Angiotensin converting enzyme gene polymorphism and activity in Turkish patients with essential hypertension

AJH

1999;12:1038 –1043

Angiotensin Converting Enzyme Gene Polymorphism and Activity in Turkish Patients With Essential Hypertension Abdulkerim Bedir, Nurol Arık, Bahattin Adam, Kag˘an Kılınc¸, Tevfik Gu¨mu¨s¸, and Ertug˘rul Gu¨ner

Studies in various ethnic groups have shown contradictory evidence on the association of the angiotensin converting enzyme (ACE) insertion/ deletion (I/D) polymorphism with essential hypertension. We conducted a case-control study in Samsun, Turkey, to examine the association between ACE genotype, ACE serum activity, and blood pressure. Serum ACE activity was measured and ACE I/D polymorphism performed in 165 hypertensive and 143 normotensive subjects. Genomic DNA was extracted from blood samples and amplified by polymerase chain reaction (PCR). PCR primers were flanking the polymorphic region in intron 16 of the ACE gene. The distribution of the DD, ID, and II ACE genotypes was 65, 77, and 23 in hypertensive patients and 42, 82, and 19 in normotensive subjects (P > .05). The estimated frequency of the insertion allele was 0.37 in hypertensive and 0.42 in normotensive subjects. Nevertheless, sensitivity analysis, based on positive family history and severity of hypertension, suggested that significant

associations existed between more homogeneous groups of hypertensives and normotensives (P < .05). ACE genotype influenced ACE activity and the highest level was in DD genotype, being the lowest in II genotype. ACE serum levels were significantly higher in hypertensives as compared with normotensives (P < .01). A modest correlation was observed between blood pressure and ACE among hypertensive persons (r ⴝ 0.25, P < .05) and this did persist in multivariate analysis (P < .05 for systolic blood pressure and P < .005 for diastolic blood pressure). These data suggest that ACE DD genotype may have predisposing effects on severe hypertensives and cases with positive family history, and that ACE may be one of the independent factors on hypertension. Am J Hypertens 1999;12:1038 –1043 © 1999 American Journal of Hypertension, Ltd.

KEY WORDS:

Angiotensin converting enzyme, genotype, blood pressure.

ngiotensin converting enzyme (ACE) is responsible for both the production of angiotensin II and the breakdown of bradykinin, which then plays an important role in cardiovascular homeostasis. An insertion (I) and

A

deletion (D) polymorphism in intron 16 of the human ACE gene has been reported to be related to the levels of the circulating enzyme.1 In addition, this I/D polymorphism has been associated with some but not all studies with increased risk of several cardiovascular

Received July 2, 1998. Accepted February 22, 1999. From the Departments of Biochemistry and Nephrology, Faculty of Medicine, Ondokuz Mayis University, Samsun, Turkey. Present affiliation: Department of Biochemistry, Faculty of Medicine, Fatih University, Ankara, Turkey.

Address correspondence and reprint requests to Abdulkerim Bedir, MD, Department of Biochemistry, Faculty of Medicine, Ondokuz Mayis University, 55139 Samsun, Turkey; e-mail: Akbedir@ hotmail.com

© 1999 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.

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AJH–OCTOBER 1999 –VOL. 12, NO. 10, PART 1

sequelae, including myocardial infarction, left ventricular hypertrophy, and dilated cardiomyopathy.2– 4 Essential hypertension is a polygenic disease indicating that several genes are involved in its pathogenesis. The renin angiotensin system, which is involved in the regulation of blood pressure, may play an important role in the development of hypertension. However, there is still controversy over the association of the ACE gene polymorphism with hypertension. Previously, Zee et al5 proposed that the insertion allele could be a marker of hypertension; however, a more recent analysis of their data has shown that this association was, in fact, a result of an age-related loss of the deletion allele.6 The most extensive study by Jeunemaitre et al7 showed no linkage among hypertensive sib pairs in Utah. Although studies in white populations failed to support the relation between ACE gene polymorphism and hypertension,8,9 Duru et al10 found that the frequency of the ACE deletion allele was greater in African-Americans with hypertension. Recently, Wilson and coworkers11 also reported a positive association of the ACE DD genotype with hypertension. It is generally understood that ethnic origin should be carefully considered in studying the association between ACE gene polymorphism and disease etiology.12 Therefore, the present study was initiated to determine whether the frequencies of DD genotype and deletion allele were greater in Turkish populations with hypertension than in those with normal blood pressure and to examine the relationship between ACE activity and blood pressure. METHODS Recruitment of Participants White adult Turks from Northern Turkey were studied. The hypertensive group consisted of 165 subjects from the Hypertension Outpatient Clinic of Ondokuz Mayis University Hospital. Hypertension was defined as systolic blood pressure (SBP) ⬎ 140 mm Hg or diastolic blood pressure (DBP) ⬎ 90 mm Hg13 or those currently receiving one or more antihypertensive drugs. Secondary forms of hypertension were excluded by clinical and laboratory examination. The normotensive controls including healthy persons from hospital staff or retired staff had negative family history for hypertension and SBP ⬍ 140 mm Hg and DBP ⬍ 90 mm Hg on three occasions spanning 2 months. Demographic and blood chemistry data were obtained from questionnaires and laboratory examinations. Experimental Procedures Venous blood samples drawn into tubes with EDTA were used to extract genomic DNA by a modified phenol extraction method.14 Aliquots of 500 ␮L of blood were added to 1.5-mL Eppendorf tubes containing 500 ␮L of phenol equilibrated with Tris-HCl, pH 8.0, and mixed by

