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Angiotensin II Type 1 Receptor Gene Polymorphism Is Associated With Increase of Left Ventricular Mass But Not With Hypertension
AJH
1998;11:316 –321
Angiotensin II Type 1 Receptor Gene Polymorphism Is Associated With Increase of Left Ventricular Mass But Not With Hypertension Seiju Takami, Tomohiro Katsuya, Hiromi Rakugi, Noriyuki Sato, Yukiko Nakata, Atsushi Kamitani, Tetsuro Miki, Jitsuo Higaki, and Toshio Ogihara
A genetic epidemiologic approach is useful to elucidate the genes responsible for hypertension. Genetic analyses of the components of the reninangiotensin system have succeeded in showing an association between their polymorphism and hypertension. Recently, two types of angiotensin II receptor were cloned and characterized. To examine the genetic contribution of angiotensin II type 1 receptor (AT1) and type 2 receptor (AT2) genes in human essential hypertension, a casecontrol study was performed in Japanese subjects. The study comprised 321 subjects with hypertension who satisfied the criteria for essential hypertension, together with 215 age and sex matched controls. The significance of the differences in genotype distribution between hypertensive and normotensive subjects was
M
ultiple environmental and genetic factors are involved in the pathogenesis of human hypertension. Genetic epidemiologic approaches have identified the genes responsible for some types of hereditary hypertension, but they are quite rare.1–5 Most cases of hu-
Received June 10, 1997. Accepted October 13, 1997. From the Department of Geriatric Medicine, Osaka University Medical School, Osaka, Japan. Address correspondence and reprint requests to Toshio Ogihara, MD, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita City, Osaka 565, Japan.
© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
examined by x2 analysis. Neither AT1 nor AT2 gene variants were associated with human essential hypertension in the Japanese subjects. However, the AT1 receptor gene polymorphism was associated with left ventricular mass index in normotensive subjects. The study results suggest that gene polymorphisms of both angiotensin II receptors are not directly involved in the increase of genetic risk for hypertension, but that the AT1 receptor gene might contribute genetically to the increase of left ventricular mass. Am J Hypertens 1998;11:316 –321 © 1998 American Journal of Hypertension, Ltd.
KEY WORDS:
Genetics, hypertension, renin angiotensin system, receptor, echocardiography.
man hypertension are classified as essential hypertension with unknown cause. The renin-angiotensin system (RAS) plays an important role in blood pressure regulation, and recent advances in molecular biology have highlighted the genetic importance of some components of RAS, such as angiotensin converting enzyme (ACE) and angiotensinogen (AGT), in the pathogenesis of cardiovascular disease. Recently, two subtypes of angiotensin II receptors, angiotensin II type 1 receptor (AT1) and angiotensin II type 2 receptor (AT2), were cloned and characterized.6 – 8 Gene targeting approaches have suggested that both genes are involved in blood pressure regu0895-7061/98/$19.00 PII S0895-7061(97)00457-3
AJH–MARCH 1998 –VOL. 11, NO. 3, PART 1
AT1 GENE AND LVM IN HYPERTENSION
317
TABLE 1. CHARACTERISTICS OF ESSENTIAL HYPERTENSIVE AND NORMOTENSIVE SUBJECTS
Family history (one first degree relative) Number Age (years) Sex (male/female) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) Brinkman index† Alcohol intake (mL/day)† CTR RV51SV1 (mV) T.Chol (mmol/L) HDL-Chol (mmol/L) TG (g/L) FPG (mmol/L) F-IRI (mU/mL) HbA1c PG/2h (mmol/L) BUN (mmol/L) Crnn (mmol/L) UA (mmol/L) LV mass index
Hypertensives
Normotensives
P Value
1 321 62.1 6 10.1 157/164 24.0 6 3.2 178.3 6 17.1 102.9 6 12.1 347 6 40 18.8 6 1.9 0.51 6 0.06 3.3 6 1.0 5.4 6 0.9 1.4 6 0.4 1.5 6 0.8 5.7 6 1.2 5.7 6 2.5 0.06 6 0.01 7.3 6 2.9 5.6 6 1.5 80.4 6 44.2 315 6 89 138 6 52
2 215 60.9 6 11.4 113/102 22.9 6 2.9 125.9 6 14.0 75.9 6 9.2 487 6 58 17.8 6 2.6 0.48 6 0.05 2.8 6 0.8 5.4 6 0.9 1.4 6 0.4 1.3 6 0.7 5.5 6 1.3 7.3 6 5.2 0.06 6 0.01 8.4 6 4.6 5.6 6 1.2 70.7 6 17.7 303 6 125 110 6 41
NS NS* ,.001 ,.0001 ,.0001 ,.05 NS ,.005 ,.0001 NS NS NS NS NS NS NS NS ,.05 NS ,.0005
Values of blood pressure are expressed as mean 6 SD. Comparison was performed by ANOVA. * Comparison was performed by x2 test. † Data are expressed as mean 6 SE. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; CTR, cardiothoracic ratio; T.Chol, total cholesterol; HDL-Chol, HDL-cholesterol; TG, triglyceride; FPG, fasting plasma glucose; F-IRI, fasting-immunoreactive insulin; HbA1c, glycosylated hemoglobin; PG/2h, plasma glucose 2 h after eating; BUN, blood urea nitrogen; Crnn, creatinine; UA, uric acid; LV mass index, left ventricular mass index.
