM, Siscovick DS, Subirana I, Freimer NB,
0.01 0.03 0.77 42.0% HTN 0.04 0.01 6.5⫻10⫺3 Hartikainen AL, McCarthy MI, O’Reilly PF, Peltonen L, Pouta A, de
Jong PE, Snieder H, van Gilst WH, Clarke R, Goel A, Hamsten A, Peden
SBP 0.05 0.08 0.53 JF, Seedorf U, Syvänen AC, Tognoni G, Lakatta EG, Sanna S, Scheet P,
DBP 0.02 0.06 0.78 Schlessinger D, Scuteri A, Dörr M, Ernst F, Felix SB, Homuth G, Lorbeer
R, Reffelmann T, Rettig R, Völker U, Galan P, Gut IG, Hercberg S,
0.03 0.06 0.63 4.5% HTN ⫺0.07 0.03 0.04
Lathrop GM, Zelenika D, Deloukas P, Soranzo N, Williams FM, Zhai G,
SBP ⫺0.14 0.19 0.48 Salomaa V, Laakso M, Elosua R, Forouhi NG, Völzke H, Uiterwaal
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Supplemental material

ASSOCIATION OF HYPERTENSION DRUG TARGET GENES WITH BLOOD

PRESSURE AND HYPERTENSION IN 86,588 INDIVIDUALS

Andrew D. Johnson, Christopher Newton-Cheh, Daniel I. Chasman, Georg B. Ehret,

Toby Johnson, Lynda Rose, Kenneth Rice, Germaine C. Verwoert, Lenore J. Launer,
Vilmundur Gudnason, Martin G. Larson, Aravinda Chakravarti, Bruce M. Psaty, Mark
Caulfield, Cornelia M. van Duijn, Paul M Ridker, Patricia B. Munroe and Daniel Levy on
Behalf of the CHARGE Consortium, Global BPgen Consortium and Women’s Genome
Health Study

Corresponding author: Daniel Levy, Framingham Heart Study and Center for Population

Studies, National Heart, Lung, & Blood Institute, 73 Mt. Wayte Ave., Suite #2,

Framingham, MA 01702, telephone: 508-935-3458, fax: 508-626-1262,. Email:

