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(Hypertension. 1999;34:430-434.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Angiotensinogen Gene Polymorphism Near Transcription Start Site and Blood Pressure

Role of a T-to-C Transition at Intron I

Tomoaki Ishigami; Kouichi Tamura; Takayuki Fujita; Izumi Kobayashi; Kiyoshi Hibi; Minoru Kihara; Yoshiyuki Toya; Hisao Ochiai; Satoshi Umemura

From Yokohama City University, Internal Medicine II, Yokohama City, Japan.

Correspondence to Tomoaki Ishigami, MD, PhD, Yokohama City University, Internal Medicine II, 3-9, Fukuura, Kanazawa-Ku, Yokohama City, Japan. E-mail utomo661{at}med.yokohama-cu.ac.jp

	Abstract

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Abstract—Molecular variants of the angiotensinogen gene, a key component of the renin-angiotensin system, are considered genetic risk factors for primary hypertension. A relation between the angiotensinogen gene locus and hypertension has been found in whites, Japanese, and African Caribbeans but not in Chinese. The lack of a consistent association between M235T polymorphism at exon 2 and hypertension has suggested that another site in linkage disequilibrium with M235T is the causal mutation. We studied the relations among plasma angiotensinogen concentrations, blood pressure, related clinical variables, and mutations of the 5' upstream core promoter region of the human angiotensinogen gene in 274 subjects recruited from our outpatient clinic. We confirmed that plasma angiotensinogen concentration was significantly correlated with A-20C mutation and percent body fat and found that systolic and diastolic blood pressures were significantly correlated with G-6A and T+68C mutations. These results suggest that mutations near the transcription start site may be associated with increased blood pressure.

Key Words: angiotensinogen • blood pressure • polymorphism • transcription, genetic • hypertension, essential

	Introduction

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Essential hypertension affects 20% of the adult population; its pathogenesis involves interactions between genetic and environmental factors.¹ Improved understanding of the molecular basis of essential hypertension may facilitate the development of new targeted forms of pharmacological therapy that can be tailored to the needs of individual patients and thereby minimize the risk of morbidity and mortality from cardiovascular diseases. The genetic analysis of complex traits and diseases such as blood pressure and hypertension is difficult because of polygenic origin, genetic heterogeneity, variable penetrance, unknown modes of inheritance, and variable effects of environmental factors.

Molecular variants of the angiotensinogen (AGT) gene, a key component of the renin-angiotensin system, are considered genetic risk factors for primary hypertension.² In animal models and humans, an association with hypertension has been confirmed only for the AGT^{2 3} and -adducin genes.⁴ Recent studies in which the AGT gene was inactivated or duplicated in transgenic mice have shown a relationship among AGT gene expression, plasma AGT, and blood pressure.⁵ A relation between the AGT gene locus and hypertension has been found in whites,⁶ Japanese,⁷ and African Caribbeans⁸ but not in Chinese.⁹ A meta-analysis of M235T with hypertension revealed that this allele contributes to hypertension¹⁰ with positive family history. The lack of a consistent association between M235T and hypertension has suggested that another site in linkage disequilibrium with M235T is the causal mutation.

Recent studies of promoter activity and DNA binding properties have suggested that a nucleotide substitution in the 5' upstream core promoter region of the human AGT gene affects the basal transcriptional rate of the gene.¹¹ Jeunemaitre et al¹² have demonstrated 3-point mutations in the 5' core promoter region of the human AGT gene, such as A to C at -20, C to T at -18, and G to A at –6. Other studies have also implicated these mutations of the 5' upstream core promoter region in the pathogenesis of hypertension.^{13 14}

To further delineate the genetic basis of essential hypertension, we studied the association of AGT gene mutations near the transcription start site, determined by direct sequencing methods, with plasma AGT concentrations and blood pressure.

	Methods

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Subjects
We studied the relations among plasma AGT concentrations, blood pressure, related clinical variables, and mutations of the 5' upstream core promoter region of the human AGT gene in 274 subjects recruited from our outpatient clinic who were part of the 188 subjects already reported.¹⁴ We excluded subjects receiving oral contraceptives; subjects with secondary hypertension, renal diseases, or severe infectious diseases; and subjects with advanced malignant disorders. Informed consent was obtained from all participants. Percent body fat was measured in each subject by the bioimpedance method.¹⁵ Expert nurses independently measured blood pressure 2 times, and average blood pressure was recorded. Hypertension was defined as an average systolic blood pressure (SBP) of >140 mm Hg, an average diastolic blood pressure (DBP) of >90 mm Hg (or both), or a previous diagnosis of hypertension in subjects receiving antihypertensive medication. All antihypertensive medication was withdrawn 24 hours before the start of the study.

