Influence of 825 C>T polymorphism of G protein�3 subunit gene (GNB3) on hemodynamicresponse during dobutamine stressechocardiography

Anna Banaœ1, Edyta P³oñska2, Barbara Larysz3, Mateusz Kurzawski1,

Marek DroŸdzik1

�Department of Experimental and Clinical Pharmacology, Pomeranian Medical University, Powstañcow Wlkp. 72,

PL 70-111, Szczecin, Poland

�Department of Cardiology, Pomeranian Medical University, Powstañcow Wlkp. 72, PL 70-111, Szczecin, Poland

�Department of Cardiology, County Hospital, Arkoñska 4, 71-455, Szczecin, Poland

Correspondence: Marek DroŸdzik, e-mail: drozdzik@sci.pam.szczecin.pl

Abstract:

There is a wide interpersonal difference to dobutamine response during dobutamine stress echocardiography (DSE). The aim of thisstudy was to determine an association between GNB3 825C>T gene polymorphism, encoding the �3-subunit of G protein, and heartrate (HR), systolic (SBP) and diastolic blood pressure (DBP) response to dobutamine during DSE. The study involved 119 patientswith clinical indications for DSE. Genotyping of GNB3 825C>T (rs5443) polymorphism was based on PCR-RFLP method withBseDI restriction enzyme. Significantly higher levels of both resting SBP and DBP in 825T allele carriers vs. 825CC patients wererevealed. HR of 825CC vs. CT + TT subjects was slower along the test period reaching marked difference at dobutamine level30 μg/kg/min (109 ± 20 vs. 116 ± 18 bpm, respectively, p = 0.047). SBP and DBP were markedly lower in 825CC homozygotes com-pared to 825T allele carriers throughout DSE. It can be concluded that GNB3 825C>T polymorphism is associated with resting SBPand DBP in Polish Caucasian patients subjected for diagnostic DSE. The polymorphism also modulate HR, SBP and DBP responseduring DSE.

Key words:

G protein, 825C>T polymorphism, dobutamine stress echocardiography

Introduction

GTP-binding proteins are very important partners formany transmembrane receptors that activate or inhibitintracellular signaling cascades. Recent studies havefocused on �-adrenergic receptors belonging to Gs

protein coupled receptor family (including �1-adre-nergic receptor – �1-AR), that play important role incardiovascular function and disease. The hemody-namic response of �1-AR is multifactorial, but in partmay depend on elements of the intracellular signalingcascade, especially heterotrimeric G protein consist-ing of �, � and � subunits.

������������� �� ����� ����� ��� ������ 123

������������� �� ����

����� ��� ������

��� � �������

��������� � ����

�� �������� �� �� �! "�#���

��#��� $" %�!� �� �"���"��

The �3-subunit of G protein is encoded by theGNB3 gene, which is located on chromosome 12p13.In 1998, a GNB3 825T>C polymorphism (thymidineto cytosine exchange) was discovered in exon 10 ofGNB3 [14]. This polymorphism does not affect theamino acid sequence. However, the T allele is in al-most complete linkage disequilibrium with a series ofother polymorphisms within the GNB3 gene, the so-called ‘T-haplotype’, which is associated with dele-tion of nucleotides 498–620 of exon 9, due to alterna-tive splicing. It has been shown that this splice variantis functional and is postulated to be associated withenhanced signaling activity [6]. The GNB3 825T al-lele was observed more frequently in patients with es-sential hypertension, obesity, left ventricular hyper-trophy and in subjects with lower renin plasma activ-ity [7, 14]. However, in contrast to initial studies itwas also reported that the polymorphism was not as-sociated with hypertension in the large LURIC study[13]. Dobutamine is an inotropic synthetic catechola-mine commonly used as a diagnostic tool for dobuta-mine stress echocardiography (DSE). Due to dobuta-mine positive inotropic and chronotropic effects DSEhas become a popular method for evaluation of coro-nary artery disease in patients unable to undergo stan-dard exercise stress tests. However, clinical observa-tions demonstrate interpersonal variations in responseto dobutamine as for heart rate (with some patients re-quiring atropine administration due to inability tocomplete the test and achieve target HR) and bloodpressure response [1, 3]. Polymorphisms within genesinvolved in regulation of the heart function mightshed light on the observed diversity in heart responseto dobutamine. One from potential candidates is theGNB3 gene.

