Author + information
- Received November 11, 2013
- Revision received April 10, 2014
- Accepted April 18, 2014
- Published online December 1, 2014.
- Dennis M. McNamara, MD, MS∗∗ (, )
- Anne L. Taylor, MD†,
- S. William Tam, PhD‡,
- Manuel Worcel, MD§,
- Clyde W. Yancy, MD, MSc‖,
- Karen Hanley-Yanez, BS∗,
- Jay N. Cohn, MD¶ and
- Arthur M. Feldman, MD, PhD#
- ∗Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
- †Columbia University College of Physicians and Surgeons, New York, New York
- ‡Independent consultant, Dover, Massachusetts
- §Consultant, Paris, France
- ‖Division of Cardiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
- ¶University of Minnesota, Minneapolis, Minnesota
- #Temple University School of Medicine, Philadelphia, Pennsylvania
- ↵∗Reprint requests and correspondence:
Dr. Dennis M. McNamara, Center for Heart Failure Research, Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh Medical Center, 566 Scaife Hall, 200 Lothrop Street, Pittsburgh, Pennsylvania 15213.
Objectives The purpose of this study was to evaluate the influence of the guanine nucleotide-binding proteins (G-proteins), beta-3 subunit (GNB3) genotype on the effectiveness of a fixed-dose combination of isosorbide dinitrate and hydralazine (FDC I/H) in A-HeFT (African American Heart Failure Trial).
Background GNB3 plays a role in alpha2-adrenergic signaling. A polymorphism (C825T) exists, and the T allele is linked to enhanced alpha-adrenergic tone and is more prevalent in African Americans.
Methods A total of 350 subjects enrolled in the genetic substudy (GRAHF [Genetic Risk Assessment of Heart Failure in African Americans]) were genotyped for the C825T polymorphism. The impact of FDC I/H on a composite score (CS) that incorporated death, hospital stay for heart failure, and change in quality of life (QoL) and on event-free survival were assessed in GNB3 genotype subsets.
Results The GRAHF cohort was 60% male, 25% ischemic, 97% New York Heart Association functional class III, age 57 ± 13 years, with a mean qualifying left ventricular ejection fraction of 0.24 ± 0.06. For GNB3 genotype, 184 subjects were TT (53%), 137 (39%) CT, and 29 (8%) were CC. In GNB3 TT subjects, FDC I/H improved the CS (FDC I/H = 0.50 ± 1.6; placebo = −0.11 ± 1.8, p = 0.02), QoL (FDC I/H = 0.69 ± 1.4; placebo = 0.24 ± 1.5, p = 0.04), and event-free survival (hazard ratio: 0.51, p = 0.047), but not in subjects with the C allele (for CS, FDC I/H = −0.05 ± 1.7; placebo = −0.09 ± 1.7, p = 0.87; for QoL, FDC I/H = 0.28 ± 1.5; placebo = 0.14 ± 1.5, p = 0.56; and for event-free survival, p = 0.35).
Conclusions The GNB3 TT genotype was associated with greater therapeutic effect of FDC I/H in A-HeFT. The role of the GNB3 genotype for targeting therapy with FDC I/H deserves further study.
As first demonstrated in V-HeFT (Veterans Affairs Vasodilator-Heart Failure Trial) treatment with isosorbide dinitrate and hydralazine (I/H) improves survival in subjects with heart failure (1). After the development of angiotensin-converting enzyme (ACE) inhibitors and the demonstration in V-HeFT II of greater survival benefit with enalapril compared with I/H (2), I/H was relegated to less common use in heart failure. Interestingly, the survival benefit for I/H differed markedly in the V-HeFT studies between black and white cohorts. Post hoc analysis demonstrated a dramatic benefit of I/H for black subjects in V-HeFT I, with a lesser impact evident in the larger white cohort (3). A similar analysis by race for V-HeFT II found that the superiority of ACE inhibitors was driven by the white subset, whereas in black subjects, the survival with enalapril and I/H appeared more equivalent. The observed enhanced impact of I/H in black subjects with heart failure in these landmark studies led to A-HeFT (African American Heart Failure Trial) (4), in which a fixed-dose combination (FDC) of I/H added to background therapy was demonstrated to improve survival in a cohort of self-designated African Americans with a reduced ejection fraction (5).
