Association of angiotensin II type 1 receptor polymorphism with resistant essential hypertension

Clinica Chimica Acta 269 (1998) 91–100

Association of angiotensin II type 1 receptor polymorphism with resistant essential hypertension ´ Szombathy a , Csaba Szalai b , Barna Katalin c , Tamas ´ Palicz a , Tamas a a, ´ ´ Romics , Albert Csaszar ´ ´ * Laszlo a

Third Department of Medicine, Semmelweis University of Medicine, Budapest, Hungary ´ ’ s Hospital for Children, Molecular Genetics Laboratory, Budapest, Hungary Heim Pal c ¨ Blood Transfusion Unit, Szentgyorgyi Albert University of Medicine, Szeged, Hungary

b

Received 22 September 1997; accepted 26 September 1997

Abstract Angiotensin II type l receptor (AT 1 ) mediates the vasoconstrictive and growth-promoting effect of angiotensin II in humans. It has been reported that a polymorphism of the AT 1 gene (an A / C transversion at position 1166: A-C 1166 ) occurs more frequently in resistant hypertensives taking two or more antihypertensive drugs. On the contrary, a recent study of the influence of the A-C 1166 polymorphism on aortic stiffness demonstrated that the distribution of the genotypes did not differ between normotensive and hypertensive subjects. In addition, a recent population-based survey of Caucasian hypertensives reported lower blood pressure values in CC homozygotes than in heterozygotes and AA homozygotes. Because of these controversial results and the lack of a sufficient amount of data the present study was designed to assess the contribution of the AT, gene A-C 1166 polymorphism to resistant essential hypertension. Forty-eight subjects with resistant essential hypertension (HT) and 48 normotensive (NT), age and sex-adjusted controls (from a population of 300 healthy blood donors) were selected. All subjects were genotyped for the A-C 1166 polymorphism in the 39-UTR of the AT 1 gene using PCR-based techniques. The influence of genotype on blood pressure (BP) was investigated using ANOVA Randomized Complete Block (ANOVA RCB) design according to sex, age and BMI. There were no significant differences in allele or genotype frequencies between HT and NT subjects (X 2 5 0.61; P 5 NS). In HT subjects higher values of systolic blood pressure were associated with the C allele of the AT 1 gene only in older and overweight patients (P , 0.001 and P , 0.001, respectively). Also in HT patients an association between the presence of the C allele of the AT 1 gene and higher values of diastolic blood pressure was present in overweight patients (P 5 0.001). These results suggest that in

*Corresponding author: Tel.: (361) 395 85 33; fax: (361) 155 7183; e-mail: [email protected] 0009-8981 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0009-8981( 97 )00184-8

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resistant hypertensive subjects the AT 1 A-C 1166 polymorphism is potentially involved in the regulation of blood pressure. As the effects of genotypes on blood pressure are pronounced in older and overweight subjects this polymorphism may amplify the effects of age and BMI on resistant essential hypertension.  1998 Elsevier Science B.V. Keywords: Angiotensin II type 1 receptor; Polymerase chain reaction; A-C 1166 polymorphism; Blood pressure (human)

1. Introduction Essential hypertension results from the combined influence of environmental and genetic factors. Although environmental determinants predisposing to hypertension are well described the genetic components increasing such susceptibility remain unknown in the vast majority of subjects [1]. With advances in molecular techniques, several loci influencing blood pressure have been identified in rodents [1,2]. According to the latest results, several genetic loci seem to be related to the renin-angiotensin system in the pathogenesis of hypertension in humans [1–4]. The main effector of this system is the AT 1 receptor through which angiotensin II (Ang II) exerts its major vasoconstrictive effect and, hence, modulating blood pressure [5,6]. The gene of the human AT 1 has recently been cloned and sequenced [7]. Several diallelic polymorphisms have been detected in the coding and 39 untranslated regions of this gene [8]; however, none of them are functional. An increase in the frequency of one variant (an A / C transversion located at the 59 end of the 39 untranslated region, position 1166) has been observed in hypertensives and a further increase in the frequency has been found in subjects with a more resistant form of essential hypertension in which two or more antihypertensive drugs are used to adequately control blood pressure [8,9]. In a recent study of the influence of the AT 1 gene polymorphisms on aortic stiffness the distribution of the AT 1 genotypes did not differ between normotensive and hypertensive subjects [10]. According to another population-based study conducted in Italy blood pressure values were lower in CC homozygotes than in heterozygotes and AA homozygotes [11]. In addition, synergistic effects of AT 1 and angiotensin-converting enzyme gene polymorphisms in myocardial infarction have been reported by Tiret et al. [12]. Due to the controversial findings and small amount of data about the AT 1 polymorphisms we aimed at further investigating the possible effects of a common AT 1 receptor gene variant on resistant essential hypertension.