The aim of this study was to determine an associa-tion between GNB3 gene polymorphism and heartrate (HR), systolic blood pressure (SBP) and diastolicblood pressure (DBP) response to dobutamine duringDSE.

Materials and Methods

Subjects

One hundred nineteen subjects of Caucasian origin(65 women and 54 men, aged 31–80 years) with clini-

cal indication for DSE were enrolled in the study.DSE was indicated for diagnosis of coronary arterydisease in subjects who were unable to undergo stan-dard exercise stress testing. Detailed characteristics ofpatients recruited for the study is presented in Table 1.The study protocol was approved by local ethics com-mittee (Pomeranian Medical University, Szczecin,Poland), and all study participants gave informedwritten consent.

Clinical assay

Dobutamine stress echocardiography was performedaccording to standard guidelines using escalatingadministration of dobutamine (from 10, 20 and30 μg/kg/min with a 3-min interval) [9]. HR (basedon 12-lead electrocardiogram) and blood pressurewere measured at baseline (after a rest period), at thestart of the dobutamine infusion period and at the endof each 3-min. Patients with atropine requirements,i.e., in subjects who were not able to reach target HRat the third dose of dobutamine (30 μg/kg/min) aswell as subject who did not complete the DSE proto-col were excluded from the study (n = 75). Each sub-ject discontinued �-blockers and/or calcium channelblockers five half-lives of the drug before the studyonset.

124 ������������� �� ����� ����� ��� ������

Tab. 1. Patients’ characteristics (% or the mean ± SD)

Parameter n = 119

Age (years) (mean ± SD) 59 ± 8

Sex

females

males

65 (54.6%)

54 (45.3%)

BMI (kg/m2) 27.8 ± 3.8

Smoking 19 (15.9%)

Hypertension

SBP

DBP

78 (65.5%)

133 ± 16

83 ± 9

Diabetes 15 (12.6%)

Medication

1. ACE

2. ASA

3. �-blockers

4. Ca2+-blockers

5. Statins

6. Diuretics

55 (46.2%)

72 (60.5%)

68 (57.1%)

23 (19.3%)

68 (57.1%)

28 (23.5%)

Genotyping

Genomic DNA was extracted from samples of periph-eral blood leukocytes according to the standard protocol.Genotyping of GNB3 825C>T (rs5443) polymorphism,based on PCR-RFLP method with BseDI restriction en-zyme was performed as described previously [12].

Statistical analysis

Based on allele frequencies, it was expected that thenumber of GNB3 825TT homozygotes would be low.As such, it was planned a priori that GNB3 825TT ho-mozygotes would be combined with heterozygotes.Categorical baseline characteristics were comparedbetween groups using the �� test (Statistica 8.0, Stat-soft Software, Warszawa, Poland). Numerical vari-ables between groups were evaluated by U Mann-Whitney test and ANOVA. A p level of less than 0.05was considered statistically significant.

Results

Genotype distributions fitted Hardy-Weinberg equilib-rium. The frequency distribution of GNB3 825C>Tpolymorphism was as follows: 53.8% for CC genotype,41.2% for CT and 5.0% for TT genotype. The T allelefrequency was estimated to 25.6%, while the C allelefrequency was 74.4%. Homozygous GNB3 825TT sub-jects were pooled with CT subjects, and then comparedto CC due to a limited number of TT homozygotes.