Hypertension is far more prevalent among blacks, and investigations of the genomic basis for this disorder have demonstrated significant differences between black and white cohorts in the prevalence of genetic variants that affect vascular tone. One example extensively studied is the G-protein beta-3 subunit (GNB3). A common GNB3 polymorphism in the coding region of exon-10, C825T, is associated with enhanced alpha2-adrenergic receptor intracellular signaling (6). The GNB3 T allele is linked to the risk of hypertension (7,8) and low plasma renin (9), and it has a much higher prevalence in black cohorts than among whites (10). Whether the T allele linked to low plasma renin is also associated with a diminished impact of ACE inhibitors remains unknown.
We hypothesized that genomic differences in GNB3-mediated alpha2-adrenergic receptor activation may underlie the racial differences in the therapeutic efficacy of FDC I/H, and that the GNB3 T allele more prevalent among African Americans would be associated with a greater impact of I/H. We investigated the interaction of drug therapy and GNB3 genotype in the genetic substudy of A-HeFT, GRAHF (Genetic Risk of Heart Failure in African Americans).
A total of 350 subjects in A-HeFT were enrolled in GRAHF, a substudy of genetic risk assessment of heart failure in African Americans. This investigation was approved by the institutional review boards at the participating institutions. Inclusion criteria for A-HeFT included self-designation as African Americans, heart failure resulting from systolic dysfunction, and standard background therapy for heart failure with neurohormonal blockade including ACE inhibitors or angiotensin receptor antagonists and beta blockers (4). Subjects were randomized to FDC I/H or placebo in addition to standard therapy. Subjects were enrolled in the GRAHF genetic substudy at the A-HeFT 6-month visits after providing informed consent. For comparisons of allele frequencies by race, the cohort from GRACE (Genetic Risk Assessment of Cardiac Events), a single-center investigation based at the heart failure clinic at the University of Pittsburgh, Pennsylvania (11,12), was used (n = 469).
DNA was isolated from peripheral blood by leukocyte centrifugation and cell lysis using the PureGene DNA purification kit (Gentra Systems, Minneapolis, Minnesota). The guanine nucleotide binding protein (G-protein) beta polypeptide-3 (GNB3) position 825 C/T polymorphism was assessed using a TaqMan SNP Genotyping Assay (Applied Biosystems, Inc., Foster City, California) with tagged primers (reporter 1 tagged dye = VIC; reporter 2 tagged dye = FAM). Context sequence for the GNB3 825 C/T polymorphism was as follows: AGAGCATCATCTGCGGCATCACGTC[C/T] GTGGCCTTCTCCCTCAGTGGCCGCC. Products were read using the Applied Biosystems 7000 (ABI).
Follow-Up and Clinical Outcomes
Subjects in A-HeFT were followed for 18 months with assessment of deaths, hospital stays for heart failure, and quality of life (QoL) as endpoints (4). QoL assessment was performed by the Minnesota Living with Heart Failure Questionnaire at baseline and at the 6-month visit. All subjects enrolled in A-HeFT had a clinical assessment of left ventricular ejection fraction (LVEF) before enrollment (qualifying LVEF). In addition, in a subset of subjects in a remodeling substudy, LVEF and left ventricular end-diastolic diameter (LVEDD) were quantified by echocardiography at a central core laboratory at baseline (n = 267) and at the 6-month follow-up visit (n = 259). The primary endpoint for A-HeFT was a composite weighted score with 3 components: mortality, hospital stay for heart failure, and change in QoL at 6 months (4,5). The change in QoL score was reported as a component of the composite score (CS) ranging from most worsening to most improvement scores of −2 to 2 (4,5).
Event-free survival was compared by genotype class by Kaplan-Meier log rank analysis. Continuous variables such as CS were compared by genotype class by analysis of variance (ANOVA). Statistical analysis was performed using SPSS version 20.0 (SPSS Inc., Chicago, Illinois). Results are presented as mean ± SD. Categorical variables were compared using a nonparametric chi-square Fisher-exact test.
