Zhang Y et al / Acta Pharmacol Sin 2003 Nov; 24 (11): 1089-1093

Angiotensin II type 2 receptor gene polymorphisms and essential hypertension¹

ZHANG Yi, ZHANG Kui-Xing, WANG Gu-Liang, HUANG Wei², ZHU Ding-Liang³

Ruijin Hospital, Shanghai Institute of Hypertension, State Key Laboratory of Medical Genomics, Shanghai Second Medical University, Shanghai 200025, China; ²Chinese National Human Genome Center at Shanghai, Shanghai 201203, China

¹ Project supported by the grants from National Natural Science Foundation of China (No 30270545), Ministry of Science and Technology of China (No Z19-01-03-01A, G1998051013, 2002BA711A05), and Science and Technology Commission of Shanghai Municipality (No 01QA14035).

³ Correspondence to Prof ZHU Ding-Liang. Phn 86-21-6437-0045, ext 610901. Fax 86-21-5465-4498. E-mail f97075@sh.cnuninet.net

Received 2003-08-25 Accepted 2003-09-19

KEY WORDS angiotensin II; angiotensin receptors; hypertension; polymorphism (genetics); single nucleotide polymorphism

ABSTRACT

AIM: To identify the genetic variants of angiotensin II type 2 receptor (AT₂R) gene in a Chinese population and to determine whether the AT₂R gene polymorphisms are associated with essential hypertension (EH). METHODS: The detection of single nucleotide polymorphisms (SNPs) was performed in 19 subjects by a direct DNA sequencing. Two hundred fifty patients with EH and 250 nomortensive controls were included in the study to assess the contribution of polymorphism of AT₂R gene to hypertension. RESULTS: We identified 9 SNPs in the promoter, intron, exons and 3' untranslated region (3'UTR) of AT₂R gene; among them 5 SNPs were novel molecular variants. A case-control study using a most frequent SNP (1334T/C) in the promoter region, showed a significant increase in allele frequency of C¹³³⁴ in male hypertensive subjects (17.5 % vs 10.3 % for normotensive subjects, P<0.05). CONCLUSION: The catalogue of SNPs of AT₂R gene in Chinese population showed ethnic difference in DNA sequence variation. A polymorphism in the promoter region (1334T/C) of AT₂R gene might be involved in the development of hypertension in Chinese population.

INTRODUCTION

Hypertension is a major risk factor for stoke, myocardial infarction, congestive heart failure, and end stage renal disease^[1,2]. The renin-angiotensin system (RAS) plays a key role in salt and water homeostasis and vascular tone regulation. It is well accepted that angiotensin II (Ang II), the central product of the system, causes its effects via at least two major receptor subtypes, Ang II typer 1 receptor (AT₁R) and type 2 receptor (AT₂R)^[3]. The AT₁R mediates almost all the known effects of Ang II in human, such as vasocon-striction, release of aldosterone, renal sodium absorption, vascular cell growth and so on^[4]. In contrast to many data on the role of AT₁R, the role of AT₂R in vascular diseases remains to be defined. Although the relationships between the polymorphisms of RAS genes and hypertension have been extensively studied, there are few reports of the association between the human AT₂R gene and essential hypertension (EH)^[5].

The aim of the present study was to screen for the genetic variants in AT₂R gene in a Chinese population and to assess the association between the polymorphisms of AT₂R gene and EH through a case-control study.

MATERIALS AND METHODS

Subjects All subjects in this study are of Han Chinese currently residing in Shanghai area. DNA samples from 19 unrelated hypertensive subjects were used for detection of single nucleotide polymorphism (SNP). Two hundred and fifty unrelated patients with EH and 250 normotensive (NT) subjects were enrolled for association study. The inclusion criteria for the cases were as follows: (1) at least three consecutive blood pressure measurements 140 mmHg for systolic blood pressure (SBP) and/or 90 mmHg for diastolic blood pressure (DBP), or currently receiving antihypertensive drugs for at least 1 year, (2) an onset of hypertension after 30 and before 60 years ago, (3) no clinical or biochemical signs of secondary hypertension. The subjects recruited for the controls had the following characteristics: (1) SBP<140 mmHg and DBP<90 mmHg, (2) age at the time of the study 40 years.

