The 825C/T Polymorphism of the G-Protein Subunit β3 Is Not Related to Hypertension
Abstract—A polymorphism at position 825 (C→T) of the cDNA that encodes the β3 subunit (GNB3) of the pertussis toxin–sensitive G protein was recently shown to be associated with human hypertension. To verify this finding and to investigate whether this polymorphism could also be associated with coronary heart disease, we analyzed the GNB3 variant in subjects from 2 previously described studies: Projet d’Etude des Gènes de l’hypertension Artérielle Sévère à modérée Essentielle (PEGASE), a case-control study of moderate to severe hypertension (681 cases and 308 controls), and Etude Cas-Témoins de l’Infarctus du Myocarde (ECTIM), a case-control study of myocardial infarction (MI) (564 cases and 633 controls). Genotyping was performed with allele-specific oligonucleotides. Genotype and allele frequencies were in Hardy-Weinberg equilibrium in all groups. Allele and genotype frequencies did not differ significantly between case patients with essential hypertension or MI and control subjects. In the ECTIM study, the 825T allele frequencies in cases and controls from Belfast, Northern Ireland, were 0.31 and 0.30 (P=0.79), respectively; the corresponding frequencies in cases and controls from France were 0.33 and 0.31 (P=0.30), respectively. In the PEGASE study, the 825T allele frequency was 0.35 in female and male cases and 0.31 in male normotensive controls (P=0.12). The odds ratios for hypertension (PEGASE) and MI (ECTIM) associated with T-allele carrying were 1.23 (95% confidence interval, 0.94 to 1.62; P=0.13) and 1.11 (95% confidence interval, 0.88 to 1.39; P=0.37), respectively. There was no association of the GNB3 polymorphism with early onset of hypertension, familial history of hypertension, or blood pressure level. We conclude that the 825C/T polymorphism of the GNB3 gene did not contribute in any important way to the risk of essential hypertension or MI in these studies.
- hypertension, essential
- myocardial infarction
- coronary artery stenosis
- body mass index
- C825T polymorphism
Genes that encode the components of the various transport systems that regulate salt and water homeostasis are obvious candidates for influencing blood pressure regulation and hypertension. The ubiquitously expressed pH-regulating ion transport system, Na+/H+ exchanger (NHE), swaps extracellular Na+ for intracellular H+. Five isoforms have been isolated in human tissues. Different investigators have demonstrated enhanced NHE isoform-1 (NHE-1) activity in several blood cell lines of patients with essential hypertension compared with normotensive control subjects,1 2 and transgenic mice that overexpress a recombinant NHE protein developed salt-sensitive hypertension.3 Siffert et al4 have demonstrated that immortalized B lymphoblasts of patients with essential hypertension, which display enhanced NHE-1 activity, also showed increased activity of pertussis toxin–sensitive G proteins. Variations in NHE-1 transcripts could not be detected,5 and polymorphisms in genes that encode the subunits of trimeric G proteins, such as Gαi2, Gαi3, Gβ1, and Gβ2,6 have been ruled out. Subsequently, Siffert et al7 were able to localize a polymorphism at position 825 (C→T) of the cDNA that encodes the β3 subunit (GNB3) of the pertussis toxin–sensitive G protein and demonstrated that the 825T allele was significantly associated with hypertension. However, given the expected small effect of susceptibility genes involved in human essential hypertension, a large number of patients must be studied before it can be definitively established that a genotype-phenotype association exists. Therefore, we investigated the 825C/T polymorphism of GNB3 in relation to hypertension and extended our study to patients with coronary heart disease (CHD).