Resting hemodynamics

Significantly lower resting SBP and resting DBP incarriers of GNB3 825CC genotype when comparedwith subjects bearing 825T allele was observed. HRwas comparable in both subsets of patients.

HR response during DSE

HR during DSE gradually increased in both sub-groups, but more prominent in GNB3 825CT+TT sub-jects, yielding significant difference in comparisonwith GNB3 825CC carriers with dobutamine infusionat 30 μg/kg/min. During the whole time of observa-tion HR in subjects characterized by GNB3 825CCwas significantly different from GNB3 825 CT/TTgenotype carriers. Acceleration of heart rate was slower,although not significant, in carriers of GNB3 825CT/TT genotype (Tab. 2, Fig. 1).

SBP response during DSE

Resting SBP was markedly lower in GNB3 wild type825CC homozygotes compared to 825T allele carri-ers, and throughout almost whole DSE testing time.Only at the third stage for dobutamine dose of 30 μg/kg/min the difference in response was not significant.For the observation time SBP in GNB3 825 CT+TTpatients was significantly higher in comparison withGNB3 825CC subjects (Tab. 2, Fig. 1).

DBP response during DSE

Resting DBP was significantly lower in GNB3 wildtype 825CC homozygotes compared to 825T allele

������������� �� ����� ����� ��� ������ 125

GNB3 polymorphism in DSE���� ����� �� ��

Tab. 2. Systolic blood pressure (SBP, mmHg) and its increase (�SBP) at consecutive stages of DSE (the mean ± SD)

Dobutamine(μg/kg/min)

SBP DBP HR

CCn = 64

CT + TTn = 55

p CCn = 64

CT + TTn = 55

p CCn = 64

CT + TTn = 55

p

0 (resting) 130n ± 15 137 ± 15 0.023 80 ± 9 86 ± 9 0.001 68 ± 10 71 ± 10 0.208

10 135n ± 15 142 ± 15 0.018 85 ± 8 87 ± 14 0.107 73 ± 10 76 ± 9 0.169

20 143n ± 20 150 ± 19 0.036 83 ± 8 84 ± 10 0.224 89 ± 18 94 ± 18 0.078

30 147n ± 21 154 ± 22 0.054 79 ± 9 82 ± 9 0.111 109 ± 20 116 ± 18 0.047

�0–30 16n ± 17 17 ± 20 0.989 –1 ± 8 –3 ± 7 0.250 41 ± 16 45 ± 17 0.212

U Mann-Whitney test

126 ������������� �� ����� ����� ��� ������

Fig. 1. HR, SPB and DBP changesduring DSE (calculated by means ofrepeated measurements ANOVA)

carriers, similarly to SBP. At other DSE time pointsDBP was comparable in CC and TT genotype carri-ers. DBP, similarly to SBP, was elevated in GNB3 Tallele carriers in comparison with GNB3 825CC pa-tients during the study period (Tab. 2, Fig. 1).

Discussion

The study was dedicated to define the role ofGNB3 825C>T polymorphism in interindividual vari-ability to dobutamine response during DSE (amongpatients with clinical indication for the test). Availabledata pointed out an association between GNB3 poly-morphisms and HR and blood pressure in restingstate. The baseline HR and blood pressure data fromthe present study suggest an association betweenGNB3 825C>T polymorphism and blood pressure.It was revealed that carriers of GNB3 825T allele(CT and TT genotypes) were characterized by signifi-cantly elevated levels of both systolic and diastolicblood pressure. This observation is in keeping withother clinical studies on GNB3 polymorphisms, e.g.,subjected to meta analysis by Bagos at al., who docu-mented significantly higher blood pressure in subjectswith GNB3 825T allele [2].