lation and modulation of the effect of angiotensin II in different manners.9 –12 An association study in a white population showed that an A to C nucleotide substitution in the 39 untranslated region of human AT1 gene (A1166C/AT1) was associated with hypertension.13 This polymorphism was also independently associated with aortic stiffness, and upregulation of AT1 receptor gene was observed in the ventricles of cardiomyopathic hamsters.14 –16 On the other hand, recent reports revealed that AT2 receptor has an antiproliferative effect on neointima formation following vascular injury, and that its mRNA level is increased after myocardial infarction.17,18 We cloned the entire region of human AT2 receptor genomic DNA and mapped it to the human X chromosome, which contains a candidate locus for rat hypertension, denoted as BP/SP-2.19,20 Furthermore, a polymorphism was identified in the 39 untranslated region of AT2 receptor gene, but how it is genetically involved in cardiovascular disease remains unclear. To assess the role of AT1 and AT2 receptor gene polymorphisms in essential hypertension, we performed an association study in the Japanese population, which has an ethnically homogeneous ge-
netic background as opposed to the white population. We further examined the possible effects of these gene polymorphisms on cardiac hypertrophy
TABLE 2. GENOTYPES AND ALLELE FREQUENCIES OF AT1 RECEPTOR GENE POLYMORPHISM AND COMPARISONS BETWEEN HYPERTENSIVE AND NORMOTENSIVE SUBJECTS AT1 (A11663C) Genotype HBP NBP
CC 4 3
AC 56 40
AA 261 172
Total 321 215
x2 5 0.15, P 5 NS Allele frequency HBP NBP
C 0.10 0.11
A 0.90 0.89
x2 5 0.15, P 5 NS A11663 C: substitution from adenine to cytosine at 1166 nucleotide position in 39 untranslated region of AT1 receptor gene. HBP, hypertensive subjects; NBP, normotensive subjects. Comparison was performed by x2 test.
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TABLE 3. SEX MATCHED COMPARISONS OF AT1 AND AT2 RECEPTOR GENE POLYMORPHISMS BETWEEN HYPERTENSIVE AND NORMOTENSIVE SUBJECTS AT1 (A11663C) Male Genotype HBP NBP
CC 2 1
AC 32 23
AA 123 89
AT2 (C31233A) Total 157 113
A2 60 36
x2 5 0.09, P 5 NS Allele frequency HBP NBP
C 0.11 0.11
A 0.89 0.89
CC 2 2
AC 24 17
A 0.38 0.32
C 0.09 0.10
C 0.62 0.68
x2 5 1.13, P 5 NS
AA 138 83
Total 164 102
AA 18 7
x2 5 0.46, P 5 NS Allele frequency HBP NBP
Total 156 112
x2 5 1.13, P 5 NS
x2 5 0.02, P 5 NS Female Genotype HBP NBP
C2 96 76
A 0.91 0.90
x2 5 0.47, P 5 NS
CA 67 42
CC 79 53
Total 164 102
x2 5 1.32, P 5 NS A 0.31 0.27
C 0.69 0.73
x2 5 0.94, P 5 NS
Comparison was performed by x2 test.
and biochemical parameters responsible for cardiovascular disease.
were obtained in the fasting state. Informed consent was obtained from each participant.
METHODS
Evaluation of Left Ventricular Mass To evaluate left ventricular mass, echocardiographic measurements (two dimensional targeted M-mode and pulsed Doppler) were performed in 110 hypertensives and 61 normotensives using an ultrasound imager (SonolayerSSA260A, Toshiba Medical Co., Tokyo, Japan) with a 2.5 MHz transducer. Left ventricular volume and thickness of the intraventricular septum and left ventricular posterior wall were measured following the Penn convention. Left ventricular mass was calculated by the formula validated by Devereux and Reichek.21 Left ventricular mass index (LVMI) was obtained by dividing left ventricular mass by body surface area. Echocardiography was performed by observers blinded to the genetic background and BP of the patients.