The ECTIM Study
Etude Cas-Témoins de l’Infarctus du Myocarde (ECTIM) is a study of 564 patients with myocardial infarction (MI) and 633 control subjects representative of geographic areas in Northern Ireland and France. Details of the study are provided elsewhere.8 9 Men aged 25 to 64 years were recruited between 1988 and 1991 from regions covered by registers for the World Health Organization Monitoring Trends and Determinants in Cardiovascular Disease (MONICA) project. In control subjects, hypertension was defined as a diastolic blood pressure (DBP) ≥95 mm Hg or current hypertensive treatment. Normotension was defined as a DBP <95 mm Hg, absence of antihypertensive treatment, and absence of personal history of CHD. Informed consent was obtained from subjects and their physicians.
The PEGASE Study
Hypertensive subjects (n=681) were recruited for the Projet d’Etude des Gènes de l’hypertension Artérielle Sévère à modérée Essentielle (PEGASE) study in 15 regions of France by 139 general practitioners who belong to the EURAXI network as previously described.10 The study was approved by a review committee, and each participant provided written informed consent. Recruitment started in October 1994 and lasted for 1 year. Hypertensive patients from the PEGASE study were compared with French normotensive subjects from the ECTIM study (n=308).
Genomic DNA was prepared from white blood cells by phenol extraction. From the published sequence of the GNB3 gene,11 a 268-bp fragment that contains the polymorphic site was amplified with the use of 2 primers as previously described.7 Each amplification was performed with 250 ng of DNA in a total volume of 50 μL containing 10 mmol/L Tris-HCl (pH 9), 50 mmol/L KCl, 2.5 mmol/L MgCl2, 0.1% Triton X-100, 0.2 mg/mL BSA, 200 μmol/L dNTP, 25 pmol/L of each primer, and 0.2 units of Taq polymerase as follows: 94°C for 5 minutes to denature, followed by 94°C for 30 seconds, 63°C for 45 seconds, and 72°C for 45 seconds for 35 cycles, and 72°C for 10 minutes.
Genotyping of all subjects participating in the ECTIM and PEGASE studies was performed with allele-specific oligonucleotides (ASOs)12 as previously described.13 For ASOs, we used the following oligonucleotides: 5′-ATC ACG TCTGTG CCT TC-3′ and 5′-ATC ACG TCC GTG CCT TC-3′ to detect the TCT (Ser) and TCC (Ser) codons, respectively. After enzymatic amplification, one-fifth of the polymerase chain reaction (PCR) product was denatured in 150 μL of 0.5 mmol/L NaOH and 1.5 mmol/L NaCl and blotted onto nylon membranes (N+). Each allele was detected after preincubation of the membranes for 2 hours with 100 pmol of unlabeled oligonucleotide probe specific for the other allele, then it was incubated for 2 hours with 100 pmol of the labeled probe specific for the allele. The melting temperature used for hybridization was calculated by adding 4°C for each C or G and 2°C for each A or T and subtracting 5°C from the total. The membranes were washed twice at room temperature in 1× SSC for 5 minutes, followed by 5 minutes in 0.5× SSC at the melting temperature −3°C.
Data were analyzed with SAS statistical software. To simplify presentation of the data, for the ECTIM Study, the 3 French centers were considered together after we verified that the results were homogeneous across centers. Hardy-Weinberg equilibrium was tested by a χ2 test with 1 df. Genotype and allele frequencies were compared between groups by a χ2 test.
GNB3 825C/T Polymorphism and MI
The ECTIM Study
GNB3 825 genotypes were available for 564 case patients with MI and 633 control subjects. Mean age of cases and controls was 54.0±8.1 and 53.2±8.4 years, respectively. Genotype frequencies were in Hardy-Weinberg equilibrium in cases with MI and controls and in Northern Ireland and France. The distribution of allele and genotype frequencies did not differ significantly between cases and controls or between countries (Table 1⇓). The 825T allele frequencies in cases and controls from Belfast were 0.31 and 0.30 (P=0.79), respectively; the corresponding frequencies in cases and controls from France were 0.33 and 0.31 (P=0.30), respectively. The 825C/T polymorphism was not associated with blood pressure, body mass index (BMI), or degree of coronary artery stenosis. Among controls, the frequency of 825T allele carriers did not differ between hypertensive (n=206; mean age, 57.3±6.2 years) and normotensive subjects (n=467; mean age, 51.3±8.9 years) (P=0.9).