The present study demonstrate significant differ-ences in HR at the late stage of DSE and its magni-tude of response during the test between carriers ofGNB3 825T allele and 825CC genotype. HR of825CC vs. CT+TT cases was slower along the test pe-riod reaching marked difference at dobutamine level30 μg/kg/min. The association of GNB3 825 T allelecarriers with HR was reported also by Dörr et al. inpatients subjected to a standard, symptom-limited ex-ercise test, however, significant only in subjects medi-cated with �-blockers [4]. Such an association was notobserved in �-blocker-free cases. Other polymor-phism of G protein, namely encoding the � subunit ofG� (GNAS1 393T>C) was evaluated by Mao et al. [10]during dobutamine stress test in healthy volunteersand Nieminen et al. [11] during exercise test. Mao etal. evaluated magnitude of HR response in volunteerswithout and with atropine. The study results revealedno significant association between the studied geno-type and magnitude of HR response during DSE, andin resting state. Nieminen et al. [11] reported an im-

pact of GNAS1 393T>C polymorphism on HR re-sponse curve.

Blood pressure levels observed in the current studypointed out higher values of both SBP and DBP in pa-tients characterized by GNB3 825T allele, with sig-nificant differences for SBP. Magnitude of blood pres-sure response did not discriminate examined group ofsubjects, i.e., GNB3 825CC and 825CT+TT carriers.There is some discrepancy concerning association be-tween GNB3 825C>T polymorphism and hyperten-sion. However, one of the recently published metaanalysis of 34 studies has associated GNB3 825Tallele with hypertension [2], which is in keeping withour findings, i.e., resting SBP and DBP were signifi-cantly higher in GNB3 825T allele carriers vs. 825CCsubjects. Dörr et al. [4] documented a lack of associa-tion between GNB3 825C>T genotypes and restingand peak SBP or DBP in patients subjected to stresstest. Effects of the � subunit of G� (GNAS1 393T>C)polymorphism were also studied by Mao et al., whorevealed an influence of GNAS1 393T>C polymor-phism on systolic blood pressure response in healthyvolunteers exposed to dobutemine during DSE bothwithout atropine and under atropine medication [10].Diastolic blood pressure response was similar in allanalyzed genotypes of the polymorphism. Contrarydata were reported by Nieminen et al., who did not re-veal an association between GNAS1 393T>C poly-morphism and blood pressure response in Caucasianpatients during standard exercise test [11]. The ob-served differences could be in part explained by eth-nic variability as Mao study recruited Chinese volun-teers.

The GNB3 825C>T variant allele frequency amongPolish subjects in the current study (25.6%) is lowercompared to the frequency reported in some otherCaucasian populations (i.e., 37.3% according to Hap-Map Project). However, T allele frequency among allpatients genotyped in course of the current study(n = 194), including patients non-compliant with thestudy criteria was slightly higher (27.1%), and similarto frequencies reported by other Polish investigators –28% among 172 healthy controls from eastern Poland[5], 32.7% among consecutive diabetes mellitus pa-tients form southern part of the country [15] and 35%among 217 syncopal patients from central part of thecountry [8].

In summary, the GNB3 825C>T polymorphism isassociated with resting blood pressure, both systolicand diastolic, in Polish Caucasian patients subjected

������������� �� ����� ����� ��� ������ 127

GNB3 polymorphism in DSE���� ����� �� ��

for diagnostic dobutamine stress echocardiography.The polymorphism also modulate heart rate, systolicand diastolic blood pressure response during dobuta-mine stress echocardiography. Further prospectivestudies are needed to confirm these associations andto explore their potential clinical relevance.

References:

1. Aquilante CL, Humma LM, Yarandi HN, Andrisin TE,Lewis JF, Hamilton KK, Johnson JA: Influence of gen-der and race on hemodynamic response to dobutamineduring dobutamine stress echocardiography. Am J Car-diol, 2004, 94, 535–538.

2. Bagos PG, Elefsinioti AL, Nikolopoulos GK, Hamo-drakas SJ: The GNB3 C825T polymorphism and essen-tial hypertension: a meta-analysis of 34 studies including14,094 cases and 17,760 controls. J Hypertens, 2007, 25,487–500.