Blood pressure was measured in the seated position after 10 min of rest using a mercury sphygmomanometer by experienced and certified examiners. Afterwards, a detailed questionnaire was filled out, a physical examination was carried-out and 10 mL blood sample was drawn from all subjects for DNA extraction and biochemical examination. Informed consent was obtained from all participants.

SNP detection and genotyping Genomic DNA was extracted from peripheral white cells using a standard phenol-chloroform method.

The detection of SNPs was performed by a direct DNA sequencing. After PCR amplification and purification, all the exons, flanking introns and the promoter region of AT₂R gene were sequenced. The primers were designed with Primer 3 software developed at Whitehead Institute and Howard Hughes Medical Institute (http://www-genome.wi.mit.edu/genome_software/other/primer3.html ). In brief, "touchdown PCR"^[6] was performed under standard conditions in a total volume of 10 µL on GeneAmp 9700 PCR System (Applied Biosystems). DNA was denatured once for 2 min at 94 ºC, following 40 cycles of denaturing (94 ºC, 30 s), annealing (58 ºC, 40 s) and extension (72 ºC, 40 s), except that in the first 10 cycles, the annealing temperatures decreased from 63 ºC to 58 ºC by 0.5 ºC a cycle. One final extension step for 7 min at 72 ºC was added. Wizard PCR Preps DNA Purification Resin (Promega) was used for purification. The PCR products were analyzed on ABI-PRISM 377 Automatic sequencer (Applied Biosystems). SNPs were detected with Polyphred Program that developed at University of Washington (http://droog.mbt.washington.edu/Polyphred.html).

Genotypes of the SNP03 in the AT₂R promoter region were determined by PCR amplification followed by direct DNA sequencing as above-mentioned.

Statistical analysis Results were presented as mean±SD. Differences in the distribution of genotypes between groups were determined by the ² test. P<0.05 was considered statistically significant.

RESULTS

Identification of AT₂R variants Nineteen hypertensive subjects were enrolled in the initial search for AT₂R gene variants. In a total length of 2938 kb sequenced, 9 SNPs were identified, of which 3 in the promoter region, 1 in intron, 2 in exons, and 3 in 3' untranslated region (3'UTR), respectively. Tab 1 shows the position and base substitutions of these SNPs.

Tab 1. SNPs detected within AT₂R gene.

Among them, 4 SNPs (SNP 04,06,07,08) were listed in the database of SNPs of the National Center of Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and the other 5 SNPs (SNP01,02,03,05,09) were detected for the first time in the Chinese population. The frequency of each allele was calculated and shown in Tab 2. The alleles of 2 SNPs (SNP01, 02) in promoter region and 2 SNPs (SNP05, 06) in exons were with lower frequency (<10 %).

Tab 2. Allele frequencies of SNPs of AT₂R gene.

Association between hypertension and AT₂R polymorphisms The genotype for a most frequent variant (SNP03, 1334T/C) located in promoter region was then performed in 250 EH and 250 NT subjects. Clinical characteristics from both groups were presented in Tab 3. The C¹³³⁴ was significantly more frequent in the male EH group compared with the male NT group (17.5 % vs 10.3 %, P<0.05, Tab 4).

Tab 3. Characteristics of hypertensive and normotensive subjects.

Tab 4. Comparison of genotype and allele frequency for variant 1334T/C in hypertensive and normotensive subjects.

EH, essential hypertension; NT, normotensive control.

Values are means±SD. EH, essential hypertension; NT, normotensive control; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FG, Fasting glucose; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoproteins; HDL, high-density lipoproteins. BUN, blood urea nitrogen; Cr, Creatinine; and UA, uric acid.

