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(Hypertension. 1997;30:321.)
© 1997 American Heart Association, Inc.
Articles |
Association of Variants in Critical Core Promoter Element of Angiotensinogen Gene With Increased Risk of Essential Hypertension in Japanese
From the Department of Geriatric Medicine, Osaka University Medical School (Japan).
Correspondence to Toshio Ogihara, MD, PhD, Professor of Medicine, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan. E-mail tkatsuya{at}yo.rim.or.jp
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Abstract |
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Abstract We examined the association between variants in the core promoter element 1 (AGCE1) of the human angiotensinogen gene (AGT), which acts as a critical regulator of AGT transcription, and the risk for hypertension. One hundred and eighty patients with documented essential hypertension and a family history of hypertension and 194 control subjects without hypertension were selected and frequency matched by age and sex. Genomic DNA from leukocytes was analyzed for genetic variants (position: -20 to -18) in AGCE1. The haplotype in AGCE1 was significantly associated with increased risk of essential hypertension (P<.05). The frequency of subjects with homozygous C allele at position -18(CC/C-18T) was significantly higher in case patients than in control subjects (P<.005), and the evaluated odds ratio for hypertension was 4.2 (95% confidence interval [CI]: 1.4 to 12.8, CC/C-18T versus CT/C-18T). The homozygous threonine allele at codon 235 (TT/M235T) in exon 2 of AGT was also associated with hypertension (P<.02; odds ratio, TT versus other genotypes, 1.8; 95% CI, 1.1 to 2.7). According to haplotype analysis between AGT polymorphisms, we identified linkage disequilibrium between M235T and A-20C and between M235T and C-18T. We conclude that C-18T polymorphism in AGCE1 is a genetic risk factor for essential hypertension in the Japanese and is more tightly and directly associated with hypertension than TT/M235T.
Key Words: genetics • renin-angiotensin system • transcription • haplotypes • polymorphism, restriction fragment length
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Introduction |
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Angiotensinogen is the precursor of angiotensin II, which plays a significant role in the regulation of blood pressure and is a potent growth factor in cardiovascular hypertrophy.1 2 3 4 5 6 Recently, variants of the human angiotensinogen gene (AGT) were found to be associated with an increased risk of essential hypertension6 7 8 9 10 and ischemic heart disease.11 12 13 These studies suggested that a homozygous threonine allele at codon 235 in AGT (TT/M235T) is more frequent among individuals with hypertension than among normotensive subjects7 8 9 10 and that TT genotype is associated with an increase of circulating angiotensinogen concentration, which may result in hypertension.7 However, the interaction between TT/M235T and the angiotensinogen concentration has not been clarified.
In a recent report, Yanai et al14 found that a cis-acting DNA element located between the TATA box and the transcription initiation site is critical in the response to the regulatory sequence in AGT and that sequence difference in this angiotensinogen core promoter element 1 (AGCE1: at -25 to -1 base region upstream from transcriptional initiation site) alters the binding affinity of a ubiquitous transcriptional factor, AGCE-binding factor 1 (AGCF1). When three haplotypes (at -20 to -18) in AGCE1 were compared, CTC and ATC showed 2.5 times higher transcriptional activity than ATT.15 Their investigation suggested that genetic variants in AGCE1 might directly affect the circulating angiotensinogen level, resulting in human hypertension. Interestingly, Jeunemaitre et al7 have already identified two polymorphisms in AGCE1 at -20 and -18. We performed a case-control study in the Japanese population to determine whether a genetic variant in AGCE1 is directly associated with an increased risk of hypertension and examined the linkage disequilibrium among the polymorphisms of AGT.