Subjects and Measurements The study population was randomly selected from the outpatients at Osaka University Hospital. These participants were all Japanese and consisted of 321 hypertensive subjects and 215 normotensive subjects (Table 1). Hypertensive patients had been diagnosed according to conventional criteria, which consisted of a systolic blood pressure (SBP) . 160 mm Hg or a diastolic blood pressure (DBP) . 95 mm Hg without antihypertensive treatment. Blood pressure (BP) was measured in each subject in the supine position, without antihypertensive treatment, on three occasions spanning at least 1 month. Patients with any secondary cause for hypertension were excluded. The prevalence of diabetes mellitus was similar in hypertensives (0.15) and normotensives (0.14). A detailed family and past history was taken for each subject. All patients had at least one first degree relative with essential hypertension. The normotensive subjects without a family history of hypertension in first degree relatives were selected from the same population, matching for age and sex and satisfying the criteria that SBP and DBP were ,140 and ,90 mm Hg, respectively. Blood samples
Determination of Genotypes AT1 Receptor Participants’ DNA was extracted from leukocytes using the QIAamp kit (Quiagen Inc., Valencia, CA). Polymerase chain reaction was performed to amplify a fragment encompassing the A 3 C polymorphic site at 1166 nucleotide position in the 39 untranslated region of human AT1 receptor gene. The design of the primers was as follows: sense, 59-ATAATGTAAGCTCATC-
75 6 9 104 6 13 177 6 15
125 6 13
79 6 8 76 6 9 108 6 19 103 6 12
SBP, systolic blood pressure; DBP, diastolic blood pressure. All values given in millimeters of mercury as mean 6 SD. * P 5 .0001 v AA & AC together (ANOVA). † P 5 .0005 v both AA and AC (ANOVA). All other P values were nonsignificant.
126 6 14 125 6 13 103 6 12 104 6 14 178 6 17 179 6 18 76 6 9 79 6 8 126 6 14 131 6 12 103 6 12 108 6 19 178 6 17 211 6 27* AA & AC CC AA AC & CC AC
AT1 GENE AND LVM IN HYPERTENSION
319
CACC-39; antisense, 59-GAGATTGCATTTCTGTCAGT-39. The reaction volume was 30 mL, which contained 100 ng genomic DNA, 10 pmol of each primer, 250 mmol/L dNTP, 1.0 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl at pH 8.3, and 0.5 unit Taq polymerase (Perkin Elmer, Branchburg, NJ). Amplification was carried out using an Omni Gene Thermal Cycler (Hybaid Limited, Teddington, Middlesex, U.K.). After a heating step of 5 min at 94°C, the main reaction, 94°C for 30 sec, 55°C for 45 sec, and 72°C for 45 sec, was repeated for 40 cycles, and then followed by an extension step at 72°C for 10 min. Polymerase chain reaction products were confirmed to show exact amplification, and were then digested with DdeI (Toyobo, Osaka, Japan) for 3 h at 37°C. The digested products were visualized on 3% Sea Kem HGT agarose gel (FMC BioProducts, Rockland, ME) by ethidium bromide staining.
76 6 9 76 6 9
211 6 27† 178 6 17
131 6 12 126 6 14
DBP SBP DBP SBP DBP SBP DBP SBP DBP SBP DBP
Hypertensive
SBP
Hypertensive Normotensive Normotensive
Hypertensive
Dominant Recessive
TABLE 4. COMPARISON OF BLOOD PRESSURE ACCORDING TO AT1 GENOTYPES
Codominant
Normotensive
AJH–MARCH 1998 –VOL. 11, NO. 3, PART 1
AT2 Receptor We previously reported a C to A mutation at the 3123 nucleotide position in the 39 untranslated region of AT2 receptor gene.19 This mutation altered the sequence of the AluI recognition site. We designed the following primers: sense, 59-GGATTCAGATTTCTCTTTGAA-39; antisense, 59-GCATAGGAGTATGATTTAATC-39. Polymerase chain reaction was performed under the same conditions as for AT1 receptor gene except for the annealing temperature, which was set at 53°C. After confirming DNA amplification, 10 mL of PCR product was digested with 12 units AluI (Takara Biotechnology, Osaka, Japan) for 3 hours at 37°C, and electrophoresed on 3% agarose gel with ethidium bromide staining. Statistical Analysis All statistical analyses were conducted using the StatView J-4.5 program (Abacus Concepts, Inc., Berkeley, CA) and JMP 3.0 (SAS Institute, Inc., Cary, NC). The significance of differences in classified values between hypertensives and normotensives was examined using x2 analysis with one or two degrees of freedom. The mean value of clinical data for each genotype was compared by one way analysis of variance (ANOVA). The association between AT1 polymorphism and BP was examined considering all the inheritance models of C allele, that is, recessive (CC v AC1AA), dominant (CC1AC v AA), and codominant (CC v AC v AA). Furthermore, multiple logistic regression analysis was performed separately in hypertensive and normotensive subjects to evaluate the effects of the AT1 and AT2 polymorphisms on LVMI after adjustment for age and sex. RESULTS The characteristics of the population are shown in Table 1. The genotype distribution and allele frequencies of AT1 polymorphism in patients and control subjects are summarized in Table 2. The genotype distribution of
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TABLE 5. COMPARISON OF ECHOCARDIOGRAPHIC MEASUREMENTS ACCORDING TO AT1 GENOTYPES Hypertensives
IVSth (mm) PWth (mm) LV mass index
Normotensives
C Allele (2) (mean 6 SD)
C Allele (1) (mean 6 SD)
P (ANOVA)
C Allele (2) (mean 6 SD)
C Allele (1) (mean 6 SD)
P (ANOVA)
10.4 6 2.2 10.6 6 2.1 137 6 54
11.3 6 2.7 11.1 6 2.1 145 6 45
NS NS NS
9.3 6 1.9 9.5 6 1.9 107 6 35
10.9 6 1.9 11.3 6 2.3 138 6 53
,.01 ,.01 ,.05
IVSth, intraventricular septum thickness; PWth, left ventricular posterior wall thickness; LV mass index, left ventricular mass index; BSA, body surface area. LV mass index 5 [1.04 3 {(LVDd 1 IVSth 1 PWth)392(LVDd))3}213.6]/BSA. BSA 5 (Body weight)0.425 3 (Height)0.725 3 7.184 3 1023 m2.
