Published online before print June 24, 2002, doi: 10.1161/01.HYP.0000025145.12159.70
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(Hypertension. 2002;40:162.)
© 2002 American Heart Association, Inc.
Scientific Contributions |
Relation of the G Protein ß3-Subunit Polymorphism With Left Ventricle Structure and Function
ekFrom the Klinik und Poliklinik für Innere Medizin II, Universität Regensburg (K.S., M.F., J.E., C.H., S.H., S.K., M.M., A.L., G.A.R., H.S.), Regensburg, Germany; and Institut für Epidemiologie und Sozialmedizin, Universität Münster (H-W.H.), Münster, Germany.
Correspondence to Heribert Schunkert, MD, Klinik und Poliklinik für Innere Medizin II, Universität Regensburg, Franz-Josef-Strauss Allee 11, D-93042 Regensburg, Germany. E-mail heribert.schunkert{at}klinik.uni-regensburg.de
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Abstract |
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The G protein ß3-subunit C825T polymorphism results in a truncated splice variant protein that is associated with enhanced transmembrane signaling, increased proliferative activity, and arterial hypertension. The aim of the present study was to further investigate the association of this polymorphism with left ventricular (LV) structure and function. A total of 2052 individuals from a large-scale population-based sample were investigated for the G protein ß3-subunit C825T polymorphism and echocardiographic parameters of LV structure and function. Complete genotyping and echocardiographic data were available in 1720 individuals (829 men and 891 women). The mean LV mass indices in men with CC (n=384) and TT (n=84) genotypes were 98.3±1.2 g/m2 and 100.0±2.8 g/m2, respectively (P=0.64). In women, the corresponding values were 83.1±1.0 g/m2 for the CC genotype (n=397) and 83.8±2.1 g/m2 for the TT genotype (n=91, P=0.32). Likewise, LV dimensions or parameters of the diastolic function and serologic markers of LV mass were not associated with the C825T variant. Finally, multivariate analyses accounting for potentially confounding factors failed to show any influence of this polymorphism on echocardiographic parameters. In conclusion, we were not able to confirm the previously published associations of the G protein ß3-subunit C825T polymorphism with LV structure and diastolic function.
Key Words: genetics • myocardium • hypertrophy • G proteins • signal transduction
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Introduction |
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The C825T polymorphism in the heterotrimeric G protein ß3-subunit (GNB3) gene was initially discovered as a putative cause of the enhanced Na+-H+ exchanger activity in hypertensive patients.1 Although this mutation does not result in an amino acid substitution, its carriers express predominantly a truncated splice variant protein called GNB3s (s indicates spliced). This aberrant domain is depleted by 41 amino acids but is still functional. In fact, the truncated protein has been shown to be associated with enhanced signal transduction and proliferative activity in response to stimuli that require the pertussis toxin sensitive pathway.1
Many, but not all, previous studies found an association of the GNB3 C825T gene polymorphism with arterial hypertension, albeit the absolute effect on blood pressure (BP) levels was small.1–9 Additional phenotypes previously described in patients with increased Na+-H+ exchanger activity include obesity,10 salt retention with low plasma renin activity,3,11 and left ventricular hypertrophy (LVH).12
The association with LVH may be of particular interest because cardiac hypertrophy is a powerful and independent cardiovascular risk marker in hypertensive patients.13 The risk for LVH is influenced by various pathophysiological states, ie, hypertension, obesity, salt intake level, and age. In addition, the genetic background may account substantially to the variability of left ventricular mass (LVM) observed in normotensive or hypertensive individuals.14,15
Therefore, the association between the GNB3 polymorphism and LVH may be plausible and of potential clinical and scientific relevance. In particular, we used data from a large population-based survey to further study the previously reported association of the C825T polymorphism with cardiac geometry and function.
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Methods |
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Study Subjects
A description of this population-based sample has been previously published.4,16 Briefly, 2098 study participants were recruited at the occasion of 2 echocardiographic studies in 1994 and 1995 that were part of the Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) surveys in Augsburg, Germany. The population was studied by means of physical examination; standardized BP measurement; an interview on the socioeconomic background, medical, and medication history; and the presence of coronary risk factors such as arterial hypertension, hypercholesterolemia, diabetes mellitus, and cigarette smoking. Arterial hypertension was defined as BP
140/90 mm Hg or the regular intake of antihypertensive medication, unless otherwise specified (eg, in subgroup analyses). Biochemical parameters (glucose, glycosylated hemoglobin, total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides) were also determined. In a subset of participants, atrial and brain natriuretic peptide determination was performed using methods described elsewhere.17 The study was approved by the local ethics committee, and all the participants gave informed consent.
