Association Between Renin–Angiotensin System Gene Polymorphism and Essential Hypertension: a Community-Based Study

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Journal of Human Hypertension (2009) 23, 176–181 & 2009 Macmillan Publishers Limited All rights reserved 0950-9240/09 $32.00 www.nature.com/jhh ORIGINAL ARTICLE Association between renin–angiotensin system gene polymorphism and essential hypertension: a community-based study

X Jiang1,6, H Sheng2,6,JLi3, P Xun4, Y Cheng1, J Huang5, H Xiao2 and Y Zhan1 1Department of Geriatrics, First Affiliated Hospital, Nanjing Medical University, Nanjing, China; 2State Key Laboratory of Genetic Engineering, National Engineering Center for Biochip at Shanghai, Shanghai, China; 3Department of Cardiology, Dongtai People’s Hospital of Jiangsu Province, Dongtai, China; 4Department of Statistics, School of Public Health, Nanjing Medical University, Nanjing, China and 5Department of Cardiology, First Affiliated Hospital, Nanjing Medical University, Nanjing, China

Renin–angiotensin (RAS) genes, a group of promising Our results revealed that AGT A-6G, T174 M and ACE-I/D candidate genes involved in essential hypertension were significantly associated with EH (AGT A-6G: (EH), play a key role in blood pressure regulation. AG þ GG vs AA; OR ¼ 1.36; 95% CI ¼ 1.04–1.77. T174M: Recently, a series of novel RAS gene polymorphisms CT þ TT vs CC; OR ¼ 1.45; 95% CI ¼ 1.15–1.90. ACE I/D: were reported, which significantly influence the rate of ID þ DD vs II; OR ¼ 1.171; 95% CI ¼ 1.00–1.37). Moreover the gene transcription. This study was designed to the logistic regression analysis suggested that the explore the association between the RAS gene poly- haplotype of AGT À6A, 174C, À217G and À20A might morphisms and EH in a remote countryside population. decrease the risk of EH (OR ¼ 0.64; 95% CI ¼ 0.49–0.83), We examined six polymorphisms in the main compo- after adjusting the confounding factors of gender, age nent genes of RAS: angiotensin-converting enzyme and BMI. In conclusion, the AGT A-6G, T174 M and ACE (ACE) (I/D), angiotensinogen (AGT) (A-6G, A-20C, I/D polymorphisms are associated with EH and the AGT G-217A and T174 M) and angiotensin type 1 receptor haplotype À6A, 174C, À217G and À20A decrease the (AT1R) (A1166C). Six polymorphisms were genotyped risk of EH in the southern Chinese population. by gene chip technology. Association studies were Journal of Human Hypertension (2009) 23, 176–181; performed in 220 EH patients and 235 normotensives. doi:10.1038/jhh.2008.123; published online 2 October 2008