levyd@nhlbi.nih.gov
Supplemental Methods: Methods expanded from the in print article
Description of cohorts, participants, genotypes and phenotypes.
The CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology)
consortium cohorts, their genotyping, SNP imputation (1) and BP and hypertension
GWAS have been previously described (2). The individual studies obtained approval
from their IRBs for consent procedures, examination, data security, and DNA collection
and use in genetic research. All participants gave written informed consent for
participation in the studies and genetic research. Participants underwent standardized
resting seated BP readings (means of two repeated measures used in analysis) and
had GWAS results available (n=29,136): from the Age, Gene/Environment
Susceptibility-Reykjavik Study (n=3,219), the Atherosclerosis Risk in Communities
Study (n=8,047), the Cardiovascular Health Study (n=3,277), the Framingham Heart
Study (n=8,096), the Rotterdam Study (n=4,737) and the Rotterdam II Study (n=1,760).
Blood pressure readings from the first exam attended were used. Participants included
both treated and untreated individuals. Hypertension was defined as SBP≥140 or
DBP≥90 mmHg or drug treatment for hypertension at the time of assessment. For
participants taking anti-hypertensive medication 10 mmHg was added to observed SBP
values and 5 mmHg to DBP values (2).
The Global BPgen consortium (GBPG) included 17 cohorts of European ancestry
with either population-based designs or controls drawn from case-control designs as
previously described (3). Each study obtained appropriate approvals for the research
conducted. Blood pressures were measured as previously described, and in most cases
the analysis was based on the mean of two resting sitting measurements (3).
Genotyping and imputation was completed in all cohorts. For individuals taking
antihypertensive treatment, 15 mmHg and 10 mmHg were added to SBP and DBP,
respectively.
The Women’s Genome Health Study (WGHS) population sample with BP and
hypertension data consisted of 23,019 female health professionals of European descent
≥45 years of age at enrollment, free of cardiovascular disease or other major chronic
illnesses, with GWAS and genotyping previously described (4). The study was approved
by the IRB of Brigham and Women’s Hospital, Boston, MA. Blood pressure was
determined by self-report in ranges, as follows (SBP: <110, 110-119, 120-129, 130-139,
140-149, 150-159, 160-169, 170-179, 180+; DBP: <65, 65-74, 75-84, 85-89, 90-94, 95-
104, 105+), with the midpoint of these ranges used in analyses, and hypertension
defined as above.
All cohorts in the current study conducted imputation using a HapMap CEU
reference panel. Cochrane’s Q and I2 were calculated for SNPs carried forward for
replication (Tables 1-3) to assess between study heterogeneity. I2 > 0.50 was
considered to be evidence for moderate heterogeneity.
Discovery in CHARGE and replication in GPBG/WGHS.
Within each cohort, regression models for BP phenotypes were fit adjusting for
sex, age, age squared, and BMI. Prior to meta-analysis, SNPs with minor allele
frequencies <0.5% (SBP, DBP) and <1.0% (HTN) were filtered out. A less stringent
threshold was applied for HTN in order to increase power (2). Genomic control (lambda)
parameter values (5) were calculated and applied, to account for within study
heterogeneity, though overall inflation rates were modest (2). Meta-analyses of the
SNP-trait association estimates were inverse-variance weighted and reflect the
combination of additive model analyses from the cohorts (2).
We identified a priori 30 candidate genes that code for proteins that are direct
targets of anti-hypertensive drugs based on general knowledge and DrugBank
(www.drugbank.ca), a database of human drug target genes (6). We analyzed all
CHARGE BP and hypertension associations within 60 kb of each of the maximum
extent of the transcript (based on RefSeq gene annotations) for each target gene. We
then applied a resampling based test (1): we compared the p-value from the most
significant in the region of each drug target gene (Table S1) against the lowest p-value
from resamplings with replacement (n=10,000 samplings) of equal numbers of
consecutive SNPs randomly distributed throughout each GWAS results dataset. An
overview figure of the procedure is given in Figure S1.
The proportion of the 10,000 resamplings which gave a more extreme region-
wide result than the original data provides a gene-specific P value. This approach
assumes that LD relationships will be relatively similar to target regions when
approximated across the genome by equal numbers of consecutive SNPs. By repeated
application to different GWAS (e.g., 7) we have found this approach provides results
between the conservatism of Bonferroni correction, but more conservative than a P <
1/n threshold, where n is the number of SNPs tested in a region. To provide a greater
number of SNPs for attempted replication, we ultimately decided to select additional
SNPs at the 1/n threshold. SNPs selected for replication are in bold in Table S2.
For selected gene regions, we examined the most significant CHARGE SNP-trait
association for the same trait in GBPG and WGHS. Replication was defined a priori as
allelic association in the same direction as in CHARGE (thresholds: SBP, P<8.3x10-3,
DBP, P<7.1x10-3, hypertension, P<8.3x10-3). Secondary analysis of the other 2 traits
was examined to see if the findings showed consistency, but this was not a criterion to
declare replication. We also conducted meta-analysis to compare to GWAS thresholds
though this was not deemed an important threshold for replication. We used SNAP (8)
to identify SNPs creating a protein change, or (based on HapMap populations) in
linkage disequilibrium (LD) with protein-changing variants or SNPs with prior
associations with SBP, DBP or hypertension.
Conditional models for association in the ADRB1 and AGT regions.
To assess whether there was strong evidence for additional variants contributing
to BP effects in the ADRB1 and AGT regions, regression models were conditioned on
the lead SNP in each locus within WGHS (SBP+DBP: rs1801253 in ADRB1, SBP:
rs2004776 in AGT, DBP: rs11122587 in AGT). WGHS was selected because it
represented to largest single cohort with the least chance for heterogeneity, and had
strong findings within both regions. A stepwise forward selection procedure was used
whereby additional SNPs were added to the model only if they were significant after a
multiple test correction (0.05/n), where n was the number of tested (either imputed or
genotyped SNPs within 60kb of gene boundaries). Analysis was alternatively performed
using either imputed SNPs or genotyped-only SNPs. After adjusting for the lead
replicated SNPs, no additional SNPs were found to be significant for either ADRB1 or
AGT for SBP or DBP, even when the additional testing and test correction was limited to
genotyped SNPs only (n=52 genotyped SNPs for AGT and n=23 genotyped SNPs for
ADRB1).