DISCUSSION

AT₂R is highly expressed in fetal life and declines rapidly after birth^[7,8]. AT₂R expression is restricted to a few tissues in adults. In the adult, AT₂R mRNA has been detected in the adrenal gland, heart, brain, and its protein has been detected in the kidney^[7,9,10]. During the past few years, studies have demonstrated that the AT₂R is responsible for vasodilation, natriuresis, and antiproliferation thus AT₂R appears to play a counter regulatory protective role opposing the AT₁R^[11-19]. Recently, animal model studies have indicated that AT₂R plays an important role in the regulation of blood pressure^[20,21].

The human AT₂R gene about 5 kb in length is located on X chromosome, it includes 3 exons and regulatory elements in the first intron and promotor region^[22-24]. According to the SNPs data of NCBI, there are 15 SNPs in human AT₂R gene. However, only 2 intron SNPs were listed in Japanese nucleotide polymorphisms database (http://snp.ims.u-tokyo.ac.jp/). It suggests that important ethnic difference in SNPs distribution might exist. Therefore, it is necessary to establish the database of SNPs in Chinese nation. In this study, we first identified the molecular variants within AT₂R gene using direct DNA sequencing, the most solid technique for mutation detection. Nine SNPs were detected in Chinese, among them 4 SNP were listed in the NCBI database, and the other 5 SNPs were detected for the first time within AT₂R gene.

We selected SNP03 (1334T/C) to test its potential association with hypertension for the following reasons (1) 3 SNPs (SNP03 in promoter region, SNP04 in intron and SNP05 in exon) were previously tested in a pilot association study including 96 hypertensive and 96 normotensive subjects. Preliminary results showed a positive association of SNP03 with hypertension (data not shown); (2) SNP03 is a most frequent variant located in the promoter region. The T to C substitution will result in the lost of a transcriptional binding site.

It was worthwhile to note that 3 transcriptional binding sites, V$COMP1.01, V$RBPJK.01, and V$HMGIY.01 were involved in this polymorphism location (http://www.genomatix.de). V$RBPJK.01, the binding site for mammalian transcriptional repressor RBP-JKappa/CBF1, would be lost when the T to C polymorphism happened. It suggests that this SNP might be related to the expression of AT₂R gene, and further evidence need to substantiate the functional significance concerned.

We observed a positive association between AT₂R 1334T/C polymorphism and hypertension only in male. A possible explanation is that the AT₂R gene is located on X chromosome, therefore it might exert differential influence on male and female. Nevertheless, the exact mechanisms remain to be evaluated.

In conclusion, this is the first study to catalog the SNPs of AT₂R gene in Chinese and a polymorphism in the promoter region of the human AT₂R gene was associated with EH in male Chinese. This results need to be verified in another large population and the functional significance of the SNP deserves to be further explored.