Echocardiography
Two-dimensionally guided M-mode tracings recorded on a strip-chart paper at 50 mm/sec were obtained by 2 expert echocardiographists using the Sonos 1500 (Hewlett-Packard). A single cardiologist analyzed all M-mode tracings. Left ventricular end-diastolic (LVEDD) and end-systolic (LVESD) diameter, interventricular septum thickness (IVS), and posterior wall thickness (PWD) were measured according to the recommendation of the American Society of Echocardiography.18 End-systolic and end-diastolic left ventricular volumes were calculated from the 2D parasternal long-axis 4-chamber views by use of an area-length method formula. Fraction shortening and ejection fraction were used as parameters of the left ventricular systolic function. Diastolic function of the left ventricle was assessed using the ratio of the early (E) and late (A) diastolic filling velocities (E/A ratio) and isovolumetric relaxation time (IVRT), ie, interval between the closure of the aortic valve and the start of the mitral inflow signal. LVM was calculated using the formula LVM (g) =0.8x[1.04(LVEDD+IVS+PWD)3-LVEDD3]+0.6, as described by Devereux et al.19 The LVM values were indexed for body surface area (LVMI-BSA) and for fat-free body mass (LVMI-FFM). LVH was defined by LVMI >116 g/m2 and >104 g/m2 in men and women, respectively.13,20 Diastolic dysfunction was considered to be present when E/A was <1.0 and/or IVRT was longer than 100 ms.21
Fat-Free Body Mass Calculation
To eliminate a need of gender-specific LVMI evaluation by indexing LVM for fat-free body mass (FFM), gender-specific equations for the FFM were used as previously described by Kuch et al22: FFM=5.1xheight1.14xweight0.41 and FFM=5.34xheight1.47xweight0.33 for men and women, respectively.
Genotype Assessment
DNA was extracted from whole blood, and the GNB3 polymorphism was genotyped according to standard protocols.3,4
Statistical Analysis
Anthropometrical, biochemical, and echocardiographic data according to the C825T allele status were compared using 1-factor ANOVA test for independent samples and 
2 test for categorical values. Data are expressed as mean±SEM. The P<0.05 was regarded as statistically significant. Multivariate analyses were calculated with LVMI-BSA, LVMI-FFM, LVEDD, LVESD, IVS, PWD, IVRT, and E/A values as dependent and with C825T genotype as an independent variable, including age, systolic BP, gender, and body mass index in the models. Considering our sample size of individuals with CC and TT genotypes, we estimated to have a 75% power to detect an LVMI-BSA difference (
) of 6.4 g/m2 in men and 6.2 g/m2 in women. This estimate is based on the 
=0.05 level of significance and a SD 
=20 g/m2 (assumption based on previous findings). The analyses were performed using SPSS version 10.0 and JMP 4 statistical software.
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Results |
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Of 2098 individuals from the MONICA surveys, 46 individuals were excluded from this study because of a history of myocardial infarction. Anthropometrical data on the whole sample have been previously published.4 From a total of 2052 studied probands (1020 men and 1032 women), complete genotyping and high-quality echocardiographic data were available in 1720 subjects (829 men and 891 women). The allele frequencies in the entire cohort and 2 subpopulations (men and women) were distributed in Hardy-Weinberg equilibrium. The T allele frequency in the whole population was 0.32 (0.32 and 0.33 in men and women, respectively). Anthropometrical and biochemical data according to the C825T genotype are listed in Table 1. Except for the previously described association of the TT genotype with arterial hypertension in the whole study population,4 there were no statistically significant differences between the groups of men and women according to genotype status. Moreover, the GNB3 C825T polymorphism did not contribute significantly to the variability of atrial and brain natriuretic peptide levels in a subgroup of 285 men and 321 women.
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Analyses of LVMI Indexed for BSA
Echocardiographic data are presented in Table 2. The mean LVMI-BSA in men with CC and TT genotypes were similar. Likewise, in women, the CC and TT genotype groups did not differ with respect to LVMI-BSA. Moreover, there was no statistically significant association of the GNB3 C825T polymorphism with other echocardiographic parameters of the left ventricle structure, systolic, or diastolic function (Tables 2 and 3).
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Furthermore, no significant differences were obtained when only men or women without antihypertensive medication were analyzed (Table 4 for the LVMI-BSA data of these subgroups). Finally, LVMI-BSA did not significantly differ by the GNB3 C825T polymorphism status in subgroups of men and women with younger and older age (
55 and >55 years, respectively), obesity (body mass index 30 kg/m2), or hypertension (Table 4).
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Accordingly, in multivariate regression analyses, the C825T polymorphism did not contribute to LVMI-BSA, IVS, LVEDD, or LVESD values after adjustment for age, systolic BP, gender, and body mass index (Table 5).
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Analyses of LVMI Indexed for FFM
LVMI-FFM for the whole study sample was 3.31±0.03 g/kg for the CC genotype and 3.35±0.07 g/kg for the TT genotype (P=0.25). Corresponding values in men and women are presented in Table 2. In a subgroup of untreated hypertensive subjects (BP 160/95 mm Hg, n=126), LVMI-FFM was 3.57±0.09 g/kg for the CC genotype and 4.05±0.26 g/kg for the TT genotype (P=0.08). By multivariate analysis, neither untreated hypertensive subjects (BP 160/95 mm Hg; n=86) nor normotensive probands (BP <140/90 mm Hg, n=521) displayed an association between the GNB3 polymorphism status with LVMI-FFM or other echocardiographic parameters (LVEDD, LVESD, IVS, PWD, E/A, and IVRT) with age, gender, systolic BP, and body mass index included in the model. Using the contemporary criterion for arterial hypertension (BP 140/90 mm Hg, n=200 for the hypertensive subgroup), similar results were obtained (Table 6).