Supplemental Results
Several additional regions showed promise in the discovery phase, but failed to
replicate in our two independent replication samples (results summarized in Table S4).
Sufficient power for replication was expected for most SNPs, though non-replication
could potentially be explained by gender-specific effects (WGHS) or treatment-specific
effects that we did not examine. Treatment profiles across our cohorts likely varied
substantially.
A SNP upstream of ADRB2 (rs2082382) was associated in CHARGE with
decreased risk for hypertension (P = 0.0006), and is in strong LD with rs1042714
(Glu27Gln, pairwise r2=0.93 in the HapMap CEU), a nonsynonymous SNP previously
studied in relation to BP and treatment outcomes (9-13). Prior studies have been
inconsistent (14), but the largest study reported nominal association of Glu27 with
increased SBP (9). In our study, the Gln27 allele was modestly associated in CHARGE
with lower hypertension odds (P = 0.0011), but not with SBP (P = 0.26) or DBP (P =
0.23). The CHARGE findings for ADRB2 did not replicate in GBPG or WGHS based on
our defined thresholds, though it is notable that the hypertension association for
rs2082382 was nominally significant in the all female cohort (WGHS: P=0.04) and in the
same direction as CHARGE. This result may be consistent with gender-specific
associations in ADRB2 which have been suggested by past studies (15-17). Further
details of current results and past studies for CACNA1C, CACNA1A, and SLC12A3 are
shown in Table S4.
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Table S1. Comparison of CHARGE meta‐analysis results for SBP and DBP for SNPs in Tables 1 and 2, respectively, with treatment
adjustment of SBP+15mmHg/DBP+10mmHG versus SBP+10mmHg/DBP+5mmHG*.

SNP Gene ‐logP (+15/+10 adjusted) ‐logP (+10/+5 adjusted)

