(Hypertension. 1999;34:779-781.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
A-6G Variant of Angiotensinogen Gene and Aldosterone Levels in Hypertensives
From the Department of Endocrinology, Internal Medicine, and the Associated Unit of Clinical Laboratories, Faculty of Medicine, Catholic University of Chile, Santiago.
Correspondence to Carlos E. Fardella, Department of Endocrinology, Faculty of Medicine, Universidad Católica de Chile, Marcoleta 391, Santiago, Chile.
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Abstract |
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Abstract—Recently, a novel mutation in the promoter region of the angiotensinogen gene that involves the presence of an adenine instead of a guanine 6 bp upstream from the transcription initiation site (A-6G) has been shown to induce an increase in gene transcription. The aim of this study was to determine the prevalence of the A-6G mutation in essential hypertensive patients and to correlate it with aldosterone and renin activity levels. We studied 191 hypertensives. We measured levels of aldosterone (plasma and urinary) and plasma renin activity. We determined the variants A and G using a mutagenically separated polymerase chain reaction technique. In 191 hypertensives, the A variant was detected in 266 of 382 (69.6%) and the G variant in 116 of 382 alleles (30.4%). Plasma aldosterone was significantly higher in patients homozygous for AA than in those homozygous for GG (369±208 versus 246±142 pmol/L). Urinary aldosterone was significantly higher in homozygous AA than in AG or GG patients (62.4±39.4 versus 50.8±25.2 and 37.4±22.3 nmol/d, respectively). When the patients were grouped according to the presence or absence of the A allele, the aldosterone levels and the plasma aldosterone/plasma renin activity ratio were significantly higher in patients with the A allele. The presence of the A variant was associated with higher levels of aldosterone. These results suggest that the presence of the A variant could determine the appearance of arterial hypertension through higher transcription activity of the angiotensinogen gene and concomitant aldosterone production.
Key Words: angiotensinogen • aldosterone • hypertension • genetics • renin
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Introduction |
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The angiotensinogen gene isolated by Gaillard et al1 and Fukamizu et al2 has been implicated in essential hypertension through both genetic linkage and allelic association.3 4 The involvement of the angiotensinogen gene was first suggested by Jeunemaitre et al,5 who established that there is a genetic linkage to essential hypertension in affected sibships, a greater prevalence of T235 variant among hypertensives than in control subjects, and higher plasma concentrations of angiotensinogen in subjects carrying T235. The T235 variant corresponds to the presence of a threonine instead of a methionine at residue 235 of the angiotensinogen gene.
Recently, we demonstrated in a Chilean population a high prevalence of the T235 variant, which was more common in patients with low-renin hypertension.6 However, it is not yet clear whether the T235 allele directly accounts for the physiological effects or just acts as a marker for other causative mutations. More recently, a common variant in the promoter region of the angiotensinogen gene has been identified that is in complete linkage disequilibrium with the T235 variant. The promoter variant corresponds to the presence of an adenine, instead of a guanine, 6 bp upstream from the initiation site of transcription (A-6G).7
In vitro experiments have already demonstrated that the guanine-to-adenine substitution at position -6 affects the basal transcription rate of the angiotensinogen gene.7 In vivo, the effect of this variant has not been evaluated. The aim of this study was to determine the prevalence of the A-6G variant in a Chilean population of essential hypertensive (EH) patients and establish its correlation with plasma aldosterone (PA) and plasma renin activity (PRA) levels. These data could provide useful information about the mechanisms involved in blood pressure regulation in patients carrying the A-6 variant.
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Methods |
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Subjects
Patients were recruited from the Outpatient Clinic Hypertension Program at the Universidad Católica de Chile in Santiago. Subjects were considered hypertensive if their diastolic blood pressure was >90 mm Hg and the systolic blood pressure was >140 mm Hg on
3 occasions on different days and
without any medication.8 The first and fifth Korotkoff
sounds were used to designate systolic and
diastolic blood pressure, respectively. The subjects rested
seated for 10 minutes before the blood pressure was measured with a
mercury sphygmomanometer. The observer was always a health
professional. All individuals with clinical evidence of secondary
hypertension, diabetes, or renal or hepatic disease were excluded.
Under these criteria, we selected 191 subjects with essential
hypertension.
The EH patients were admitted to our Metabolic Ward between
8 and 9 AM after a 12-hour fast. All subjects were allowed
free sodium intake. Weight and height were measured at the time of
admission. On admission, a catheter was placed in an antecubital vein,
and the subjects remained recumbent for 
1 hour. After 1 hour of
supine posture, free-flowing blood was withdrawn to measure sodium,
potassium, calcium, albumin, blood urea nitrogen,
creatinine, 24-hour urinary sodium and potassium, PA and
urinary aldosterone (UA), and PRA. The serum
aldosterone and UA were measured by radioimmunoassay with
antiserum from Diagnostic Products Corp. The PRA was
determined as previously described by Menard and Catt,9
and the normal PRA value in our population is 2.5±0.75 µg ·
L-1 ·
h-1.10 A blood sample was also
obtained to analyze the genomic DNA.
The Chilean population originated through a biracial mixture with white genes coming from the Spanish conquerors and a gene pool derived from the native Amerindians (Mapuches).11 The Amerindian admixture in the population studied was estimated by ABO blood group distribution, assuming a hybrid population of biparenteral origin.12 13 14 Informed consent was obtained from all participants according to the guidelines of the Declaration of Helsinki, and the protocol was approved by the Research Commission of the School of Medicine at Catholic University of Chile.