GNB3 825C/T Polymorphism and Hypertension
The PEGASE Study
GNB3 825 genotypes were available for 681 case patients with essential hypertension (394 males and 287 females). Mean age of hypertensive subjects was 48.0±8.9 years. Genotype frequencies were in Hardy-Weinberg equilibrium. The distribution of allele and genotype frequencies did not differ significantly between male and female hypertensives (Table 2⇓). Allele and genotype frequencies were also similar in hypertensive patients from the PEGASE study and normotensive control subjects from the ECTIM study (n=308; mean age, 50.8±9.0 years) (Table 2⇓). The 825T allele frequency was 0.35 in female and male cases and 0.31 in male normotensive controls (P=0.12). The 825C/T polymorphism was not related to age, systolic blood pressure (SBP), DBP, or BMI in hypertensive subjects (Table 3⇓).
In the PEGASE and ECTIM studies, the GNB3 825C/T polymorphism was not associated with hypertension or MI. Moreover, there was no association between this polymorphism and early onset of hypertension, familial history of hypertension, blood pressure, or BMI. Siffert et al7 recently demonstrated a significant association of the 825T allele with essential hypertension in a study of 426 hypertensive and 427 normotensive control subjects; the T-allele frequency was higher in hypertensive (0.31) than in normotensive subjects (0.25), and the odds ratio for hypertension in the presence of the 825T allele (CT + TT versus CC) was 1.44 (95% confidence interval, 1.09 to 1.88; P=0.025). The hypertensive subjects investigated by Siffert et al (56.6±13.7 years) were older than those in the PEGASE study (48.0±8.9 years). According to previous reports that suggest that genetic factors have a stronger effect at younger ages and in more severe forms of hypertension,14 participants in the PEGASE study were recruited on the basis of both a strong elevation of DBP (moderate or severe hypertension) and a relatively young age of onset of hypertension.10 Moreover, as reported before, patients in the PEGASE study had a high prevalence of parental history of CHD (30.4%) and stroke (20.2%). Our negative results, however, cannot rule out that the 825C/T polymorphism might be specifically related to hypertension in a subgroup of hypertensives with salt-sensitive hypertension. This will have to be checked in specifically designed studies.
The method used for genotyping in the original study (restriction enzyme digestion) and the ASO technique used for the ECTIM and PEGASE studies are different. Genotyping with ASO is highly reliable, and Studencki et al15 have demonstrated that for β-globin gene mutations, the ASO method is superior to classic restriction fragment length polymorphism analysis and direct restriction enzyme digestion.
In conclusion, our results suggest that it is unlikely that the GNB3 825C/T polymorphism contributes in any important way to the risk of essential hypertension or MI. Conversely, because increased NHE-1 activity is also associated with left ventricular hypertrophy and increased susceptibility to nephropathy in insulin-dependent diabetes,16 17 it might be of interest to investigate the 825C/T polymorphism in relation to these phenotypes.
This work was supported by grants from Squibb Laboratory, British Heart Foundation, Institut National de la Santé et de la Recherche Médicale, and Institut Pasteur–Lille. Eva Brand is supported by a grant from Deutsche Forschungsgemeinschaft (Br 1592/1-1). Stefan-Martin Herrmann is supported by a grant from Deutsche Forschungsgemeinschaft (HE 2852/1-1). Recruitment of patients in the PEGASE study was supported by Parke-Davis France. We thank the practitioners of the EURAXI network for their collaboration and Christiane Souriau for DNA extractions.
Drs Eva Brand and Stefan-Martin Herrmann contributed equally to this work.
- Received August 24, 1998.
- Revision received October 13, 1998.
- Accepted January 18, 1999.
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