Polymerase Chain Reaction Direct Sequencing and Restriction Fragment Length Polymorphism Methods and Measuring Plasma AGT Concentrations
Blood samples were collected for isolation of genomic DNA and plasma. Polymerase chain reaction (PCR) direct sequencing methods were used to determine the mutations. The PCR and PCR-direct sequencing methods were performed as previously described.^{14 16} PCR and subsequent digestion of the products with SfaNI were performed to determine M235T AGT genotypes.⁶ Plasma AGT concentrations were measured as described previously.¹⁴

Statistical Analysis
The correlations among plasma AGT concentrations, age, percent body fat, SBP, DBP, and lipids concentrations were examined. Continuous variables are expressed as mean±SEM. Allele frequencies were estimated by the gene-counting method. For the regression model, the genotype effect was assumed to be additive; ie, scores of 0, 1, and 2 were assigned for genotype wild homozygotes, heterozygotes, and mutant homozygotes of each mutation, respectively. Stepwise multiple regression analyses were conducted to determine the percentage of explained variance in dependent variables that is accounted for by genotypes and other variables. The relations between plasma AGT concentrations and independent variables (genotypes, percent body fat, lipids concentrations, blood pressure, body mass index, age, and gender [men=1, women=0]) were evaluated. In addition, the relations between blood pressure and independent variables (genotypes, percent body fat, lipids concentrations, body mass index, history of hypertension [with =1, without =0], and plasma AGT concentrations) were evaluated. We set the inclusion criterion as F=3.84 and the exclusion criterion as F=2.71. Statistical analyses were performed with SPSS 6.1 statistical software (SPSS, Inc) on a Macintosh computer.¹⁷ P<0.05 was considered to indicate statistical significance.

Pairwise linkage disequilibrium coefficients were estimated by the maximum-likelihood method, and the extent of disequilibrium was expressed as D'=D/D_max or D/D_min, according to Hill¹⁸ and Thompson et al.¹⁹

	Results

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Table 1 shows the general characteristics of the 118 men and 156 women (average age, 58.9±0.6 years). We found A-20C and G-6A mutations at the 5' upstream core promoter region but no C-18T mutation in our subjects, which is similar to the results of Morgan et al.²⁰ We also found a novel T-to-C transition at +68 from the transcription start site in intron 1 of the AGT gene. By use of the consensus motif sequence search program (TFSEARCH), we found that a T-to-C transition at +68 generated the consensus sequence of a member of the GATA family of transcription factors. Their genotype and allele frequencies are summarized in Table 2.

View this table: [in this window] [in a new window]   Table 1. General Characteristics of Subjects

View this table: [in this window] [in a new window]   Table 2. Genotype and Allele Frequencies of Subjects

Univariate regression analysis reconfirmed positive correlations between plasma AGT concentrations and SBP and DBP (Table 3). After adjustment for these factors by stepwise multivariate linear regression analysis, plasma AGT concentrations significantly correlated with A-20C, HDL cholesterol, and percent body fat (Table 4). In addition, SBP and DBP (data not shown) in all subjects significantly correlated with G-6A and T+68C mutations on stepwise multivariate linear regression analysis (Table 5).

View this table: [in this window] [in a new window]   Table 3. Correlation Matrix Among Blood Pressure, Plasma AGT Levels, and Related Variables by Univariate Regression Analysis

View this table: [in this window] [in a new window]   Table 4. Multiple Linear Regression Analysis of Independent Variables to AGT Concentration

View this table: [in this window] [in a new window]   Table 5. Multiple Linear Regression Analysis of Independent Variables to SBP in All Subjects

Haplotype analysis between AGT polymorphisms revealed highly significant linkage disequilibrium among M235T, G-6A, and T+68C and marginally significant linkage disequilibrium among A-20C and the other polymorphisms, such as M235T, T+68C, and G-6A (Table 6). The estimated C-T haplotype is 90% of subjects with the C allele; this suggested allele C (-20) occurs almost always on genes with T235, as observed in whites.