REFERENCES

• 1 Mcsterd A, D'Agostino RB, Silbershatz H, Sytkowski PA, Kannel WB, Grobbee DE, et al. Trends in the prevalence of hypertension, antihypertensive therapy, and left ventricular hypertrophy from 1950 to 1989. N Engl J Med 1999; 340:1221-7.
• 2 Kannel WB. Elevated systolic blood pressure as a cardiovascular risk factor. Am J Cardiol 2000; 85: 251-5.
• 3 Clin AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Duncia JV, et al. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun 1989; 165: 196-203.
• 4 Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, et al. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 1993; 45: 205-51.
• 5 Schmieder RE, Erdmann J, Delles C, Jacobi J, Fleck E, Hilgers K, et al. Effect of the angiotensin II type 2-receptor gene (+1675 G/A) on left ventricular structure in humans. J Am Coll Cardiol 2001; 37: 175-82.
• 6 Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. `Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 1991; 19: 4008.
• 7 Shanmugam S, Llorens-Cortes C, Clauser E, Corvol P, Gase JM. Expression of angiotensin II AT₂ receptor mRNA during development of the rat kidney and adrenal gland. Am J Physiol 1995; 268: F922-F930.
• 8 Sechi LA, Griffin CA, Grady EF, Kalinyak JE, Schambelan M. Characterization of angiotensin II receptor subtypes in rat heart. Circ Res 1992; 11:1482-9.
• 9 Shanmugam S, Corvol P, Gase JM. Angiotensin II type-2 receptor mRNA expression in the developing cardiopulmonary system of the rat. Hypertension 1996; 28: 91-7.
• 10 Ozono R, Wang ZQ, Moore AF, Inagami T, Siragy HM, Carey RM. Expression of the subtype-2 angiotensin II (AT₂) receptor protein in rat kidney. Hypertension 1997; 30: 1238-46.
• 11 Siragy HM, Carey RM. Protective role of the angiotensin AT₂ receptor in a renal wrap hypertension model. Hypertension 1999; 33: 1237-42.
• 12 Siragy HM, Inagami T, Ichiki T, Carey RM. Sustained hypertensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT₂) angiotensin receptor. Proc Natl Acad Sci USA 1999; 96: 6506-10.
• 13 Haithcock D, Jiao H, Cui XL, Hopfer U, Douglas JG. Renal proximal tubular AT₂ receptor: signaling and transport. J Am Soc Nephrol 1999;10: S69-S74.
• 14 Carey RM, Wang ZQ, Siragy HM. Role of the angiotensin type 2 receptor in the regulation of blood pressure and renal function. Hypertension 2000; 35 (Pt 2) :155-63.
• 15 Carey RM, Howell NL, Jin XH, Siragy HM. Angiotensin type 2 receptor-mediated hypotension in angiotensin type-1 receptor-blocked rats. Hypertension 2002; 39: E27-28.
• 16 Carey RM, Wang ZQ, Siragy HM. Update: role of the angiotensin type-2 (AT(2)) receptor in blood pressure regulation. Curr Hypertens Rep 2000; 2: 198-201.
• 17 Siragy HM, Carey RM. The subtype-2 (AT₂) angiotensin receptor regulates renal guanosine cyclic 3', 5' monophosphate and AT1 receptor-mediated prostaglandin E₂ production in conscious rats. J Clin Invest 1996; 97: 1978-82.
• 18 Siragy HM, Jaffa AA, Margolius HS. Bradykinin B₂ receptor modulates renal prostaglandin E₂ and nitric oxide. Hypertension 1997; 29: 757-62.
• 19 Tanaka M, Tsuchida S, Imai T, Fujii N, Miyazaki H, Ichiki T, et al. Vascular response to angiotensin II is exaggerated through an upregulation of AT₁ receptor in AT₂ knockout mice. Biochem Biophys Commun 199; 258: 194-8.
• 20 Gigante B, Piras O, De Paolis P, Porcellini A, Natale A, Volpe M. Role of the angiotensin II AT₂-subtype receptors in the blood pressure lowering effect of losartan in salt-restricted rats. J Hypertens 1999; 16: 2039-43.
• 21 Tsutsumi Y, Matsubara H, Masaki H, Kurihara T, Murasawa S, Takai S, et al. Vascular smooth muscle-targeted over expression of angiotensin II type 2 receptor causes endothelium-dependent depressor and vasodilative effects via activation of the vascular kinin system. J Clin Invest 1999; 104: 855-64.
• 22 Koike G, Horiuchi M, Yamada T, Szpirer C, Jacob HJ, Dzau VJ. Human type 2 angiotensin II receptor gene: cloned, mapped to the X chromosome, and its Mrna is expressed in the human lung. Biochem Biophys Res Commun 1994; 203: 1842-50.
• 23 Martin MM, Elton TS. The sequence and genetic organization of the human type 2 angiotensin II receptor. Biophys Res Commun 1995; 209: 554-62.
• 24 Warnecke C, Willich T, Holzmeister J, Bottari S, Fleck E, Regitz-Zagrosek V. Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements. Biochem J 1999; 340: 17-24.