Systolic blood pressure (SBP)
rs1801253 ADRB1 2.95039 3.13668
rs2004776 AGT 3.58670 3.02687
rs1985579 CACNA1A 4.98005 4.58503
rs6580586 ADRB2 2.35566 2.79588
rs2239101 CACNA1C 3.36663 3.01773
rs11064160 SCNN1A 1.92482 2.10791
Correlation coefficient=0.95
Diastolic blood pressure (DBP)
rs1801253 ADRB1 2.61816 3.12494
rs11122587 AGT 4.32624 3.95861
rs2399594 SLC12A3 3.62342 3.19382
rs4149570 SCNN1A 2.10991 2.12494
rs1985579 CACNA1A 3.89313 3.82391
rs12089381 REN 2.36957 2.43180
rs13278559 CA1 2.25321 2.60206
Correlation coefficient=0.94
* all associations have the same direction of effect relative to the same allele regardless of treatment adjustment applied
Table S2. SNP associations with lowest p‐value within 60 kb of drug target gene results for SBP, DBP and HTN from a GWAS meta‐
analysis of 29,135 individuals in the CHARGE consortium.
Discovery region information Systolic Blood Pressure (SBP) Diastolic Blood Pressure (DBP) Hypertension (HTN)
Gene Drug class # snps Marker ID P value* Resampling Marker ID P value* Resampling Marker ID P value* Resampling
P value P value P value
ACE ACE inhibitors 62 rs9894286 0.05 0.52 rs4459609 0.06 0.55 rs4305 0.01 0.14
ADRA1A Alpha blockers 383 rs11782159 0.01 0.32 rs6985931 0.01 0.53 rs4416829 0.01 0.54
ADRA1B Clonindine 147 rs3729604 0.02 0.42 rs3729604 0.02 0.40 rs6892282 0.03 0.60
ADRA2A Beta blockers 121 rs17128451 0.07 0.78 rs17128451 0.12 0.91 rs9663346 0.03 0.54
-4 -4
ADRB1 Beta blockers 142 rs1801253 7.3x10 0.031 rs1801253 7.5x10 0.032 rs7902859 0.04 0.68
-3 -4
ADRB2 ACE inhibitors 142 rs6580586 1.6x10 0.059 rs1800888 0.01 0.34 rs2082382 7.0x10 0.029
-4 -4 -4
AGT ARBs 271 rs2004776 9.5x10 0.07 rs11122587 1.4x10 0.011 rs12046196 5.6x10 0.042
AGTR1 ARBs 224 rs11710606 0.02 0.49 rs10935726 0.04 0.73 rs6803439 0.05 0.84
-3
CA1 Diuretics 105 rs13278559 0.12 0.89 rs13278559 2.5x10 0.07 rs17814075 0.02 0.33
CA2 Diuretics 75 rs11782151 0.13 0.85 rs10106305 0.09 0.74 rs168600 0.07 0.68
CA4 Diuretics 46 rs4968405 0.05 0.43 rs345164 0.08 0.59 rs11656267 0.03 0.28
-5 -3 -4 -3
CACNA1A CCBs 331 rs1985579 2.6x10 7.8x10 rs1985579 1.5x10 0.014 rs1985579 2.9x10 0.19
-3 -3 -4
CACNA1C CCBs 810 rs2239101 1.0x10 0.16 rs2239101 2.7x10 0.35 rs16929470 1.3x10 0.030
-5 -3
CACNA1H CCBs 80 rs4984796 0.01 0.17 rs4984796 0.02 0.26 rs4984796 8.0x10 3.9x10
CACNA2D1 CCBs 698 rs10280428 0.01 0.59 rs3823920 0.01 0.63 rs10280428 0.02 0.86
CACNG1 CCBs 106 rs3785588 0.03 0.42 rs3785581 0.03 0.43 rs9892538 0.04 0.60
KCNE1 Diuretics 198 rs11700993 0.07 0.88 rs1012599 0.09 0.93 rs2834508 0.03 0.60
-3 -3 -3
KCNMA1 Diuretics 1,146 rs184090 4.7x10 0.62 rs12219498 1.6x10 0.30 rs7893394 2.0x10 0.37
-3 -3
MME Endopeptidase 158 rs3821719 4.2x10 0.14 rs16824558 1.9x10 0.07 rs16824558 0.01 0.31
inhibitors
NPR1 Vasodilator 53 rs913859 0.09 0.65 rs913859 0.07 0.57 rs3891075 0.10 0.70
-3 -3
NR3C2 Aldosterone 401 rs1021956 3.8x10 0.27 rs1021956 0.01 0.51 rs10026568 4.5x10 0.31
antagonists
-3
REN Renin inhibitors 119 rs2692010 0.02 0.39 rs12089381 3.7x10 0.10 rs11240692 0.04 0.58
SCNN1A Amiloride 93 rs11064160 0.01 0.16 rs4149570 0.01 0.15 rs3782724 0.07 0.69
SCNN1B Amiloride 104 rs8057712 0.07 0.72 rs2301601 0.02 0.37 rs9646258 0.01 0.18
SCNN1D Amiloride 27 rs4018608 0.10 0.54 rs4018608 0.13 0.65 rs6603783 0.18 0.75

SCNN1G Amiloride 88 rs17797149 0.04 0.53 rs17797149 0.10 0.80 rs9929781 0.02 0.31
 SLC12A1 Diuretics 100 rs11636115 0.14 0.91 rs2459394 0.15 0.92 rs2459394 0.27 0.99
-4
SLC12A3 Diuretics 195 rs11645870 0.01 0.22 rs2399594 6.5x10 0.035 rs1347590 0.01 0.21
SLC18A2 Reserpine 187 rs11812488 0.05 0.75 rs363294 0.01 0.34 rs363264 0.04 0.72
-3
SLC9A1 Amiloride 71 rs834201 0.13 0.84 rs12410656 0.06 0.62 rs484677 3.3x10 0.06
5
*p-values have been taken from GWAS results corrected by genomic control . The λ values for each trait and group are given in footnotes of the respective trait Tables 1-3. SNPs in
bold were carried forward for replication based on either resampling and/or having a P < 1/n, where n=the number of SNPs tested in that gene region.
Table S3. Comparison of results from the current study with past studies and meta‐analyses on the same gene regions.