View this table: [in this window] [in a new window]   Table 6. Disequilibrium Statistics Among AGT Gene Mutations

	Discussion

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In this study, we found a new T-to-C transition at +68 from the transcription start site in intron 1 of the AGT gene and clarified positive correlations between blood pressure and human AGT gene mutations such as G-6A and T+68C. We also found significant linkage disequilibrium among human AGT gene mutations such as G-6A, T+68C, and M235T. Our study reconfirmed positive correlations between A-20C mutations and plasma AGT concentrations as previously reported.¹⁴ In addition, we found a positive correlation between plasma AGT concentrations and percent body fat.

Previous in vitro studies showed that G-6A mutations affect the transcriptional activity of mRNA of the AGT gene; through this mechanism, G-6A was suggested to contribute to the development of hypertension in humans.¹¹ Our results suggested that G-6A acts as a functional mutation in humans, and T+68C, which was completely linked with G-6A, was suggested to act as a functional mutation or 1 of the genetic markers of G-6A. Although these 2 mutations did not positively correlate with plasma AGT concentrations, our findings suggest that the altered transcriptional activity of the human AGT gene may affect local production of AGT in vivo and thereby alter vascular tone and blood pressure. It is important to note that the G-6A, M235T, and T+68C polymorphisms were in complete linkage disequilibrium with each other and occurred with the same frequency. Consequently, although previous analyses refer to M235T, all associations pertaining to the T235 allele can be directly extrapolated to the A-6 or C+68 polymorphism.

Previously, a C-to-T transition at -18 was reported to be associated with essential hypertension.¹³ We did not find the mutation in our samples, a finding similar to that of Morgan et al.²⁰ These inconsistent results suggest that C-18T mutation has only a minor role in essential hypertension.

We used multiple linear regression analysis to study the correlation between several quantitative phenotypes of essential hypertension and AGT gene polymorphism. Results of multiple regression analyses showed the quantitative effectiveness of the studied polymorphisms of the AGT gene. Multiple R² for plasma AGT concentrations was 0.1004; values for blood pressure ranged from 0.1161 to 0.2787. These values represent the quantitative contributions of these polymorphisms to phenotype; ie, the A-20C mutation and other variables account for 10% of the plasma AGT concentration, and the G-6A and T+68C mutations and other variables account for 11.6% to 27.9% of blood pressure. Although in vitro studies have shown moderate differences in transcriptional activity of mRNA of the AGT gene between these mutations, the net contributions of these mutations to the development of hypertension must be confirmed in humans.

Our findings described here are only statistical observations and do not imply causation. This emphasizes the need for advanced analysis of a better-defined population-based sample. Interestingly, Zhao et al²¹ recently reported the possible involvement of the A-to-C transition at –20 in the increasing transcriptional activity of human AGT gene, and other data^{11 22} have suggested the importance of the cis factor near the AGT gene transcription start site.

In our study, to minimize unknown effects of antihypertensive drugs on plasma AGT concentrations, all subjects who received antihypertensive drugs were requested to discontinue such medication 24 hours before enrollment. We analyzed the relation between genotype and plasma AGT concentrations by multivariate regression analysis to adjust for several variables, including antihypertensive medication. However, a major limitation of the interpretation of our data is the possible interference of drug treatment. In addition, the mixed sample used in our study, including a small number of hypertensive patients and normotensive control subjects, remains another study limitation, although we statistically adjusted for several variables with multivariate stepwise regression analysis. Confirmation of our results requires further studies.

The mechanisms of the positive correlations between plasma AGT concentrations and percent body fat were unknown. A possible explanation is that adipose tissue may be a source of plasma AGT. Because next to the liver, adipose tissue produces the second greatest amount of AGT in humans. Previously, we confirmed adipogenic activation of the AGT gene in cultured cell experiments.²³ In rats fasted for 3 days, the amount of AGT released per adipose cell fell to 33% of the control level. Resumption of feeding increased AGT release from 41% to 83% higher than control. This increase in AGT release was apparently derived from local adipose tissue, because liver mRNA or central plasma levels of AGT²⁴ were not affected by fasting or overfeeding.

In conclusion, available evidence, including this study, suggests that mutations near the transcription start site may be associated with increased blood pressure.

	Acknowledgments

 
This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan (No. 09770492), the Health Science Research Grants Special Research, and the Uehara Memorial Foundation.

Received February 18, 1999; first decision March 3, 1999; accepted May 11, 1999.

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