(Hypertension. 1997;29:1078-1082.)
© 1997 American Heart Association, Inc.
Articles |
Association of the Angiotensinogen Gene to Serum Angiotensinogen in Blacks and Whites
From the Departments of Medicine and Medical and Molecular Genetics, Indiana University School of Medicine, and the VA Hospital, Indianapolis; and the Marshfield (Wis) Medical Research Foundation (D.A.T.).
Correspondence to J. Howard Pratt, MD, Indiana University Medical Center, 541 Clinical Dr, Indianapolis, IN 46202-5111. E-mail howardp{at}medicine.dmed.iupui.edu
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Abstract |
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Abstract A variant of the angiotensinogen gene (AGT) that encodes for threonine at codon 235 (T235) has been associated with a higher serum angiotensinogen concentration and with hypertension in white subjects. The frequency of T235 is about two times higher in blacks than whites, suggesting that AGT may contribute to the susceptibility to hypertension in blacks more than it does in whites. However, an association of T235 with angiotensinogen level or blood pressure has not been observed in blacks, possibly because the high prevalence of T235 makes it insufficiently informative as a marker. For this reason, we undertook to further differentiate the T235 carrier state by constructing haplotypes with alleles in the 5' upstream region of AGT. One such haplotype, -1074t;T235, showed a significant association with angiotensinogen level in a cohort of black and white children and adolescents (76 blacks, mean age= 12.3±2.0 [SD] years; 139 whites, mean age=12.4±1.8 years). With a linear regression model, the level of serum angiotensinogen was significantly related to body mass index (P=.0017) and the haplotype (P=.0001). Within specific race groups, the haplotype was significantly related to serum angiotensinogen in both the blacks (P=.0277) and whites (P=.0001). The mean level of angiotensinogen was higher in the blacks carrying a single copy of the haplotype than in those without the haplotype (1472.2±68.4 versus 1274.9±46.7 nmol angiotensin I/L), a difference that was marginally significant (P=.0609). In the whites, the level of angiotensinogen was also higher in carriers of a single copy than in those with no copy (1527.9±71.2 versus 1099.2±20.1 nmol angiotensin I/L) (P=.0003). Serum angiotensinogen level did not increase with two copies of the haplotype, but in each racial group, there were only four individuals who were homozygous. The haplotype showed a marginally significant relation (P=.0757) to the mean of longitudinally determined diastolic pressures adjusted for body mass index, race, sex, and age. In summary, using a haplotype to differentiate further the T235 carrier state, we observed an association of genotype with serum angiotensinogen level and blood pressure in blacks and whites. The findings suggest that AGT may play an important role in blood pressure regulation in both racial groups.
Key Words: angiotensinogen • race • angiotensinogen genes • haplotypes • blood pressure
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Introduction |
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Angiotensin II (Ang II) is derived from angiotensinogen, a large protein synthesized in liver. The rate at which Ang II is synthesized is governed in part by the availability of angiotensinogen, whose concentration is nearly equal to the Km for the reaction of angiotensinogen with renin1 2 to form Ang I, the precursor to Ang II. Ang II regulates vascular resistance and sodium homeostasis, and thus it is important for determining BP. In studies of targeted gene duplication and disruption, angiotensinogen concentration and BP were proportional to the number of AGT genes.3 4 In human studies, BP was related to the level of serum angiotensinogen,1 5 6 perhaps because of the dependence of Ang II synthesis on angiotensinogen concentration. The AGT gene has been linked to human hypertension,7 8 9 and a variant that encodes for threonine in place of methionine at amino acid position 235 (T235) has been associated with a higher serum angiotensinogen level7 10 and with primary7 and pregnancy-induced11 12 hypertension. It is generally thought that a variation in the angiotensinogen molecule at position 235 does not itself influence the serum angiotensinogen concentration, but rather that T235 is a marker in linkage disequilibrium with the active locus.
The frequency of T235 is high in blacks—approximately 80% in American blacks versus approximately 40% in whites10 13 14 ; conceivably, AGT contributes to the susceptibility to hypertension in blacks more than it does in whites. Although AGT showed linkage to hypertension in a population of Caribbean blacks,9 T235 was not significantly related to either serum angiotensinogen level or BP in other studies in blacks.10 13 However, because of its high frequency, T235 may not be a sufficiently informative marker in blacks. Therefore, we undertook a study in which the T235 carrier state was further differentiated through construction of haplotypes that might serve as useful markers for identifying individuals with a locus that increases the level of serum angiotensinogen. Jeunemaitre et al (unpublished observations, 1997) have shown that such haplotypes are associated with hypertension in a population of white adults. Subjects for the present study were from a cohort of black and white normotensive children and adolescents who participate in a longitudinal study of BP regulation.15
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Methods |
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Subjects
Subjects were recruited from an ongoing longitudinal BP study in children.15 The study was approved by the Institutional Review Board of Indiana University–Purdue University of Indianapolis. Informed consent was obtained from each child and also from a parent or legal guardian. Children with a history of hypertension, renal disease, cardiac disease, or diabetes mellitus and those taking medication (including birth control pills) that could affect BP or the renin-angiotensin system were excluded from the study. Subjects provided a blood sample for isolation of DNA and plasma measurements of angiotensinogen. Their characteristics are presented in Table
1.