GNB3 Genotype Frequencies
A total of 350 subjects from GRAHF were genotyped for the GNB3 C825T polymorphism. Of the GRAHF cohort, 184 subjects (53%) were homozygous for the T allele, and 166 (47%) had a least 1 copy of the C allele: 137 (39%) were heterozygous and 29 (8%) were homozygous for the C allele. Comparison with the GRACE study demonstrated that the prevalence of the GNB3 T allele in GRAHF was similar to that seen in the black cohort from GRACE, but it was significantly greater than in the white cohort (% TT/CT/CC GRAHF = 53/39/8; GRACE (black cohort) = 54/27/19; GRACE (white cohort) = 14/41/45, p < 0.001) (Figure 1).
The GRAHF population was 60% male, 25% ischemic, and 97% New York Heart Association (NYHA) class III, with a mean age of 57 ± 13 years (Table 1). Most subjects were taking beta blockers (85%) and ACE inhibitors (75%) at time of study entry, and more than one third (36%) were taking aldosterone receptor antagonists. Comparisons of baseline characteristics by GNB3 genotype class revealed no significant differences. Medical therapy, blood pressure, and NYHA class were all similar in subjects homozygous with the T allele compared with subjects with 1 or 2 copies of the C allele.
Composite Score, GNB3 Genotype, and Impact of Therapy
Although in A-HeFT, treatment with FDC I/H significantly improved both CS and QoL (5), in the smaller GRAHF subset, treatment with FDC I/H was associated with a trend toward improved CS and QoL that failed to reach significance (CS: placebo = −0.10 ± 1.8; FDC I/H = 0.23 ± 1.6, p = 0.07; and for QoL: placebo = 0.19 ± 1.5; FDC I/H = 0.49 ± 1.4, p = 0.07). When analyzed by GNB3 genotype, FDC I/H improved the CS among TT homozygotes (placebo = −0.11 ± 1.8; FDC I/H = 0.50 ± 1.6, p = 0.02) (Figure 2), but it had no impact among subjects with the C allele (placebo = −0.09 ± 1.7; FDC I/H= −0.05 ± 1.7, p = 0.87). Improvement for QoL score with FDC I/H was also evident for GNB3 TT subjects (QoL component for GNB3 TT subset: placebo = 0.24 ± 1.5; FDC I/H = 0.69 ± 1.4, p = 0.04) (Figure 3) but not among those with the C allele (placebo = 0.14 ± 1.5; FDC I/H = 0.28 ± 1.5, p = 0.56).
Over the course of follow-up, there were 61 (17.3%) hospital stays for heart failure and 12 deaths (3.4%). When evaluating the combined endpoint of death or hospital stays for heart failure, the percentages of event-free survival at 6, 12, and 18 months were 91%, 81%, and 73%, respectively. Overall in GRAHF, treatment with FDC I/H did not improve event-free survival (FDC I/H event-free survival at 6, 12 and 18 months = 91%, 83%, 76%; placebo = 90%, 78%, 69%, p = 0.41) (Figure 4A). However, when evaluated in the GNB3 TT subset (n = 184), a significant impact of treatment was evident (FDC I/H event-free survival at 6, 12, and 18 months = 92%, 86%, 79%; placebo = 89%, 74%, 61%, p = 0.047) (Figure 4B) that was not apparent for subjects with the C allele (FDC I/H event-free survival at 6, 12 and 18 months = 90%, 80%, 73%; placebo = 92%, 82%, 78%, p = 0.35) (Figure 4C). When evaluating the impact of genotype by treatment subset, poorer event-free survival was evident with the GNB3 TT genotype for subjects receiving placebo (p = 0.048) (Figure 5A); this impact was not apparent in subjects treated with FDC I/H (p = 0.35) (Figure 5B).