Current study meta‐analysis Past studies

Gene Variant Trait Sample Beta S.E. p‐value Variant Trait Ancestry Design Sample Effect
‐10 18,†
ADRB1 rs1801253 SBP 86,558 ‐0.57 0.09 4.7x10 rs1801253 HTN EA C, CC 6499 ↓
‐10 18
DBP 86,558 ‐0.36 0.06 9.5x10 SBP/DBP EA C, CC 5279 ↓‡/↓‡
HTN* 86,558 ‐0.06 0.02 3.3x10‐4 SBP/DBP19 EA C 1881 ↓/↓‡
HTN20,* EA CC 557 ↓‡
DBP20 EA sibships 135 ↓‡
SBP/DBP21 Asian CC, m.s. 1348 ↑/↑

AGT rs2004776 SBP 86,558 0.42 0.09 3.8x10‐6 rs4762 HTN (meta)22,* Mixed meta 26818 ↑‡
DBP 86,558 0.32 0.06 5.0x10‐8 rs699 HTN (meta)23,† Mixed meta 25055 ↑‡
HTN* 86,558 0.08 0.02 3.7x10‐7 rs699 SBP/DBP in women24 EA C 6182 ↑‡/↑‡
rs699 SBP/DBP in men24 EA C 5202 ↑/↑
22,*
rs5049 HTN (meta) Mixed meta 2514 ↑‡
rs5051 HTN (meta)22,* Mixed meta 14937 ↓
22,*
rs5050 HTN (meta) Mixed meta 4984 ↓‡
rs699 SBP/DBP (meta)23 Mixed meta 10315 n.r.

ACE rs4305 SBP 86,558 0.06 0.01 3.0x10‐5 I/D (indel) SBP/DBP (meta)25 EA meta 15942 ↓
DBP 86,558 0.29 0.08 4.6x10‐4 SBP/DBP26 Mixed RCT, m.s. 37939 n.r.
‐5
HTN* 86,558 0.21 0.05 6.0x10
All results are reported relative to the effect of the minor alleles of the variants mentioned as determined by the current study. Beta reflects the unit change in mmHG in SBP/DBP or in log odds of
HTN per allele dose. S.E indicates standard error of the mean. Betas generally were not given in past studies so Effect indicates either an increase or decrease in mean BP or an increase or decrease in
odds of HTN. *HTN defined as SBP≥140 mmHg and/or DBP≥90 mmHg. †HTN defined as SBP≥160 mmHg and/or DBP≥ 90 mmHg.
‡
indicates significance in the cited study
Abbreviations: EA indicates European ancestry. C indicates “cohort” design. CC indicates “case‐control”. m.s. indicates “multi‐site”. meta indicates “meta‐analysis”. RCT indicates “randomized
controlled trial”. n.r. indicates the direction of effect was not reported
Table S4. Significant discovery results that did not show replication evidence and related information from past studies.

Current study meta‐analysis Past studies

Gene Variant Trait Sample Beta S.E. p‐value Variants Description of related studies
ADRB2 rs2082382 SBP 86,558 ‐0.02 0.08 0.84 rs1042713 Several ADRB2 variants have been the subject of prior study:
DBP 86,558 ‐0.04 0.05 0.43 rs1042714 Gly16Arg (rs1042713), Gln27Glu (rs1042714) and Thr164Ile
‐3 (rs1800888)9‐13. Past evidence is mixed14. rs2082382 (current
HTN* 86,558 ‐0.04 0.01 5.1x10 rs1800888
study) and rs1042714 are in high LD (r2=0.93). rs2082382 showed
nominal association (P=0.04) in WGHS, possibly consistent with
prior reports of gender differential ADRB2 effects15‐17.