AT1 polymorphism did not significantly deviate from the expected genotype frequencies, which were deduced from Hardy-Weinberg equilibrium. Because the AT2 receptor gene is located on the human X chromosome,8 AT2 receptor gene polymorphism was examined in the male and female groups separately (Table 3). The gene distribution also satisfied Hardy-Weinberg’s law. No significant differences in the genotype distribution of the AT1 and AT2 genes were observed between hypertensives and controls in total, or in the male or the female group (Tables 2 and 3). Blood pressure values in the study population according to AT1 receptor genotype are summarized in Table 4. The hypertensive subjects who were AT1/CC homozygous had significantly higher SBP than the subjects who were heterozygous or AT1/AA homozygous in the recessive model. There was no significant difference in BP among the subgroups of AT2 receptor genotypes (data not shown). Moreover, the subjects with AT1/C allele in the normotensive but not the hypertensive group had significantly thicker intraventricular septum and left ventricular posterior wall than those without AT1/C allele (Table 5). In the normotensive subjects, a higher LVMI was also significantly associated with AT1/C allele (Table 5). Concordantly, multiple logistic regression analysis showed that AT1 had a significant effect on LVMI (R2 5 0.21, F ratio 5 4.7, P 5 .03). DISCUSSION Recently, both AT1 and AT2 gene disrupted mouse models have suggested the involvement of these genes in blood pressure regulation.10 –12 Our results suggest that the C allele of AT1 receptor gene has a hypertensive effect in the recessive model of inheritance. However, the number of subjects with AT1/CC genotype was too small to confirm this effect. Apparently, neither AT1 nor AT2 gene polymorphism was associated with essential hypertension in the present case-control study. In whites, Bonnardeaux et al13 reported a positive association between AT1/C allele and essential hyper-
tension in a case-control study, whereas they failed to detect such an association using affected sibling pair analysis. In the present case-control study in Japanese subjects, no association between AT1/C allele and hypertension was detected. Both the white sibling pairs and the Japanese population are genetically homogeneous, whereas the general white population is not. Considering the power of statistical analysis, the result of genetic analysis using a genetically homogeneous population is more reliable. Therefore, we conclude that AT1/C allele is not associated with the occurrence of essential hypertension even though C allele frequency is obviously rarer in Japanese than in whites. On the other hand, AT1 receptor gene polymorphism showed a positive association with LVMI in normotensives. Statistical analysis of our data suggests that the AT1 receptor gene contributes to left ventricular mass and that its genetic effect is independent of sex, age, and BP. The reason why the association was not confirmed in hypertensives may have been that this effect was masked in the hypertensive group by the influence of high BP, which is the highest risk factor for left ventricular hypertrophy. Similarly, ACE/DD genotype was also reported as a risk factor for left ventricular hypertrophy in normotensive subjects but not in hypertensive subjects.25,26 Recently, Castellano et al27 reported the lack of association between AT1/A1166C polymorphism and LVMI in the randomly selected general population. However, as they stated, the number of subjects was probably not sufficient for the confident exclusion of the possibility of a b-type error. Another possible interpretation of the conflicting results is that their study population consisted of both normotensive and hypertensive subjects. In summary, we conclude that the AT1 and AT2 receptor genes are not involved in the occurrence of essential hypertension, and that AT1/C allele may be a candidate to affect left ventricular mass in the Japanese normotensive population. Further investigation, however, such as examination of the gene regulatory mechanisms, is required to elucidate the genetic con-
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angiotensin II type 1 receptor polymorphism on aortic stiffness in never-treated hypertensive patients. Hypertension 1995;26:44 – 47.