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Discussion |
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GNB3 C825T polymorphism is an attractive candidate for the genetic modulation of the LVM. Indeed, Poch et al23 recently provided data from 86 previously untreated hypertensive patients, suggesting an association of this polymorphism with LVH. In particular, carriers of the T allele displayed a 3-fold increased risk to present with LVH. By contrast, the present study evaluated a substantially larger and epidemiologically phenotyped population sample and failed to show any association between the GNB3 T allele and cardiac structure.
Some features of the previous Spanish population and our study sample may be of relevance for the discrepant findings. With respect to the genetic structure of the 2 populations, the T allele frequency was 0.34 in Spain and 0.32 in our population, suggesting a similar distribution of the genetic variant under investigation. However, if the causative allele responsible for LVH is not identical with the T allele of the GNB3 gene but merely in linkage disequilibrium, even slight differences between the populations may be important and explain different findings.23
Moreover, the phenotypes under investigation may be different in the 2 studies. In this respect, Poch et al23 focused their work on untreated hypertensive patients. Such selected patients may be more informative to detect minor effects of a given candidate gene with respect to LVH compared with the normal population sample. However, in the present study, analysis of multiple subgroups potentially related to the effects of the GNB3 C825T polymorphism, including hypertensive subjects without antihypertensive medication, revealed no differences according to the genotype status. Furthermore, other phenotypes related to LVH, independent from echocardiographic measurements such as the serum levels of atrial and brain natriuretic peptide, did not display an association with the GNB3 polymorphism. Thus, we have no indication for having missed a positive finding owing to imprecise characterization of our sample.
Another explanation for the differences between the Spanish and the present study may be that the clinically based ascertainment strategy in Spain favored a higher prevalence of the T allele. In fact, previous studies documented an association of T allele particularly with severe hypertension, including combination antihypertensive therapy3,4 and end-organ damage.5 Thus, a positive association of the GNB3 T allele may merely reflect not only a causative nature of this polymorphism for LVH, but also differences in ascertainment strategies, resulting in differences in underlying mechanisms such as more severe hypertension in some selected patient groups.
Diastolic left ventricle dysfunction is yet another finding related to hypertension and LVH. Jacobi et al24 have reported in 34 mild to moderate hypertensive patients an association of the GNB3 T allele with the impaired diastolic filling (CC versus TC/TT) and velocity time integral A/E (CC versus TC/TT) and concluded that this polymorphism contributes to the development of hypertensive heart disease. In the present study, however, 2 parameters of diastolic left ventricle function (E/A and IVRT) and indices of systolic left ventricle function failed to show any association with the T allele status, even in subgroups of untreated hypertensive subjects.
The most likely explanation of the present negative study is that the GNB3 T allele has no strong and clinically relevant effect on the variability of LVM. Indeed, LVH is a complex genetic trait, and a single gene variant, albeit functionally relevant, may carry a minute contribution to the final phenotype. Therefore, it is inherently complicated to demonstrate its influence in even large-scale and well-powered association studies. By contrast, smaller studies have been shown to carry a risk of an -error.25 Thus, a cautious interpretation of such studies may be advisable.
A limitation of the present work may be that we used a standardized office-based BP measurement, whereas in the study of Poch et al,23 a 24-hour ambulatory BP measurement was performed. It has been consistently shown that ambulatory BP measurements are more powerful to predict LVH than is office BP.26,27 However, multiple subgroup analyses with and without adjustment for BP levels failed to show in the present study an association between the GNB3 polymorphism and LVM. Thus, it is unlikely that a more precise technique for BP measurement would have affected our results.
Perspectives
Common and complex phenotypes such as LVH and diastolic dysfunction have multiple etiologic components. In this scenario, effects conferred by a single gene variant either must be very strong or found at a very high frequency to be statistically detectable, even if the sample of the association study is large.28 In this respect, lack of association of the GNB3 C825T polymorphism with LVH and left ventricle diastolic function in this large Western European population sample represents another example of failure to reproduce small positive association studies. Recently, more stringent criteria for the evaluation of these studies have been proposed to avoid such confusion.25 Ultimately, studies that meet these criteria will help to dissect clinically relevant genetic variants from single nucleotide polymorphisms without biological function in the system under investigation.
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (DFG grants Schu672/9-1, Schu672/10-1, Schu672/12-1, and Ho1073/8-1), the Bundesministerium für Forschung und Technologie (KBF-FKZ 01GB9403 to Drs Schunkert and Hense), the Wilhelm-Vaillant-Stiftung (to Drs Hengstenberg and Schunkert), Ernst-und-Berta-Grimmke-Stiftung (to Drs Hengstenberg and Schunkert), and the Deutsche Stiftung für Herzforschung (to Drs Hengstenberg and Schunkert).
Received February 22, 2002; first decision March 22, 2002; accepted May 24, 2002.
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