3. Daly AL, Linares OA, Smith MJ, Starling MR, SupianoMA: Dobutamine pharmacokinetics during dobutaminestress echocardiography. Am J Cardiol, 1997, 79,1381–1386.

4. Dörr M, Schmidt CO, Spielhagen T, Bornhorst A, Hent-schel K, Franz C, Empen K et al.: �-Blocker therapy andheart rate control during exercise testing in the generalpopulation: role of a common G-protein �-3 subunit vari-ant. Pharmacogenomics, 2010, 11, 1209–1221.

5. Dzida G, Golon-Siekierska P, PuŸniak A, Sobstyl J,Bi³an A, Mosiewicz J, Hanzlik J: G-protein �3 subunitgene C825T polymorphism is associated with arterial hy-pertension in Polish patients with type 2 diabetes melli-tus. Med Sci Monit, 2002, 8, CR597–602.

6. Hayakawa T, Takamura T, Abe T, Kaneko S: Associationof the C825T polymorphism of the G-protein �3 subunitgene with hypertension, obesity, hyperlipidemia, insulinresistance, diabetes, diabetic complications, and diabetictherapies among Japanese. Metabolism, 2007, 56, 44–48.

7. Kiani JG, Saeed M, Parvez SH, Frossard PM: Associa-tion of G-protein beta-3 subunit gene(GNB3) T825 allelewith type II diabetes. Neuro Endocrinol Lett, 2005, 26,87–88.

8. Lelonek M, Pietrucha T, Matyjaszczyk M, Goch JH:A novel approach to syncopal patients: associationanalysis of polymorphisms in G-protein genes and tiltoutcome. Europace, 2009, 11, 89–93.

9. Lewandowski TJ, Armstrong WF, Bach DS.: Reducedtest time by early identification of patients requiring at-ropine during dobutamine stress echocardiography. J AmSoc Echocardiogr, 1998, 11, 236–242.

10. Mao YM, Liu ZQ, Chen BL, Guo D, Han CT, Yang LJ,Wang SY et al.: Effect of 393T>C polymorphism ofGNAS1 gene on dobutamine response in Chinesehealthy subjects. J Clin Pharmacol, 2009, 49, 929–936.

11. Nieminen T, Lehtimäki T, Laiho J, Rontu R, Niemelä K,Kööbi T, Lehtinen R et al.: Effects of polymorphisms in��-adrenoceptor and á-subunit of G protein on heart rateand blood pressure during exercise test. The Finnish Car-diovascular Study. J Appl Physiol, 2006, 100, 507–511.

12. Poch E, Giner V, González-Núñez D, Coll E, Oriola J,de la Sierra A: Association of the G protein �3 subunit Tallele with insulin resistance in essential hypertension.Clin Exp Hypertens, 2002, 24, 345–353.

13. Renner W, Hoffmann MM, Grünbacher G, WinkelmannBR, Boehm BO, März W: G-protein �3 subunit (GNB3)gene polymorphisms and cardiovascular disease: theLudwigshafen Risk and Cardiovascular Health (LURIC)study. Atherosclerosis, 2007, 192, 108–112.

14. Siffert W, Rosskopf D, Siffert G, Busch S, Moritz A,Erbel R, Sharma AM et al.: Association of a humanG-protein �3 subunit variant with hypertension. NatGenet, 1998, 18, 45–48.

15. Zychma MJ, Zukowska-Szczechowska E, Ossowska-Szymkowicz I, Trautsolt W, Grzeszczak W: G-Protein ��

subunit C825T variant, nephropathy and hypertension inpatients with type 2 (Non-insulin-dependent) diabetesmellitus. Am J Nephrol, 2000, 20, 305–310.

Received: April 16, 2011; in the revised form: September 13,

2011; accepted: September 30, 2011.

128 ������������� �� ����� ����� ��� ������