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1998;11:316 –321
Angiotensin II Type 1 Receptor Gene Polymorphism Is Associated With Increase of Left Ventricular Mass But Not With Hypertension Seiju Takami, Tomohiro Katsuya, Hiromi Rakugi, Noriyuki Sato, Yukiko Nakata, Atsushi Kamitani, Tetsuro Miki, Jitsuo Higaki, and Toshio Ogihara
A genetic epidemiologic approach is useful to elucidate the genes responsible for hypertension. Genetic analyses of the components of the reninangiotensin system have succeeded in showing an association between their polymorphism and hypertension. Recently, two types of angiotensin II receptor were cloned and characterized. To examine the genetic contribution of angiotensin II type 1 receptor (AT1) and type 2 receptor (AT2) genes in human essential hypertension, a casecontrol study was performed in Japanese subjects. The study comprised 321 subjects with hypertension who satisfied the criteria for essential hypertension, together with 215 age and sex matched controls. The significance of the differences in genotype distribution between hypertensive and normotensive subjects was
M
ultiple environmental and genetic factors are involved in the pathogenesis of human hypertension. Genetic epidemiologic approaches have identified the genes responsible for some types of hereditary hypertension, but they are quite rare.1–5 Most cases of hu-
Received June 10, 1997. Accepted October 13, 1997. From the Department of Geriatric Medicine, Osaka University Medical School, Osaka, Japan. Address correspondence and reprint requests to Toshio Ogihara, MD, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita City, Osaka 565, Japan.
© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
examined by x2 analysis. Neither AT1 nor AT2 gene variants were associated with human essential hypertension in the Japanese subjects. However, the AT1 receptor gene polymorphism was associated with left ventricular mass index in normotensive subjects. The study results suggest that gene polymorphisms of both angiotensin II receptors are not directly involved in the increase of genetic risk for hypertension, but that the AT1 receptor gene might contribute genetically to the increase of left ventricular mass. Am J Hypertens 1998;11:316 –321 © 1998 American Journal of Hypertension, Ltd.
KEY WORDS:
Genetics, hypertension, renin angiotensin system, receptor, echocardiography.
man hypertension are classified as essential hypertension with unknown cause. The renin-angiotensin system (RAS) plays an important role in blood pressure regulation, and recent advances in molecular biology have highlighted the genetic importance of some components of RAS, such as angiotensin converting enzyme (ACE) and angiotensinogen (AGT), in the pathogenesis of cardiovascular disease. Recently, two subtypes of angiotensin II receptors, angiotensin II type 1 receptor (AT1) and angiotensin II type 2 receptor (AT2), were cloned and characterized.6 – 8 Gene targeting approaches have suggested that both genes are involved in blood pressure regu0895-7061/98/$19.00 PII S0895-7061(97)00457-3
AJH–MARCH 1998 –VOL. 11, NO. 3, PART 1
AT1 GENE AND LVM IN HYPERTENSION
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TABLE 1. CHARACTERISTICS OF ESSENTIAL HYPERTENSIVE AND NORMOTENSIVE SUBJECTS
Family history (one first degree relative) Number Age (years) Sex (male/female) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) Brinkman index† Alcohol intake (mL/day)† CTR RV51SV1 (mV) T.Chol (mmol/L) HDL-Chol (mmol/L) TG (g/L) FPG (mmol/L) F-IRI (mU/mL) HbA1c PG/2h (mmol/L) BUN (mmol/L) Crnn (mmol/L) UA (mmol/L) LV mass index
Hypertensives
Normotensives
P Value
1 321 62.1 6 10.1 157/164 24.0 6 3.2 178.3 6 17.1 102.9 6 12.1 347 6 40 18.8 6 1.9 0.51 6 0.06 3.3 6 1.0 5.4 6 0.9 1.4 6 0.4 1.5 6 0.8 5.7 6 1.2 5.7 6 2.5 0.06 6 0.01 7.3 6 2.9 5.6 6 1.5 80.4 6 44.2 315 6 89 138 6 52
2 215 60.9 6 11.4 113/102 22.9 6 2.9 125.9 6 14.0 75.9 6 9.2 487 6 58 17.8 6 2.6 0.48 6 0.05 2.8 6 0.8 5.4 6 0.9 1.4 6 0.4 1.3 6 0.7 5.5 6 1.3 7.3 6 5.2 0.06 6 0.01 8.4 6 4.6 5.6 6 1.2 70.7 6 17.7 303 6 125 110 6 41
NS NS* ,.001 ,.0001 ,.0001 ,.05 NS ,.005 ,.0001 NS NS NS NS NS NS NS NS ,.05 NS ,.0005
Values of blood pressure are expressed as mean 6 SD. Comparison was performed by ANOVA. * Comparison was performed by x2 test. † Data are expressed as mean 6 SE. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; CTR, cardiothoracic ratio; T.Chol, total cholesterol; HDL-Chol, HDL-cholesterol; TG, triglyceride; FPG, fasting plasma glucose; F-IRI, fasting-immunoreactive insulin; HbA1c, glycosylated hemoglobin; PG/2h, plasma glucose 2 h after eating; BUN, blood urea nitrogen; Crnn, creatinine; UA, uric acid; LV mass index, left ventricular mass index.