Keywords: renin–angiotensin system; gene polymorphism; gene chip; Southern China

Introduction reported that AGT gene polymorphism might contribute to the prevalence of EH in Caucasians, which Essential hypertension (EH) is a common complex energized an intensive research effort focusing on disease caused by both genetic and environmental 1 the EH-associated genes. factors. High blood pressure (BP) often leads to a Up-to-date, many EH candidate genes have been series of pathologies, such as insulin resistance, characterized, which are associated with the renin– abnormal metabolism of blood glucose and lipids as angiotensin system (RAS), the sympathetic nervous well as increased risks of various cerebrovascular system, sodium and electrolyte balance and intracel- and cardiovascular diseases, all of which are lular messengers. The proteins encoded by these genes detrimental to human health condition. Although have been demonstrated to participate in BP regula- the precise aetiology of EH remains unclear, accu- tion by various biochemical and physiological path- mulating evidence has suggested that genetic ways in the development and progression of EH. susceptibility is an important factor for the devel- 2 Among all these genes which play a role in the opment of EH. In 1992, Jeunemaitre et al. first aetiology of EH, those encoding for the main compo- nents of the RAS are deemed the most probable candidate genes of EH as the RAS system plays an Correspondence: Dr Y Zhan, Department of Geriatrics, First Affiliated Hospital, Nanjing Medical University, Nanjing essential role in BP regulation by maintaining vascular 3 210029, China. tone and water-to-sodium balance. As a result, RAS E-mail: [email protected] or Dr H Xiao, National Engineering genes encoding for angiotensinogen (AGT), angioten- Center for Biochip at Shanghai, Shanghai 201203, China. sin type 1 receptor (AT1R) and angiotensin-converting E-mail: [email protected] 6 enzyme (ACE) have been widely investigated in These authors contributed equally to this work. 4–8 Received 1 April 2008; revised 4 September 2008; accepted different ethnic populations. In addition, recent 6 September 2008; published online 2 October 2008 studies have shown that the promoter variants of AGT RAS gene polymorphism and essential hypertension X Jiang et al 177 gene significantly influence the rate of AGT transcrip- history of hypertension in the normotensive tion and plasma AGT.9,10 group were excluded from the study. Conflicting or even contradictory reports have (2) Those with chronic liver and kidney diseases, been seen regarding the association between RAS and diabetes mellitus in both hypertensive and gene polymorphisms and EH incidence, of EH. It is normotensive groups were excluded from the possible that patient selection, environmental fac- study following a full biochemical and assistant tors, genetic allele combinations and antihyperten- examination. sive therapy may contribute to the inconsistent data from clinical studies on genetic polymorphisms of The study was conducted in accordance with the EH.11 Given these limitations, this study was principle outlined in the Helsinki declaration for the designed and performed in the countryside of investigation of human subjects. The study protocol Dongtai area of Southern China. All subjects had was approved by our Institutional Review Board of homogeneity of environments, which should mini- Nanjing Medical University. Informed consent was mize the potential influence of mixed factors on EH obtained from each participant of the study. occurrence to the greatest extent. On the basis of previous arguments and potential functional polymorphisms,1–11 we hypothesized that Genotype genetic polymorphisms in RAS genes, including The genotyping procedure was described earlier.12 AGT A-6G, A-20C, G-217A and T174 M, AT1R Briefly, genomic DNA was extracted from human A1166C and ACE I/D, was associated with the peripheral blood with FlexiGene DNA Kit (250) occurrence of EH. The genotype, allele and haplo- (Qiagen, cat. no. 51206, Frankfurt, Germany). The type frequencies were compared between the hy- Primer3 software (http://www.genome.wi.mit.edu/ pertensive and normotensive individuals, in an cgi-bin/primer/primer3.cgi) was applied to design effort to explore the association between the six primers prior to multiplex polymerase chain reac- SNPs of RAS genes and EH in the population of tion (PCR). Two allele perfect match probes and one Southern China. mismatch probe were designed respectively for each SNP. The chips were prepared with the Omni- GridTM 100 TLC sample (GeneMachine, San Carlos, Methods CA, USA). Genotype was judged by calculating the allele score of the SNP. DNA direct sequencing was Subjects conducted to verify the results. The study sample was selected from the countryside of Dongtai country, Yancheng city, Jiangsu province, China. The subjects of the study comprised of 455 Statistical analysis men and women (40–80 years of age), randomly These data were statistically analysed with the SPSS selected from the countryside registry and stratified 11.5 software (SPSS Inc., USA). by gender and age in 5-year intervals. A case–control One-way analysis of variance (ANOVA) was used study was performed between 220 subjects in the to compare mean values between groups. Frequen- hypertensive group and 235 subjects in the normo- cies were compared by a w2 analysis. The relation- tensive group. ship between genotypes and the risk of hypertension Inclusion criteria: was expressed in terms of the odds ratios (OR) adjusted for possible confounding effects including (1) Hypertension was diagnosed according to the gender, age and BMI. diagnostic standard of hypertension set by WHO/ For multivariate risk predictors, the forward ISH in 1999 (systolic blood pressure X140 mm Hg stepwise multiple logistic regression analysis was and/or diastolic blood pressure X90 mm Hg). performed. All independent factors, including gen- (2) Normotensives were selected from the same otypes and clinical risk factors of EH with nominal population with SBP o140 mm Hg and DBP Po0.2 were presented in the initial regression mode o90 mm Hg and the absence of hypertension as doubtful risk factors and forward stepwise disease history. regression analysis was used to assess the risk (3) The enrolled participants completed standard factors using a nominal Po0.05. questionnaires by a face-to-face interview. The A value of Po0.05 was considered statistically subjects were Han Chinese ethnicity with a long- significant. Data were presented as mean±s.d. term local residence rather than immigrants from other areas. All subjects had not administered any antihypertensive drug treatment to lower Results blood pressure prior to study enrolment. Clinical characteristics of the participants Exclusion criteria: Six SNPs (AGT A-6G, A-20C, G-217A and T174 M, AT1R A1166C and ACE I/D) in RAS genes were (1) The subjects with secondary hypertension in the genotyped successfully in 220 EH and 235 normo- hypertensive group and subjects with a family tensive patients. Clinical characteristics of all 455