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2. Materials and methods

2.1. Subjects Forty-eight resistant essential hypertensives (HT) on chronic treatment for hypertension were selected according to following criteria: (1) aged 20–70 years, (2) onset of hypertension less than 60 years, (3) absence of secondary forms of hypertension through an extensive workup, (4) family history of hypertension with at least one parent affected, (5) elevated blood pressure ( . 140 / 90 mmHg) measured on the day of admission and on two other occasions, (6) administration of two or more antihypertensive drugs (calcium antagonist, angiotensin converting enzyme inhibitor, b-blocker and diuretic) for more than six months, (7) the presence of hypertensive retinopathy. Exclusion criteria were as follows: (1) treatment with adrenal steroid, nonsteroidal anti-inflammatory drugs, oral contraceptives, exogenous thyroid hormone replacement or sympathomimetics, (2) diabetes mellitus, (3) renal failure, (4) ethanol intake more than 40 g day, (5) physical exercise and / or pain prior to or during clinic blood pressure reading, (6) caffeine intake or smoking within one hour of blood pressure reading, (7) intercurrent disease and / or fever, (8) non-adherence to therapy. Forty-eight age and sex-adjusted, normotensive control subjects were selected from a population of 300 healthy blood donors. Selection was made on the following criteria: (1) aged 20–70 years, (2) blood pressure less than 140 / 90 mmHg, (3) absence of antihypertensive treatment. All the subjects gave informed consent to the study procedures.

2.2. Assessment of cardiovascular risk factors In each subject, a careful medical history was taken mainly concerning family history of hypertension, stroke, ischemic heart disease, diabetes mellitus and dyslipidaemia. Total serum cholesterol, serum triglyceride and HDL were measured by conventional assays. In each subject body mass index was calculated.

2.3. BP measurements Blood pressure was measured on admission followed by BP reading on the next three consecutive days with subjects in the supine position using a sphygmomanometer. Fifth phase Korotkoff sounds were taken as diastolic blood pressure.

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2.4. Genotyping Using genomic DNA, extracted from peripheral blood leucocytes by standard methods, genotypes for the A / C 1166 polymorphism of the AT 1 receptor gene were determined by a mismatch-PCR / RFLP strategy. The primers used for polymerase chain reaction amplification of the AT 1 receptor region including the A-C 1166 polymorphism were those used by Hingorani and Brown [13]. The PCR amplification was performed in a volume of 25 ml containing 100 ng of genomic DNA, 25 pmol of each primer, 0.2 mmol / l of each dNTP, 10 mmol / l Tris–HCl (pH 9.0), 50 mmol / l KCl, 1,5 mmol / l of MgCl 2 , 0.1% Triton X-100 and 1 U Taq DNA polymerase (Boehringer). After an initial denaturation step of 2 min at 958C, 40 cycles of PCR at 948C for 60 sec, annealing at 508C for 60 sec and extension at 728C for 2.5 min were carried out. One microgram of the amplified product was incubated with 5 U of Afl II and the manufacturer’s buffer (Sigma) for 16 h at 378C. Digested products were separated by electrophoresis on 2.5% agarose gels with ethidium bromide staining and genotypes were designated as AA, AC and CC.

2.5. Statistical analysis The effects of the AT 1 A-C 1166 polymorphism on blood pressure was tested using ANOVA randomized complete block (ANOVA RCB) design with sex, age and BMI as grouping variables. Interaction tests were performed to assess the homogeneity of genotype–phenotype association in hypertensive subjects. Analysis of differences in proportions between genotypes was conducted by x 2 test. Statistical analysis was performed with the Statistica for Windows 5.0 (Statsoft, Inc.) software package. Probability (P) values are reported for each test.