Analysis of Genomic DNA
Genomic DNA was prepared from the citrated blood of 191 EH
patients as previously described.15 We determined the
presence of A-6 or G-6
allele variants in all 191 EH patients using a mutagenically
separated polymerase chain reaction (MS-PCR) technique.16
The genomic DNA was amplified with 50 nmol of the following primers:
5'-GTGTCGCTTCTGGCATCTGTCCTTCTGG-3' (sense),
5'-TACCCAGAACAACGGCAGCTTCCACT-3' (short antisense), and
5'-CCGGTTACCTTCTCGAGAGCCCAGAACG- GCAGCT TCCACC-3', in a total volume
of 50 µL of reactant containing 50 mmol/L KCl, 2.5 mmol/L
MgCl, 100 µmol/L of each triphosphate nucleotide,
10 mmol/L Tris, and 1.5 U Taq polymerase. The
conditions for amplification were as follows: denaturation at 95°C
for 4 minutes, followed by 36 cycles of 1 minute at 94°C, 1 minute at
68°C, and 1 minute at 72°C. The G-6 variant
was identified when the MS-PCR gave a 190-bp product. Conversely,
if the A-6 variant was present, the PCR
product was only 170 bp long. The products of the MS-PCR were
seen on 7.5% polyacrylamide gel stained with ethidium
bromide.
Statistical Analysis
Values are expressed as mean±SD. Means were compared by
Student's t test for independent samples. One-way ANOVA was
used to compare differences for a given parameter between
the 3 genotypes studied. When differences were found, a Tukey
test was used to compare the 3 groups in a paired system. Statistical
significance was assumed if the computed 2-tailed probability value was
<5% (P<0.05).
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Results |
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Subjects
We studied 191 EH patients without evidence of renal or hepatic failure or other secondary causes of hypertension. The clinical and laboratory findings are shown in Table 1. The Amerindian admixture index in the populations studied, estimated by ABO blood group distribution, was hypertensives=0.38 (38% of aboriginal admixture).
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MS-PCR Amplification
The results showed that the A-6 variant was
present in 266 of 382 alleles (69.6%) and the G variant in 116
of 382 alleles (30.4%). Table 2
shows our results in comparison with those found in other populations
with different genetic backgrounds. The genotype distribution
was the following: homozygous AA, 95 of 191 (49.7%); heterozygous AG,
76 of 191 (39.8%); and homozygous GG, 20 of 191 (10.5%).
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The PA levels were significantly higher in homozygous AA than GG, but no significant differences were found between heterozygous AG and homozygous AA or GG (Table 3). The UA levels were significantly higher in homozygous AA than in AG or GG patients, but no significant differences were found between AG and GG patients (Table 3). The PRA, PA/PRA ratio, urinary sodium, and urinary potassium values were not different in the 3 genotypes studied, as is shown in Table 3.
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When the EH patients were grouped according to the presence of A variant (homozygous AA+heterozygous AG) or absence of A variant (homozygous GG), the aldosterone levels were significantly higher in patients with the A variant in both plasma (353±197 versus 246±142 pmol/L; P<0.01) and urine (20.6±12.5 versus 13.5±9.1 nmol/d; P<0.01). The PA/PRA ratio was also higher in patients with the A variant than in patients without the A variant (14.7±16.3 versus 8.1±8.0; P<0.01). The PRA, urinary sodium, and urinary potassium levels were not different in patients with the A variant from those in patients without the A variant.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This study showed a high frequency of the A-6 variant of the angiotensinogen gene in the sample of the Chilean population that we analyzed. More than 69% of hypertensive subjects had the A-6 variant, a frequency higher than those reported in whites but similar to that found in Japanese populations, suggesting that the A-6G variant shows a significant ethnic difference.7 17
The presence of the A variant was associated with high levels of aldosterone and PA/PRA ratio. In vitro evidence exists that an A for G substitution 6 bp upstream from the transcription site in the promoter region of the angiotensinogen gene affects the binding of a nuclear protein, resulting in increased gene transcription between 20% and 70%.7 In vivo, the physiological consequences of this change are unknown, but it is possible to speculate that the increase in the gene transcription might be related to an activation of the renin-angiotensin system. The activation of the system could determine an increase in angiotensin II and aldosterone levels that secondarily could increase the blood pressure. This hypothesis could be supported by our data, which showed the highest levels of aldosterone in patients with the A-6 variant.
The highest levels of aldosterone, found in homozygous AA rather than GG subjects, may explain why patients with genotypes AA and AG respond more favorably to salt reduction.18 Moreover, this finding may also explain a previous communication from our group that found a high incidence of low-renin hypertension in patients homozygous for T235.6 In the same study, we could not establish a significant association between T235 and the highest levels of aldosterone, probably because only a small number of hypertensives were studied. The T235 variant had been demonstrated to be in complete linkage disequilibrium with the A-6G polymorphism,7 and then the A-6 variant could represent the causative mutation explaining the finding described in association with the T235 variant.
The high frequency of the A-6 variant found in our population was similar to that described in the Japanese population, which may be explained by the Asian ancestry of our South Amerindian population. Similarities in the genotype distributions between Amerindian and Asian populations have also been described recently in Chilean patients with 21-hydroxylase deficiency,19 as well as in other studies.20 21
In summary, in this study we found a high prevalence of the A-6 variant in EH patients from a Chilean Hispanic population. The A-6 variant was associated with higher levels of aldosterone. These results suggest that the presence of the A-6 variant could determine the appearance of arterial hypertension through higher aldosterone production.
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Acknowledgments |
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This work was supported by grant 1980999 from the Fondo Nacional de Desarrollo Científico y Tecnológico (Chile), FONDECYT.
Received May 20, 1999; first decision July 16, 1999; accepted August 2, 1999.
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