Journal of Human Hypertension RAS gene polymorphism and essential hypertension X Jiang et al 178 Table 1 Comparison of clinical characteristics between hyper- Table 2 Distribution of genotype frequencies in hypertensive and tensive and normotensive groups normotensive groups

Variables Hypertensive Normotensive P Genotypes Hyper- Normo- P OR tensives tensives (95% CI) Gender, M/F 114/106 118/117 0.732 (n ¼ 220) (n ¼ 235) Age, year 62.2±6.1 61.1±7.7 0.091 BMI, kg/m2 24.37±2.69 23.34±2.60 o0.000 AT1R AA 201 212 0.672 — SBP, mm Hg 159.0±12.9 119.7±10.1 o0.000 A1166C DBP, mm Hg 94.8±8.5 77.7±6.8 o0.000 AC 19 23 Bun, mmol lÀ1 5.4±1.2 5.1±1.2 0.009 CC 0 0 UA, mmol lÀ1 317.2±111.1 298.8±182.4 0.198 AGT A-6G AA 136 169 0.035 — TC, mmol lÀ1 4.6±0.9 4.7±0.8 0.652 AG 74 62 TG, mmol lÀ1 1.6±0.9 1.7±1.1 0.330 GG 10 4 HDL 1.4±0.3 1.4±0.4 0.410 AA 136 169 — 1 (reference) AG+GG 84 66 1.36 (1.04–1.77) Abbreviations: BMI, body mass index; BUN, blood urea nitrogen; DBP, AGT AA 160 168 0.672 — diastolic blood pressure; HDL, high density lipoprotein; SBP, systolic A-20C blood pressure; TC, total cholesterol; TG, triglyceride; UA, uric acid. AC 54 63 Values are mean±s.d. CC 6 4 AGT GG 148 167 0.407 — G-217A subjects are presented in Table 1. The hypertensive AG 63 63 AA 9 5 group exhibited a significantly higher systolic blood AGT CC 126 167 0.008 — pressure, diastolic blood pressure, BMI and BUN T174M than the normotensive group. There were no CT 85 63 significant differences in other indices between the TT 9 5 CC 126 167 — 1 (reference) two groups. CT+TT 94 68 1.45 (1.15–1.90) ACE I/D II 83 110 0.009 — ID 108 112 DD 29 13 Association analysis between polymorphisms II 83 110 — 1 (reference) and essential hypertension ID+DD 137 125 1.17 (1.00–1.37) Three SNPs (AGT A-6G, AGT T174 M and ACE I/D) displayed significant differences in the allele and Abbreviations: AGT, angiotensinogen; ACE, angiotensin-converting genotype frequencies between hypertensive and enzyme; AT1R, angiotensin type 1 receptor; CI, confidence interval; OR, odds radio. normotensive groups (Tables 2 and 3). The allele OR is adjusted for gender, age and BMI. frequencies of AGT A-6G with G allele, T174 M with T allele and ACE I/D with D allele were much higher in the hypertensive group (Po0.05). Table 3 Distribution of allele frequencies in hypertensive and The haplotypes composed of the four SNPs in the normotensive groups AGT gene are shown in Figure 1. Five inferred haplotypes with frequencies of more than 6% were Alleles Hypertensive Normotensives P examined to determine their association with hyper- AT1R A1166C A 421 447 0.798 tension in all subjects. The frequency of haplo- C1923 type H1(-6A, 174C, -217G and -20A) in the AGT A-6G A 346 400 0.014 hypertensive group was significantly lower than G9470 that in the normotensive group (w2 ¼ 10.92, AGT A-20C A 374 399 0.962 C6671 P ¼ 0.001). Furthermore, the logistic regression AGT G-217A G 359 397 0.285 analysis suggested that the H1 overtly reduced the A8173 risk of EH (OR ¼ 0.64; 95% CI ¼ 0.49–0.83), follow- AGT T174M C 337 397 0.003 ing adjusting the confounding factors of gender, age T 103 73 and BMI. ACE I/D I 274 332 0.009 D 166 138 On further analysis, it was revealed that AGT A-6G, T174 M and ACE I/D were significantly Abbreviations: ACE, angiotensin-converting enzyme; AGT, angioten- associated with EH (AGT A-6G: AG þ GG vs AA; sinogen; AT1R, angiotensin type 1 receptor. OR ¼ 1.36; 95% CI ¼ 1.04–1.77. T174M: CT þ TT vs CC; OR ¼ 1.45; 95% CI ¼ 1.15–1.90. ACE I/D: ID þ DD vs II; OR ¼ 1.171; 95% CI ¼ 1.00–1.37) indicated that BMI and ACE-I/D DD genotype might (Table 2). There was no significant difference in be positively correlated with the prevalence of EH. the pattern of genotype distribution of AT1R A1166C, AGT A-20C and G-217A. Discussion In addition, the forward stepwise multiple logistic regression analysis revealed that ACE-I/D DD and In this study, we investigated the association BMI might effect EH independently. The results between six polymorphisms in RAS genes and the