3. Results Among hypertensive subjects, 28 (58%) received a combination of ACE inhibitor and calcium antagonist, six (12%) were treated with a combination of b-blocker and diuretic, 14 (30%) with a triple combination of ACE inhibitor, calcium antagonist and diuretic. The main characteristics of the hypertensive and normotensive subjects are shown in Table 1. There was no significant age-difference between males and females in the study groups. The differences in SBP between hypertensive and normotensive subjects were 42 and 47 mmHg in male and female, respectively, whereas for DBP the differences were 18 and 21 mmHg, respectively. The distribution of AT 1 A-C 1166 genotypes and allele frequencies in nor-

Age, years SBP, mmHg DBP, mmHg

Hypertensive Subjects

Normotensive Subjects

P by ANOVA

Male (n 5 28)

Female (n 5 20)

Male (n 5 23)

Female (n 5 25)

Male vs. Female

Hypertensive vs. Normotensive

48.8611.66 169.1616.91 99.268.8

51.7610.64 172.5616.04 100.166.44

46.2610.3 127.766.73 81.667.74

49.469.7 125.766.36 79.566.64

NS NS NS

NS , 0.001 , 0.001

Values are expressed as mean6SD. SBP indicates systolic blood pressure; DBP diastolic blood pressure.

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Table 1 Main Characteristics of Hypertensive and Normotensive Subjects

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Table 2 Distribution of AT 1 A-C 1166 Genotypes in Normotensive and Hypertensive Subjects AT 1 A-C 1166

Normotensive

Genotype

n (%)

Male / Female

n (%)

Male / Female

AA AC CC A/C

23(48) 19(40) 6(12) 0.67 / 0.33

7 / 16 12 / 7 4/2

26(54) 18(38) 4(8) 0.73 / 0.27

16 / 10 10 / 8 2/2

Hypertensive

Values indicate the number of observations (total percentage).The ratio of allele frequencies (A / C) are indicated in fraction. ‘A’ and ‘C’ represent the two alleles of a polymorphism. No deviation from Hardy–Weinberg equilibrium was found. The distribution of the AT 1 receptor genotypes do not differ between normotensive and hypertensive groups.

motensive and hypertensive subjects are listed in Table 2. In both groups, genotype frequencies did not deviate significantly from the Hardy–Weinberg equilibrium and there were no significant differences in allele or genotype frequencies between HT and NT subjects (X 2 5 0.61; P 5 NS). There was a variation in sex ratio of the AA genotype between hypertensive and normotensive subjects. The main characteristics of hypertensive subjects according to AT 1 A-C 1166 polymorphism are shown in Table 3. In hypertensive subjects, age was a variable to differ between genotypes. The mean values of BMI, serum levels of total cholesterol, triglycerides and HDL were similar in the three genotype categories. Because of the variation in age and sex ratio of the various groups of genotypes the homogeneity of effect of the AT 1 A-C 1166 polymorphism on systolic and diastolic blood pressure values, according to sex, the median of age and BMI, was also investigated (Table 4 and Table 5). Overall, the effect of the AT 1 A-C 1166 polymorphism was not similar across the different categories. Table 3 The Characteristics of Hypertensive Subjects According to AT 1 Genotype Parameter

Age,years BMI, kg / m 2 TC, mmol / l TG, mmol / l HDL, mmol / l

Genotype AA (n 5 26)

AC (n 5 18)

CC (n 5 4)

48.7610.40 29.363.60 5.760.6 1.660.6 1.060.28

49.7612.10 31.264.61 5.961.0 2.562.11 1.060.34

60.269.17 33.064.80 5.760.32 1.660.52 1.1360.05

M / F, male / female; BMI, body mass index; TC, total serum cholesterol; TG, serum triglycerides; HDL, high density lipoprotein.

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Table 4 Effects of the AT 1 A-C 1166 polymorphism on Systolic Blood Pressure according to Sex, Median of Age and BMI Pa

Grouping

Genotype

variable

AA

AC

161616.9 161.7614.57

179.969.22 18369

161.3620.19 161.267.28 162.160.66 160.364.88

Sex Men Women Age , Median $ Median BMI , Median $ Median

Homogeneity, P

CC 180614.14 18567.07

.008 .004

NS

181.1612.27 181.465.94

18066.72 183.3611.54

NS , .001

.001

178.564.84 183.5611.05

19062.7 180610

NS , .001

, .001

Values are mean6SD. a Test of codominant effect.