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Methods |
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Population
Patients with essential hypertension and control subjects were recruited from outpatients at the Department of Geriatric Medicine, Osaka University Medical School. All case patients and control subjects were Japanese and gave informed consent before participating in the research protocol, which was approved by the Hospital Ethics Committee. All case patients (n=180) had a family history of hypertension in first-degree relatives and were diagnosed as having primary hypertension (those with secondary hypertension, diabetes mellitus, or apparent ischemic heart disease were excluded). The criteria for hypertension were defined as systolic blood pressure higher than 160 mm Hg, diastolic blood pressure higher than 95 mm Hg, or under antihypertensive therapy. Control subjects (n=194) without a history of hypertension and without diabetes mellitus were recruited from the same population and were matched to case patients for sex and age. We also excluded from the control subject group those with first-degree relatives who had hypertension. Participants completed a standard questionnaire on personal medical history and family history of hypertension. Blood pressure was measured twice, with the subject seated after 5 minutes of rest.
Determination of Genotypes
Blood was drawn to obtain the buffy coat. DNA was extracted from
200 µL of buffy coat using QIAamp Kit (QIAGEN). We designed two
sets of primers to amplify the flanking region of AGCE1 (Fig 1).14 16 To confirm the
nucleotide sequence of the amplified region in the
Japanese, we sequenced the flanking region of AGCE1 using volunteers’
DNA (n=3) and confirmed that there was no sequence difference in this
region between Caucasians16 and Japanese (data not shown).
The haplotypes (at -20 to -18) in AGCE1 were directly determined
using a combination of three sets of restriction fragment length
polymorphisms (RFLPs) (Table 1).
Polymerase chain reaction (PCR) was carried out with 100 ng of genomic
DNA as a template using a thermal cycler, Omni Gene (Hybaid). DNA was
amplified with initial denaturation at 94°C for 5 minutes followed by
35 cycles (94°C for 45 seconds, 56°C for 30 seconds, 72°C for 45
seconds). PCR products were digested with 1 U of Hae III
(Takara), Mnl I, or SfaN I (New England Biolabs)
at 37°C for a period that varied between 3 hours and overnight. The
protocol for detecting the M235T polymorphism was
performed according to the method of Russ et al17 with
minor modification.11 All digested products were
separated on 3.0% MetaPhor agarose gel (FMC BioProducts) and
visualized with ethidium bromide staining (Fig 2).
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Statistical Analysis
For all the statistical analyses, we used the computer
software application StatView version 4.5J (Abacus Concepts) or JMP
version 3.0 (SAS Inc). The difference in genotype distribution
between case patients and control subjects was examined by
2 analysis. The association between
AGT polymorphism and each value of the classic risk
factors for hypertension was examined by one-way ANOVA. To assess the
quantitative effects of the covariates (sex, age, body mass index,
fasting plasma glucose level, triglyceride, and
C-18T polymorphism), we carried out multiple logistic
regression analysis using JMP. Linkage disequilibrium between
polymorphisms of AGT was evaluated according to the
classic method developed by Hill et al18 19 and Thompson
et al.20
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Results |
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Compared with control subjects, case patients had higher values of three established risk factors for hypertension: Body mass index, fasting plasma glucose level, and triglyceride concentration of case patients were significantly higher than those of control subjects (Table 2). However, no significant difference was observed in the values of other risk factors, such as total cholesterol, HDL cholesterol, and glucohemoglobin A1C (Table 2).
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Using the PCR-RFLP technique, we determined the genotypes of
all the participants. To confirm the genotype, we selected 1
sample out of 10 at random and carried out genotyping using another
primer set. The haplotype distributions in AGCE1 were significantly
different between case patients and control subjects
(2=9.51, P<.05) (Table 3). Interestingly, no significant linkage
disequilibrium was observed between A to C base substitution at
position -20 (A-20C) and C to T substitution at
-18(C-18T) (Table 4).
Therefore, we also examined the association between hypertension and
each polymorphism separately. Since the T/C-18T
allele frequency was very low, no TT homozygote was
observed in the present study. The frequency of CC/C-18T
genotype was significantly higher in case patients than in
control subjects (2=7.54, P<.005)
(Table 3). The odds ratio for essential hypertension among individuals
with CC/C-18T compared with those with CT/C-18T was
calculated as 4.2 (95% CI: 1.4 to 12.8). Furthermore, multiple
logistic regression analysis revealed that body mass index
(Wald 21=19.0, P<.0001),
fasting plasma glucose level (Wald
21=5.6, P<.02), and
C-18T polymorphism (Wald
21=4.4, P<.04) showed a
significant effect on the onset of hypertension, whereas sex, age, and
triglyceride did not. In contrast, the distribution of
A-20C genotype was almost identical in the case
patients and control subjects (Table 3).