Journal of Human Hypertension RAS gene polymorphism and essential hypertension X Jiang et al 179 60 and expression.17–19 In a recent study, Dickson * et al.10 further testified the hypothesis that the 50 polymorphisms in the AGT promoter may affect cell Hypertensive function, specifically regulation in the level of AGT 40 Normotensive transcription in AGT-producing tissues. We can assume that AGT gene transcription may be induced 30 by the activity of several AGT gene variants, leading to the increased level of plasma AGT. As a result, the 20 Percent (%) Percent RAS system is further activated to exert a potent regulatory effect on BP. If the promoter variants in 10 the AGT gene are functional as reported,9,10 we 0 would expect an immediate association between H1 H2 H3 H4 H5 one or more polymorphisms in the AGT gene and ACGA ATGA GCGA ACAA ACGC EH occurrence. Evidence has been provided that the Figure 1 Haplotype frequency of AGT gene and logistic regres- polymorphism at A-6G site is associated with the 20 sion analyses. H ¼ haplotype; The allele order is (-6), 174 (-217) level of plasma AGT and the value of BP. However, and (-20); OR ¼ odds ratio; CI ¼ confidence interval; *Adjusted in another study, an association between A-6G and OR ¼ 0.64, 95% CI ¼ 0.49–0.83; Adjusted OR is estimated by EH was not identified in Caucasians or African logistic regression with adjustment for gender, age and BMI. The 5 other haplotypes, which are less than 6% are not included in the Americans. analysis. To evaluate the potential influence of the AGT gene on EH, we performed a haplotype analysis between the four SNPs of the AGT gene and the risk of EH. It might be more significant to detect the risk of EH in a subpopulation from Southern China. genetic factors because of the larger number of With regard to biases that may lead to potential alleles as compared with the number of genotypes. false-positive or false-negative results, our study Our analyses identified a haplotype, which was was carefully designed and performed in a some- significantly more common among normotensives, what unique population. All participants reside in indicating a protective role against hypertension. the regions with a lower level of economical (OR ¼ 0.64; 95% CI ¼ 0.49–0.83). The haplotype of development and less migration. The population AGT is composed of -6A, 174C, -217G and -20A had homogeneity of environments and never took alleles, all of which are considered wild types, any antihypertensive intervention (for normotensive indicating a recessive influence on EH. We may subjects). Therefore, these subjects were more postulate that the haplotype composed by four suitable to our study purpose than the common variant alleles increases the risk of EH, although source from hospital outpatients for evaluating the convincing supporting data are still lacking. In our genetic background of EH. Our results showed that study, it is interesting to note that -6A and 174C are the genetic frequencies at AGT A-6G, T174 M and more frequently distributed among normotensives ACE I/D sites were significantly different between using both allelic and haplotype analysis. This the hypertensive and normotensive groups. How- reasserts the protective role of these alleles. The ever, there was no significant difference at AT1R number of cases for haplotype analysis is somewhat A1166C, AGT G-217A and A-20C loci between the insufficient to draw any reliable conclusion. This two groups. hypothesis requires consolidation by larger sample The AGT gene encodes for the precursor molecule studies. Nevertheless, the results support the pos- for angiotensin II (Ang II), which causes profound tulation that multiple variants in the AGT gene vasoconstriction with an increased BP. As an influence BP regulation. Last but not the least, the association has been identified between the com- ACE I/D is a common variant in the ACE gene. There mon variant of M235 T in the AGT gene and EH has been some evidence suggesting that the ACE D occurrence,2 several studies were performed in an allele significantly influences ACE plasma levels,21 effort to identify other genetic polymorphisms especially in males.22 Consistently, our data showed which may influence BP.13–16 A community-based that the ACE I/D polymorphism was associated with study showed that the T174 M and M235 T poly- EH in this population. morphisms were associated with the onset of EH in We also combine the genetic and environmental Japanese, especially in those consuming high-salt factors (for example, sex, age, BMI, total cholesterol, diet,15 and this was consistent with the findings triglyceride and high density lipoprotein). Our data from our current study. suggested that BMI and ACE-I/D DD genotype might Recently, an intense attention has been made independently determine the risk of EH. However, towards the physiological relevance of the promoter no polymorphism in the AGT gene entered the of the AGT gene.9,10,13,14 Several functional studies regression mode, which may be caused by potential have indicated that AGT A-6G, A-20C and G-217A gene–gene interaction or small sample size. polymorphisms in the promoter region play impor- EH is a disease involving multiple genes and tant roles in regulating the AGT gene transcription genetic polymorphisms. It is believed that any