There was an association between the presence of the C allele and systolic blood pressure both in men and women (P 5 0.008 and P 5 0.004, respectively). For diastolic blood pressure, this association was present only in women (P 5 0.008), but there was no statistical heterogeneity between sexes. The A-C 1166 polymorphism had a stronger effect on systolic blood pressure in older patients (heterogeneity according to median of age, P 5 0.001). There was a heterogeneity in categories of BMI (P , 0.001 for SBP and P 5 0.02 for DBP), indicating that the effect of A-C 1166 polymorphisrn on blood pressure was most pronounced in overweight hypertensive subjects. Table 5 Effects of the AT 1 A-C 1166 polymorphism on Diastolic Blood Pressure according to Sex, Median of Age and BMI Grouping

Genotype

variable

AA

AC

95.969.53 96.766.36

103.565.77 101.963.56

10562.88 11061.75

96.269.9 96.166.0

103.664.3 102.165.38

97.569.8 94.465.3

105.464.27 100.764.42

Sex Men Women Age , Median $ Median BMI , Median $ Median

Values are mean6SD. a Test of codominant effect.

P*

Homogeneity, P

NS 0.008

NS

11061.54 106.662.88

NS 0.01

NS

110 106.762.88

NS 0.001

0.02

CC

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4. Discussion The aim of the present study was to assess the influence of the AT 1 receptor A-C 1166 polymorphism on the hypertensive status and the severity of hypertension in subjects on multiple-drug treatment. Our study confirms that the presence of the C allele of the AT 1 receptor gene is associated with a tendency towards more severe hypertension in older and overweight patients. The presence of this polymorphism in these subjects might contribute to a more pronounced potentiation of angiotensin II effects related to alterations in the activity or number of the AT 1 receptor [14–16]. In a recent study of angiotensin II type 1 receptor gene polymorphisms the frequency of the C 1166 allele was higher in hypertensives compared with normotensive controls [8]. In addition, a further increase in the frequency of C 1166 was observed in the severe hypertensive group including subjects with higher diastolic blood pressure values ( $ 105 mmHg) or on multiple-drug treatment for hypertension [8]. Using the criteria of ‘severe hypertension’ defined as two or more antihypertensive drugs are administered [8–10] we found that the presence of the C allele tracks with higher blood pressure values within the hypertensive group. This observation suggests that this polymorphism might be close to a locus (or loci) influencing patient response to antihypertensive treatment. To find evidence of such loci more selected groups of resistant hypertensives should be investigated. In accordance with more recent data [11], we did not confirm an increase in the frequency of C 1166 allele in the hypertensive group compared with normotensive controls. In our study the CC genotype was also present in a relatively small proportion of normotensives (12%) and hypertensives (8%), similar to previous reports [12,17,18]. On the other hand, we found that the A-C 1166 variant was a predictor of higher blood pressure values in the hypertensive group which is the opposite of a recent observation that subjects with the C allele present lower blood pressure values [11]. As the mean blood pressure values in AC heterozygotes of the hypertensive group were intermediate between those of the two groups of homozygotes the effect of the A-C 1166 polymorphism on blood pressure appears codominant, similar to previous reports [8,10]. According to a recent population-based survey, the angiotensinogen T235 variant is associated with the use of two or more antihypertensive drugs to control blood pressure [19]. In addition, the presence of the C allele of the AT 1 receptor gene has been reported to influence the efficacy of nitrendipine or perindopril treatment in hypertensives [12]. These observations support our finding that the A-C 1166 polymorphism may be of particular interest in the development of resistance to antihypertensive therapy. Although the AT 1 A-C 1166 polymorphism occurs in a noncoding region of the

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gene, its association with higher blood pressure values suggests that it might be considered as a candidate marker of the severity of hypertension in a subgroup of hypertensives on multiple-drug treatment. In addition, this genetic variant may be in linkage disequilibrium with other functionally relevant genetic polymorphisms altering the structure and expression of the AT 1 receptor. Independent confirmation of the present findings will be of critical value with regard to the relevance of this variant in other populations and in the prediction of patient response to antihypertensive therapy.

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