lation and modulation of the effect of angiotensin II in different manners.9 –12 An association study in a white population showed that an A to C nucleotide substitution in the 39 untranslated region of human AT1 gene (A1166C/AT1) was associated with hypertension.13 This polymorphism was also independently associated with aortic stiffness, and upregulation of AT1 receptor gene was observed in the ventricles of cardiomyopathic hamsters.14 –16 On the other hand, recent reports revealed that AT2 receptor has an antiproliferative effect on neointima formation following vascular injury, and that its mRNA level is increased after myocardial infarction.17,18 We cloned the entire region of human AT2 receptor genomic DNA and mapped it to the human X chromosome, which contains a candidate locus for rat hypertension, denoted as BP/SP-2.19,20 Furthermore, a polymorphism was identified in the 39 untranslated region of AT2 receptor gene, but how it is genetically involved in cardiovascular disease remains unclear. To assess the role of AT1 and AT2 receptor gene polymorphisms in essential hypertension, we performed an association study in the Japanese population, which has an ethnically homogeneous ge-
netic background as opposed to the white population. We further examined the possible effects of these gene polymorphisms on cardiac hypertrophy
TABLE 2. GENOTYPES AND ALLELE FREQUENCIES OF AT1 RECEPTOR GENE POLYMORPHISM AND COMPARISONS BETWEEN HYPERTENSIVE AND NORMOTENSIVE SUBJECTS AT1 (A11663C) Genotype HBP NBP
CC 4 3
AC 56 40
AA 261 172
Total 321 215
x2 5 0.15, P 5 NS Allele frequency HBP NBP
C 0.10 0.11
A 0.90 0.89
x2 5 0.15, P 5 NS A11663 C: substitution from adenine to cytosine at 1166 nucleotide position in 39 untranslated region of AT1 receptor gene. HBP, hypertensive subjects; NBP, normotensive subjects. Comparison was performed by x2 test.
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TABLE 3. SEX MATCHED COMPARISONS OF AT1 AND AT2 RECEPTOR GENE POLYMORPHISMS BETWEEN HYPERTENSIVE AND NORMOTENSIVE SUBJECTS AT1 (A11663C) Male Genotype HBP NBP
CC 2 1
AC 32 23
AA 123 89
AT2 (C31233A) Total 157 113
A2 60 36
x2 5 0.09, P 5 NS Allele frequency HBP NBP
C 0.11 0.11
A 0.89 0.89
CC 2 2
AC 24 17
A 0.38 0.32
C 0.09 0.10
C 0.62 0.68
x2 5 1.13, P 5 NS
AA 138 83
Total 164 102
AA 18 7
x2 5 0.46, P 5 NS Allele frequency HBP NBP
Total 156 112
x2 5 1.13, P 5 NS
x2 5 0.02, P 5 NS Female Genotype HBP NBP
C2 96 76
A 0.91 0.90
x2 5 0.47, P 5 NS
CA 67 42
CC 79 53
Total 164 102
x2 5 1.32, P 5 NS A 0.31 0.27
C 0.69 0.73
x2 5 0.94, P 5 NS
Comparison was performed by x2 test.
and biochemical parameters responsible for cardiovascular disease.
were obtained in the fasting state. Informed consent was obtained from each participant.
METHODS
Evaluation of Left Ventricular Mass To evaluate left ventricular mass, echocardiographic measurements (two dimensional targeted M-mode and pulsed Doppler) were performed in 110 hypertensives and 61 normotensives using an ultrasound imager (SonolayerSSA260A, Toshiba Medical Co., Tokyo, Japan) with a 2.5 MHz transducer. Left ventricular volume and thickness of the intraventricular septum and left ventricular posterior wall were measured following the Penn convention. Left ventricular mass was calculated by the formula validated by Devereux and Reichek.21 Left ventricular mass index (LVMI) was obtained by dividing left ventricular mass by body surface area. Echocardiography was performed by observers blinded to the genetic background and BP of the patients.