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On the other hand, TT/M235T was associated with an increased
risk of essential hypertension (2=6.37,
P<.05). The calculated odds ratio of TT/M235T
(versus MT and MM/M235T) for hypertension was 1.8
(95% CI: 1.1 to 2.7). When the frequencies of the rare alleles of
the T/C-18T and the M/M235T polymorphism were
compared between hypertensives and normotensives, we observed that the
allele frequency difference of T/C-18T (0.033) was half
that of M/M235T (0.07). According to the haplotype
analysis between AGT polymorphisms, significant
linkage disequilibrium was detected strongly between M235T
and A-20C (D’=0.91, P<.0001) and weakly between
M235T and C-18T (D’=0.49, P<.001)
(Table 4). In addition, we could not detect any significant association
between genetic variants in AGCE1 and each value of other risk factors
(data not shown).
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Discussion |
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The renin-angiotensin system plays a role in regulating vascular resistance, sodium reabsorption, promotion of vascular growth, and the pathobiology of vascular disease.21 22 23 24 25 26 Previous reports suggest that AGT itself may have a causal role in the development of hypertension, especially since the M235T polymorphism is associated with increased concentration of circulating angiotensinogen.7 9 In transgenic or gene targeting experiments in which animal models were used, the importance of the relation between the angiotensinogen gene and its genetic regulation of circulating angiotensinogen level has also been emphasized as a risk for hypertension.27 28 29 30 However, there is no direct evidence to confirm the interaction between genetic variants of AGT and its transcriptional activation.
Recently, two reports from Yanai et al14 15 revealed the direct interaction between genetic variants in AGCE1 and angiotensinogen gene transcription.14 15 They reported that subjects with CTC or ATC haplotype (at -20 to -18) have 2.5 times higher transcriptional activity of AGT than those with ATT haplotype. In the present study, we examined the genetic epidemiological role of polymorphisms in AGCE1 by a case-control study in Japanese. We revealed that C-18T is an independent genetic risk factor for hypertension, although we could not find a subject with TT/C-18T in this study since the frequency of the T allele was very low. Even though there was no TT homozygote, the frequency of CC heterozygotes in case patients was significantly higher than that in control subjects. On the basis of our results and those of Yanai et al, we speculate that CTC or ATC haplotype should be a cause of hypertension in humans because it increases the binding affinity of AGCF1 to AGCE1, which results in the increase of circulating angiotensinogen through activation of AGT transcription. However, we should also consider the possibility of the hypotensive effect of T/C-18T allele (ATT or CTT haplotype). The attenuated AGT transcription level in subjects with T/C-18T allele may protect against hypertension. However, there was no significant difference in the allele frequency of C-18T polymorphism in the Utah population, which suggests that this association needs to be confirmed in various ethnic populations.7
In the present study, significant linkage disequilibrium was strongly observed between M235T and A-20C (D’=0.91) and weakly between M235T and C-18T (D’=0.49), and the allele frequency difference of T/C-18T was half that of M/M235T. On the other hand, all subjects with CTC/CTC genotype had TT genotype of M235T. These results suggest that a part of the previously reported genetic risk of hypertension associated with M235T might be explained by the increase of transcriptional regulation of AGT that is induced by the AGCE1 polymorphism. To confirm this speculation, measurement of the circulating angiotensinogen level among AGT genotypes should be performed in a future study.
We conclude that the determination of polymorphism in AGCE1 may be useful in the assessment of risk for essential hypertension. However, there still remains some possibility that this polymorphism may be only a marker of another true causal genetic variant of angiotensinogen. All this study shows is an increased odds ratio for the presence of hypertension in individuals with the CC/C-18T genotype. Prospective studies will be required to test whether or not this genetic marker can indeed serve as a prognostic marker for an increased risk of developing hypertension.
Received November 18, 1996; first decision December 17, 1996; accepted February 10, 1997.
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