CACNA1C rs16929470 SBP 86,558 ‐0.14 0.19 0.48 Multiple CACNA1C mutations are associated with Long QT syndrome.
DBP 86,558 0.02 0.12 0.85 SNPs have been associated with response to calcium channel
blockers in 2 populations27,28 (n=120 and n=161, respectively),
HTN* 86,558 ‐0.07 0.03 0.04
though associated SNPs across all 3 studies including the current
one are not in high LD.

CACNA1A rs1985579 SBP 86,558 ‐0.29 0.08 4.4x10‐4 n/a CACNA1A has not been the subject of previous genetic studies.
DBP 86,558 ‐0.14 0.05 8.7x10‐3 Functional studies indicate this P/Q type channel is brain‐
expressed29 and may have a role cranial vasodilatation30.
HTN* 86,558 ‐0.04 0.01 2.5x10‐3

SLC12A3 rs2399594 SBP 86,558 ‐0.15 0.08 0.07 rs11643718 SLC12A3 mutations cause Gitelman’s syndrome, an autosomal
DBP 86,558 ‐0.16 0.05 2.7x10 ‐3 recessive salt wasting disorder31. Homozygotes for Arg904Gln
(rs11643718) were shown to be over‐represented in those with
HTN* 86,558 ‐0.001 0.01 0.93
Gitelman’s syndrome32. rs2399594 and rs11643718 are in
modest LD (r2=0.23). The Gln allele of rs11643718 was modestly
associated with with decreased DBP in CHARGE (P=0.004).
*HTN defined as SBP≥140 mmHg and/or DBP≥90 mmHg
Figure S1. Example of resampling-based approach (previously described in [7]) for
candidate SNP selection for replication. This approach was applied to each region and
phenotype (SBP, DBP, HTN) listed in Table S1. Step 1: Associations within 60kb of
ADRB1 are examined and the best association established (p=7.3x10-4). Step 2: The
same number of SNPs examined in Step 1 (n=142 in this example) are examined from
a random draw of n [142] SNPs that have consecutive physical positions on the genome
and have results within the GWAS scan. If the most significant association in this case
has a P value more extreme than the most extreme P value in Step 1 than this is
recorded as one instance. Step 3: Step 2 is repeated for a total of 10,000 times and all
instances with more extreme P values are tallied for a total (310 in this example). A
resampling statistic is derived by dividing the number of instances that produce more
extreme statistics than the true case (Step 1) by the total number of samplings (10,000).
Figure S2. Comparison of different treatment effect adjustments on SBP GWAS analysis in CHARGE. Left panel:
Comparative QQ’ plot of GWAS associations with +10 mm Hg SBP adjustment (y-axis) versus +15 mm Hg SBP
adjustment (x-axis). Right panel: Scatterplot of associations for each SNP in GWAS analysis with +10 mm Hg SBP
adjustment (y-axis) versus +15 mm Hg SBP adjustment (x-axis).
Figure S3. Comparison of different treatment effect adjustments on DBP GWAS analysis in CHARGE. Left panel:
Comparative QQ’ plot of GWAS associations with +5 mm Hg SBP adjustment (y-axis) versus +10 mm Hg SBP adjustment
(x-axis). Right panel: Scatterplot of associations for each SNP in GWAS analysis with +5 mm Hg SBP adjustment (y-axis)
versus +10 mm Hg SBP adjustment (x-axis).
Association of Hypertension Drug Target Genes With Blood Pressure and Hypertension in
86 588 Individuals
Andrew D. Johnson, Christopher Newton-Cheh, Daniel I. Chasman, Georg B. Ehret, Toby
Johnson, Lynda Rose, Kenneth Rice, Germaine C. Verwoert, Lenore J. Launer, Vilmundur
Gudnason, Martin G. Larson, Aravinda Chakravarti, Bruce M. Psaty, Mark Caulfield, Cornelia
M. van Duijn, Paul M. Ridker, Patricia B. Munroe and Daniel Levy
on Behalf of the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium,
Global BPgen Consortium, and Women's Genome Health Study

Hypertension. 2011;57:903-910; originally published online March 28, 2011;

doi: 10.1161/HYPERTENSIONAHA.110.158667
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2011 American Heart Association, Inc. All rights reserved.
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