Left Ventricular Remodeling, Treatment and GNB3 Genotype
Overall in A-HeFT, treatment with FDC I/H was associated with significant reverse remodeling, as assessed by the change in LVEF by echocardiography at 6 months (13). In the GRAHF substudy, improvements in LVEF at 6 months were not significantly different by treatment group (LVEF at 6 months: placebo = 0.36 ± 0.10; FDC I/H = 0.38 ± 0.09, p = 0.16; change from baseline at 6 months: placebo = 0.02 ± 0.09; FDC I/H = 0.03 ± 0.08, p = 0.18). No clear interaction of GNB3 genotype with remodeling was evident. For subjects with the GNB3 TT genotype the mean LVEF was slightly higher at baseline (LVEF GNB3 TT: 0.36 ± 0.08 versus C allele: 0.34 ± 0.09, p = 0.052); however, this difference at baseline was not evident at 6 months (GNB3 TT: LVEF = 0.37 ± 0.10; C allele: = 0.37 ± 0.09, p = 0.64). No clear interaction of GNB3 genotype and the impact of treatment with FDC I/H on remodeling was apparent (Table 2).
In GRAHF, subjects with the GNB3 TT genotype received therapeutic benefit from FDC I/H, as demonstrated by an improved event-free survival and QoL and a higher CS. In contrast, no therapeutic impact was evident in subjects with 1 or 2 copies of the C allele. The prevalence of the T allele differs markedly by race; more than 50% of black cohorts have the homozygous GNB3 TT genotype associated with enhanced benefit of FDC I/H, compared with less than 15% of whites. These findings suggest that racial differences in the prevalence of the GNB3 T allele may underlie the apparent racial differences in the impact of therapy.
The GNB3 C825T polymorphism is a functionally silent single nucleotide polymorphism (SNP) in exon 10 that does not change the amino acid sequence (14). Common SNPs are also evident in the GNB3 promoter region (G-350A), intron 9 (A3882C and G5249A), the 3′ untranslated region (C1429T), in addition to an insertion/deletion polymorphism of 4 base pairs (6496 CACA) in intron 10. These polymorphisms are in near complete linkage disequilibrium with the −350A, 3882C, 5249A, 1429T and 6496 CACA insertion alleles co-inherited with the 825T allele as the “T haplotype” (15). This haplotype is strongly associated with a splicing variant of GNB3, GNB3s, which is evident only in cell lines with the 825T allele (7). When compared with the wild-type protein, GNB3s is missing 41 amino acids, a complete repeated loop domain, but it remains functionally active. Enhanced activation of G proteins is evident in immortalized cell lines from hypertensive compared with normotensive subjects (16), and in these same cell lines it is tightly linked to the presence of the 825T allele and GNB3s (7).
The role of the GNB3 polymorphism in the risk of hypertension has been studied extensively (17–19), and pharmacogenetic investigations have suggested increased alpha2-adrenergic activation with the T allele as a potential mechanism. In subjects with hypertension, the GNB3 T allele has been associated with an increased response to clonidine, a centrally acting alpha2-adrenergic receptor antagonist (20). In investigation of coronary vasomotor response, subjects with the T allele also demonstrate enhanced vasoconstriction (8,21), and this enhanced impact can be eliminated by selective blockade of the alpha2, but not the alpha1, adrenergic receptor (22). Although investigations of the role of GNB3 in cardiovascular disease have focused on vascular reactivity, duplication of the GNB3 locus has been implicated as a cause of obesity both in murine models and in human syndromes of chromosomal translocation (23). In contrast, genome-wide association studies (GWAS) evaluating the genomic basis of obesity (24) and hypertension (25) have not identified GNB3 as a disease-associated locus.
Pharmacogenetic investigations suggest that the GNB3 C825T polymorphism influences therapeutics directed at the nitric oxide (NO) pathway. The enhanced vasoconstriction in subjects with the GNB3 T allele can be eliminated by pretreatment with the NO synthase (NOS) inhibitor L-NMMA (26). Carriers of the T allele also have an enhanced vasodilator response to nitroglycerin (27), as well as an enhanced therapeutic response to the phosphodiesterase type 5 inhibitor (PDE5) sildenafil in subjects with erectile dysfunction (28) and in those with pulmonary arterial hypertension (29). Insulin-induced venodilation, a response mediated by the NO pathway, is diminished in healthy carriers of the GNB3 T allele (30). Together with GRAHF, these studies suggest that the GNB3 TT genotype may identify subjects who would obtain maximal benefit from therapeutic strategies that enhance NO.