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Measurements
Weight and height were measured at the time blood samples were
drawn. In addition, because participants were from an ongoing
longitudinal study, their BPs measured during the previous 5 years
(measured twice annually) were used in the analyses. BP was
measured in the right arm with a random-zero sphygmomanometer (Hawksley
& Sons) while the subject was seated. The first and fifth Korotkoff
sounds were used to designate systolic and
diastolic BPs, respectively. Three BP readings were
obtained, and the average of the last two readings was used as the
final BP measurement.
Detection of AGT Variants
DNA was extracted from white blood cells by a standard
procedure.16 Each variant was characterized using
allele-specific oligonucleotide hybridization. In
brief, appropriate genomic fragments were amplified by polymerase chain
reaction. DNA products were denatured with 0.4N NaOH, dot-blotted
in duplicate onto Nytran (Schleicher & Schuell) membranes, and
neutralized with 10 mmol/L Tris-HCl (pH 7.4) and 1.0 mmol/L
EDTA (pH 8). The filters were subsequently hybridized to the
appropriate 32P-labeled oligonucleotide in
a solution containing 6x SSC, 5x Denhardt's, 0.5% sodium
dodecyl sulfate, and 0.1 mg/mL denatured salmon sperm DNA for
12 hours at 42°C and then washed in 2x SSC at the appropriate
temperature for each deoxyoligonucleotide. Two of the
variants examined, ACG
ATG at codon 235 and an AC at -20 of the
5' upstream region of AGT, have been described
previously.7 Two additional variants, GT at -1074 and
CT at -775, have been identified in the laboratory of J.-M. Lalouel
(personal communication).
Construction of Haplotypes
Two-allele haplotypes were constructed using the
T235 allele and sequentially each of the new
AGT variants. For individuals homozygous at either
T235 or the additional variant or at both loci, the
haplotype could be established unambiguously. Individuals heterozygous
at both loci could be haplotyped only if the parental genotypes
were available.
Angiotensinogen Assay
Angiotensinogen was measured with a two-step
procedure that consisted first of conversion of
angiotensinogen to Ang I by human renin followed by
measurement of Ang I by radioimmunoassay.17
Angiotensinogen concentration was expressed as
nanomoles Ang I per liter. The intra-assay coefficient of variation
for this assay was 5.8% (n=24), and the interassay coefficient of
variation was 12% (n=81). The mean recovery of Ang I (8 or 4 ng) added
to the assay of control plasma samples at the start of the incubation
was 92% (range, 86% to 101%; n=4).
Statistical Methods
Comparisons of systolic and diastolic BPs,
age, BMI, and serum angiotensinogen levels unadjusted for
other covariates were made by two-sample t tests. Weighted
least squares was used to model both angiotensinogen level
and BP. The response variable used was the average
angiotensinogen level or average BP, and the weights used
were the number of individual measurements that went into the average
measurement. The independent variables included the factor of
interest (number of copies of -1074t;T235 haplotype) as
well as age, sex, race, and BMI as covariates. An effect was deemed to
be statistically significant if the probability value was less than 5%
and marginally significant if it was less than 10%. Bonferroni's
method was used for adjustments for multiple comparisons among the
different numbers of haplotypes.
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Results |
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Subject Characteristics
Characteristics of black and white subjects are shown in Table 1
.
Although the mean ages were not significantly different in the blacks
and whites, BMI (P<.0001), longitudinal unadjusted
systolic (P=.0175) and diastolic
(P=.0006) BPs, and serum angiotensinogen
concentration (P<.0001) were all higher in the blacks than
whites.
Frequencies of Variant Alleles
The three variants appeared to be in complete linkage
disequilibrium with T235, as evidenced by their absence in
individuals homozygous for M235. Frequencies of the variants
in blacks and whites, respectively, were 0.20 and 0.12 for T-1074,
0.12 and 0.15 for C-20, and 0.27 and 0.06 for T-775.