Subjects and Measurements The study population was randomly selected from the outpatients at Osaka University Hospital. These participants were all Japanese and consisted of 321 hypertensive subjects and 215 normotensive subjects (Table 1). Hypertensive patients had been diagnosed according to conventional criteria, which consisted of a systolic blood pressure (SBP) . 160 mm Hg or a diastolic blood pressure (DBP) . 95 mm Hg without antihypertensive treatment. Blood pressure (BP) was measured in each subject in the supine position, without antihypertensive treatment, on three occasions spanning at least 1 month. Patients with any secondary cause for hypertension were excluded. The prevalence of diabetes mellitus was similar in hypertensives (0.15) and normotensives (0.14). A detailed family and past history was taken for each subject. All patients had at least one first degree relative with essential hypertension. The normotensive subjects without a family history of hypertension in first degree relatives were selected from the same population, matching for age and sex and satisfying the criteria that SBP and DBP were ,140 and ,90 mm Hg, respectively. Blood samples
Determination of Genotypes AT1 Receptor Participants’ DNA was extracted from leukocytes using the QIAamp kit (Quiagen Inc., Valencia, CA). Polymerase chain reaction was performed to amplify a fragment encompassing the A 3 C polymorphic site at 1166 nucleotide position in the 39 untranslated region of human AT1 receptor gene. The design of the primers was as follows: sense, 59-ATAATGTAAGCTCATC-
75 6 9 104 6 13 177 6 15
125 6 13
79 6 8 76 6 9 108 6 19 103 6 12
SBP, systolic blood pressure; DBP, diastolic blood pressure. All values given in millimeters of mercury as mean 6 SD. * P 5 .0001 v AA & AC together (ANOVA). † P 5 .0005 v both AA and AC (ANOVA). All other P values were nonsignificant.
126 6 14 125 6 13 103 6 12 104 6 14 178 6 17 179 6 18 76 6 9 79 6 8 126 6 14 131 6 12 103 6 12 108 6 19 178 6 17 211 6 27* AA & AC CC AA AC & CC AC
AT1 GENE AND LVM IN HYPERTENSION
319
CACC-39; antisense, 59-GAGATTGCATTTCTGTCAGT-39. The reaction volume was 30 mL, which contained 100 ng genomic DNA, 10 pmol of each primer, 250 mmol/L dNTP, 1.0 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl at pH 8.3, and 0.5 unit Taq polymerase (Perkin Elmer, Branchburg, NJ). Amplification was carried out using an Omni Gene Thermal Cycler (Hybaid Limited, Teddington, Middlesex, U.K.). After a heating step of 5 min at 94°C, the main reaction, 94°C for 30 sec, 55°C for 45 sec, and 72°C for 45 sec, was repeated for 40 cycles, and then followed by an extension step at 72°C for 10 min. Polymerase chain reaction products were confirmed to show exact amplification, and were then digested with DdeI (Toyobo, Osaka, Japan) for 3 h at 37°C. The digested products were visualized on 3% Sea Kem HGT agarose gel (FMC BioProducts, Rockland, ME) by ethidium bromide staining.
76 6 9 76 6 9
211 6 27† 178 6 17
131 6 12 126 6 14
DBP SBP DBP SBP DBP SBP DBP SBP DBP SBP DBP
Hypertensive
SBP
Hypertensive Normotensive Normotensive
Hypertensive
Dominant Recessive
TABLE 4. COMPARISON OF BLOOD PRESSURE ACCORDING TO AT1 GENOTYPES
Codominant
Normotensive
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AT2 Receptor We previously reported a C to A mutation at the 3123 nucleotide position in the 39 untranslated region of AT2 receptor gene.19 This mutation altered the sequence of the AluI recognition site. We designed the following primers: sense, 59-GGATTCAGATTTCTCTTTGAA-39; antisense, 59-GCATAGGAGTATGATTTAATC-39. Polymerase chain reaction was performed under the same conditions as for AT1 receptor gene except for the annealing temperature, which was set at 53°C. After confirming DNA amplification, 10 mL of PCR product was digested with 12 units AluI (Takara Biotechnology, Osaka, Japan) for 3 hours at 37°C, and electrophoresed on 3% agarose gel with ethidium bromide staining. Statistical Analysis All statistical analyses were conducted using the StatView J-4.5 program (Abacus Concepts, Inc., Berkeley, CA) and JMP 3.0 (SAS Institute, Inc., Cary, NC). The significance of differences in classified values between hypertensives and normotensives was examined using x2 analysis with one or two degrees of freedom. The mean value of clinical data for each genotype was compared by one way analysis of variance (ANOVA). The association between AT1 polymorphism and BP was examined considering all the inheritance models of C allele, that is, recessive (CC v AC1AA), dominant (CC1AC v AA), and codominant (CC v AC v AA). Furthermore, multiple logistic regression analysis was performed separately in hypertensive and normotensive subjects to evaluate the effects of the AT1 and AT2 polymorphisms on LVMI after adjustment for age and sex. RESULTS The characteristics of the population are shown in Table 1. The genotype distribution and allele frequencies of AT1 polymorphism in patients and control subjects are summarized in Table 2. The genotype distribution of
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TABLE 5. COMPARISON OF ECHOCARDIOGRAPHIC MEASUREMENTS ACCORDING TO AT1 GENOTYPES Hypertensives
IVSth (mm) PWth (mm) LV mass index
Normotensives
C Allele (2) (mean 6 SD)
C Allele (1) (mean 6 SD)
P (ANOVA)
C Allele (2) (mean 6 SD)
C Allele (1) (mean 6 SD)
P (ANOVA)
10.4 6 2.2 10.6 6 2.1 137 6 54
11.3 6 2.7 11.1 6 2.1 145 6 45
NS NS NS
9.3 6 1.9 9.5 6 1.9 107 6 35
10.9 6 1.9 11.3 6 2.3 138 6 53
,.01 ,.01 ,.05
IVSth, intraventricular septum thickness; PWth, left ventricular posterior wall thickness; LV mass index, left ventricular mass index; BSA, body surface area. LV mass index 5 [1.04 3 {(LVDd 1 IVSth 1 PWth)392(LVDd))3}213.6]/BSA. BSA 5 (Body weight)0.425 3 (Height)0.725 3 7.184 3 1023 m2.