Recent reports from 2 separate longitudinal population studies suggest a higher risk of cardiovascular or cerebrovascular events in subjects with the GNB3 TT genotype. In the Fungata study of a rural Japanese cohort of 1,524 subjects, those with the GNB3 TT genotype had a higher incidence of cardiovascular disease (hazard ratio [HR]: 1.82, 95% confidence interval [CI]: 1.14 to 2.89) and stroke (HR: 1.76, 95% CI: 1.01 to 3.07) despite no differences in blood pressure or hypertension (31). In the Italian LEOGRA study, Last Evidence of Genetic Risk in the Aged, a cohort of subjects from 2 small towns was followed for 10 years. Subjects with the GNB3 TT genotype had a significantly higher risk of cerebral vascular events (HR: 2.22, 95% CI: 1.31 to 3.79) which remained significant when adjusting for blood pressure and other cardiovascular risk factors (32).
In a small Brazilian cohort with systolic heart failure, the GNB3 825T and the Arg389 β1 alleles were associated with increased cardioverter-defibrillator therapies (33). In these previous studies, the GNB3 TT genotype subset represented only 10% to 20% of the entire cohort, whereas in the GRAHF study in African Americans, this high-risk genotype was present in more than 50% of subjects. Consistent with these previous reports, GRAHF subjects with the GNB3 TT genotype who received standard heart failure therapy plus placebo demonstrated the lowest CS and poorest event-free survival. Importantly, treatment with FDC I/H appeared to eliminate this genetic risk.
The current study is limited by study number, which diminishes the power to address pharmacogenetic interactions. In particular, the small number of subjects homozygous for the C allele limits the ability to evaluate the impact of gene dose on the outcomes observed. In addition, although the T allele has been previously linked to low plasma renin, renin and neurohormones were not measured as part of the GRAHF analysis. Finally, whereas the current analysis is focused on GNB3, the influence of genomics on the effectiveness of heart failure therapy is almost certainly polygenic. Previous analysis in GRAHF suggests that genetic heterogeneity of NOS3 (34) influences the benefit of FDC I/H, and variation of the aldosterone synthase promoter affects event-free survival (35); however, the limited study number in GRAHF prevents analysis of gene-gene interactions. An individual’s response to pharmacological intervention almost certainly involves multiple genomic loci, and as a result many previous attempts to replicate other pharmacogenetic reports of single SNP interactions have not succeeded.
A-HeFT demonstrated that treatment with FDC I/H improved survival for a cohort of self-designated African Americans with heart failure, and it remains a pivotal study that for the first time resulted in the approval by the Food and Drug Administration of a heart failure therapy in a specific racial or ethnic group (36,37). The ability of race to predict the therapeutic impact of I/H likely reflects its role as a marker of functional genomic differences. Pharmacogenomic analysis should be more effective than race in predicting efficacy and targeting heart failure therapeutics. Although the current investigation suggests that the GNB3 genotype predicts the therapeutic impact of FDC I/H for an individual, these findings should be considered hypothesis generating and will require prospective validation. Further investigation is warranted to explore its potential utility for targeting heart failure therapeutics.
This study was funded in part by a research grant from NitroMed, Inc., of Lexington, Massachusetts, which funded the A-HeFT investigation. It was also supported in part by grants from the National Heart, Lung, and Blood Institute (contracts K24 HL69912 and RO1 HL75038). Drs. Tam and Worcel are former employees of NitroMed. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- angiotensin-converting enzyme
- composite score
- FDC I/H
- fixed-dose combination of isosorbide dinitrates and hydralazine
- guanine nucleotide-binding proteins (G-proteins), beta-3 subunit
- isosorbide dinitrate and hydralazine
- left ventricular ejection fraction
- left ventricular end-diastolic diameter
- quality of life
- single nucleotide polymorphism
- Received November 11, 2013.
- Revision received April 10, 2014.
- Accepted April 18, 2014.
- American College of Cardiology Foundation
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