Journal of Human Hypertension RAS gene polymorphism and essential hypertension X Jiang et al 180 single gene or its polymorphism is capable of angiotensin II type 1 receptor gene polymorphism and affecting, albeit weakly, the onset and progression essential hypertension: the Ohasama Study. Hypertens of EH.23 It is probably one of the main reasons Res 2004; 27: 551–556. contributing to the inconsistent results from various 8 Bae Y, Park C, Han J, Hong YJ, Song HH, Shin ES et al. studies. Interaction between GNB3 C825 T and ACE I/D polymorphisms in essential hypertension in Koreans. In conclusion, this study has provided further J Hum Hypertens 2007; 21: 159–166. evidence that ACE-I/D DD genotype and BMI 9 Jain S, Tang X, Narayanan CS, Agarwal Y, Peterson SM, may act independently on the prevalence of EH, Brown CD et al. Angiotensinogen gene polymorphism and the AGT T174 M and A-6G are closely asso- at -217 affects basal promoter activity and is associated ciated with EH. Although, the haplotype of AGT with hypertension in African-Americans. Biol Chem composed of -6A, 174C, -217G and -20A alleles may 2002; 277: 36889–36896. be negatively associated with the risk of EH. The 10 Dickson ME, Zimmerman MB, Rahmouni K, Sigmund main limitation of this study includes relatively low CD. The -20 and -217 promoter variants dominate sample size, which did not allow a gender-based differential angiotensinogen haplotype regulation in analysis. Nonetheless, our study still indicated angiotensinogen-expressing cells. Hypertension 2007; 49: 631–639. that genetic polymorphisms in RAS genes might 11 Farahani P, Dolovich L, Levine M. Exploring design- be associated with EH. A larger scale study related bias in clinical studies on receptor genetic with independent samples is warranted to further polymorphism of hypertension. J Clin Epidemiol 2007; validate our findings. 60: 1–7. 12 Jiang X, Sheng HH, Lin G, Li J, Lu XZ, Cheng YL et al. 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