AT1 polymorphism did not significantly deviate from the expected genotype frequencies, which were deduced from Hardy-Weinberg equilibrium. Because the AT2 receptor gene is located on the human X chromosome,8 AT2 receptor gene polymorphism was examined in the male and female groups separately (Table 3). The gene distribution also satisfied Hardy-Weinberg’s law. No significant differences in the genotype distribution of the AT1 and AT2 genes were observed between hypertensives and controls in total, or in the male or the female group (Tables 2 and 3). Blood pressure values in the study population according to AT1 receptor genotype are summarized in Table 4. The hypertensive subjects who were AT1/CC homozygous had significantly higher SBP than the subjects who were heterozygous or AT1/AA homozygous in the recessive model. There was no significant difference in BP among the subgroups of AT2 receptor genotypes (data not shown). Moreover, the subjects with AT1/C allele in the normotensive but not the hypertensive group had significantly thicker intraventricular septum and left ventricular posterior wall than those without AT1/C allele (Table 5). In the normotensive subjects, a higher LVMI was also significantly associated with AT1/C allele (Table 5). Concordantly, multiple logistic regression analysis showed that AT1 had a significant effect on LVMI (R2 5 0.21, F ratio 5 4.7, P 5 .03). DISCUSSION Recently, both AT1 and AT2 gene disrupted mouse models have suggested the involvement of these genes in blood pressure regulation.10 –12 Our results suggest that the C allele of AT1 receptor gene has a hypertensive effect in the recessive model of inheritance. However, the number of subjects with AT1/CC genotype was too small to confirm this effect. Apparently, neither AT1 nor AT2 gene polymorphism was associated with essential hypertension in the present case-control study. In whites, Bonnardeaux et al13 reported a positive association between AT1/C allele and essential hyper-
tension in a case-control study, whereas they failed to detect such an association using affected sibling pair analysis. In the present case-control study in Japanese subjects, no association between AT1/C allele and hypertension was detected. Both the white sibling pairs and the Japanese population are genetically homogeneous, whereas the general white population is not. Considering the power of statistical analysis, the result of genetic analysis using a genetically homogeneous population is more reliable. Therefore, we conclude that AT1/C allele is not associated with the occurrence of essential hypertension even though C allele frequency is obviously rarer in Japanese than in whites. On the other hand, AT1 receptor gene polymorphism showed a positive association with LVMI in normotensives. Statistical analysis of our data suggests that the AT1 receptor gene contributes to left ventricular mass and that its genetic effect is independent of sex, age, and BP. The reason why the association was not confirmed in hypertensives may have been that this effect was masked in the hypertensive group by the influence of high BP, which is the highest risk factor for left ventricular hypertrophy. Similarly, ACE/DD genotype was also reported as a risk factor for left ventricular hypertrophy in normotensive subjects but not in hypertensive subjects.25,26 Recently, Castellano et al27 reported the lack of association between AT1/A1166C polymorphism and LVMI in the randomly selected general population. However, as they stated, the number of subjects was probably not sufficient for the confident exclusion of the possibility of a b-type error. Another possible interpretation of the conflicting results is that their study population consisted of both normotensive and hypertensive subjects. In summary, we conclude that the AT1 and AT2 receptor genes are not involved in the occurrence of essential hypertension, and that AT1/C allele may be a candidate to affect left ventricular mass in the Japanese normotensive population. Further investigation, however, such as examination of the gene regulatory mechanisms, is required to elucidate the genetic con-
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