Relationships to Serum Angiotensinogen
Each haplotype was analyzed by a linear model, with
angiotensinogen level as the dependent variable and
race, sex, age, BMI, and the number of copies of the haplotype (2
df) as independent variables. Significance levels and
parameter estimates for the variable tested are
presented in Table 2. Of the three haplotypes
examined, only -1074t;T235 showed a significant association
with serum angiotensinogen level. In the case of
-1074t;T235, BMI (P=.0017) and the haplotype
(P=.0001) showed a significant relationship with
angiotensinogen level (Table 2). When the analysis
was carried out within specific race groups using the identical model,
in the blacks, serum angiotensinogen was significantly
related to age (P=.0092), BMI (P=.0197), and
-1074t;T235 (P=.0277). In the whites, serum
angiotensinogen was significantly related to
-1074t;T235 (P=.0001). The proportion of the
variance in the serum angiotensinogen level
(R2) that could be explained by the model was
.31 (Table 2). The positive relationships of age and BMI to serum
angiotensinogen level in this cohort were noted
previously.10
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As shown in the Figure, the mean level of
angiotensinogen was significantly higher in whites carrying
a single copy of -1074t;T235 than in whites carrying no
copy (1527.9±71.2 versus 1099.2±20.1 nmol Ang I/L)
(P=.0003). In blacks, those carrying one copy of
-1074t;T235 had a higher level of
angiotensinogen than those with no copy (1472.2±68.4
versus 1274.9±46.7 nmol Ang I/L), a difference that was marginally
significant (P=.0609). Only four whites and four blacks were
homozygous for -1074t;T235, and in these groups, the
angiotensinogen levels were not significantly different
from levels of the other genotype groups.
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Relationships to Longitudinal BP
For each subject, the mean of multiple BPs measured every 6
months for 3 to 5 years was used in the analysis. A weighted
ANOVA was used with systolic or diastolic BP as the
dependent variable and race, sex, age, BMI, and
-1074t;T235 as the independent variables. The BP means
were weighted by the number of readings used to calculate the average
BP reading. As shown in Table 3, systolic BP was
significantly related to sex (P=.0009), age
(P=.0001), and BMI (P=.0001); the relation to
-1074t;T235 was not significant (P=.260). On the
other hand, diastolic BP was significantly related to age
(P=.0001) and BMI (P=.0001), and there was a
marginally significant relationship to -1074t;T235
(P=.0757). The proportion of the variance explained by the
model (R2) was .38 for systolic and .23
for diastolic BPs (Table 3).
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Discussion |
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In an earlier study of the present cohort, we found an association of T235 with angiotensinogen level in whites but not blacks.10 However, the high prevalence of T235 in blacks may have compromised an ability to detect an association if indeed one existed. In the present study, we showed that a haplotype that further differentiates the T235 carrier state was significantly associated with the serum level of angiotensinogen in blacks and whites. The findings suggest that AGT contributes to the variability in serum angiotensinogen level in both racial groups.
The current study used normotensive children and adolescents, some of whom will ultimately become hypertensive. The positive association of the AGT haplotype to serum angiotensinogen at this young age suggests that AGT may participate at an early stage in the development of hypertension. Indeed, -1074t;T235 did show a marginally significant association with diastolic BP. In this regard, Fasola et al5 showed that the angiotensinogen concentration was higher in offspring of hypertensive people—individuals with a greater chance of developing hypertension.
Hypertension is a formidable health problem in the black population. The prevalence of hypertension is much higher in blacks than whites,18 19 and complication rates, particularly for renal failure, are many times higher in blacks than whites.20 21 22 Substantial evidence suggests a role for AGT in the pathophysiology of hypertension in white and Asian populations,7 8 23 and recently, hypertension was shown to be genetically linked to AGT in a population of Caribbean blacks.9 T235 is two times more prevalent in blacks than whites,10 13 14 and in the present study, the -1074t;T235 haplotype was also more prevalent in blacks. AGT may in fact be a more important contributor to hypertension in blacks than it is to hypertension in whites.
An increase in AGT expression in blacks that results in a higher level of Ang II activity would appear to be inconsistent with the suppression of plasma renin activity commonly observed in blacks24 25 26 and with the lower aldosterone production that we have observed in young blacks.27 28 To explain this apparent paradox, we suggest that greater expression of AGT may occur within the kidney, leading to overproduction of Ang II and an increase in Ang II–mediated reabsorption of sodium29 ; ultimately, plasma volume would expand with suppression of renin secretion. Alternatively, an increase in AGT expression in blacks might augment an already heightened activity of another system that also serves to retain sodium—for example, an increase in the function of the epithelial sodium channel in the renal collecting duct.30
In summary, using a haplotype that further differentiates the T235 carrier state, we found an association of genotype with serum angiotensinogen concentration in both blacks and whites. The findings implicate AGT in the regulation of BP in both racial groups.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (R01-HL-35795 and MO1-RR00750); the Veterans Administration; the American Heart Association, Indiana (L.J.B.) and Wisconsin (D.A.T.) Affiliates; and the American Heart Association National Center (D.A.T.). The authors are grateful to Mary Anne Wagner and Chunlu Guo for technical assistance.
Received August 29, 1996; first decision September 25, 1996; accepted November 6, 1996.
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References |
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