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gentle inversion. After this inactivation step, the samples were stored at 2 to 8°C until further processing. Polymerase chain reaction (PCR) was performed by using primers that flank the I/D region in intron 16 of the ACE gene, as described by Rigat et al15 with modification, in a Delta II thermal cycler (Ericomp Inc., San Diego, CA). The PCR mixture contained at least 1 ␮g of DNA template, 10 pmol of each primer, 0.5 mmol/L of each dNTP, 2 U of Taq polymerase, and 2.5 mmol/L MgCl2 in a final volume of 100 ␮L. DNA was amplified for 30 cycles (denaturation at 94°C for 1 min, annealing at 63°C for 1 min, extension at 72°C for 2 min), with a final extension time of 10 min. The PCR products were separated by electrophoresis on 2% agarose gel followed by ethidium bromide staining. Determination of serum ACE activity was carried out on a Hitachi 747 autoanalyser (Hitachi Ltd., Tokyo, Japan) with the procedure described by Buttery and Stuart16 using the substrate FAPGG. The intraand interassay variabilities were 6% and 8% in the normal range, respectively. Substrate and quality control materials were purchased from Sigma Chemical Co. (St. Louis, MO). To assess the influence of certain risk factors on the estimated strength of the association, sensitivity analyses were performed on the basis of the following criteria: family history of hypertension, severity of hypertension, body mass index, age at onset, and current age. Statistical Analysis For comparison of allelic and genotype frequencies, we analyzed the data by the ␹2 test. The clinical characteristics of two groups were expressed as mean ⫾ SD and were compared by unpaired Student’s t test. One-way ANOVA was used to test for differences in means of phenotypic characteristics between the three ACE genotypes. Plasma triglycerides and lipoprotein(a) were logarithmically transformed (log10) before the analysis to approach normal distribution. P ⬍ .05 was considered statistically significant. RESULTS In total, 143 normotensives (64 men, 79 women) and 165 hypertensives (50 men, 115 women) were enrolled in this study. All of the hypertensives were receiving antihypertensive drug treatment. Clinical and laboratory parameters were listed in Table 1. We selected controls older than hypertensives to decrease the possibility that hypertension would develop after the study (mean ⫾ SD, 58.9 ⫾ 12.2 years and 49.8 ⫾ 11.4 years, respectively; P ⬍ .001). Figure 1 shows a representative agarose gel electrophoresis in which PCR products were amplified from genomic DNA. The PCR product is a 190-bp fragment in the absence of the insertion and a 490-bp fragment

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BEDIR ET AL

TABLE 1. CLINICAL AND LABORATORY FEATURES OF HYPERTENSIVE AND NORMOTENSIVE GROUPS Normotensive Hypertensive Group Group n Age (y) Male (%) Positive family history of hypertension (%) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) Triglycerides (mg/dL) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) ApoA-I (mg/dL) ApoB (mg/dL) Lp(a) (mg/dL) ACE (U/L)

143 58.9 ⫾ 12.2 45

P

165 49.8 ⫾ 11.4 .0001 30

0 25.4 ⫾ 4.3 114.7 ⫾ 10.2 72.6 ⫾ 7.4 147.4 ⫾ 95.3

51 29.5 ⫾ 4.9 155.6 ⫾ 23.8 96.7 ⫾ 12.9 163.5 ⫾ 85.7

173.0 ⫾ 52.7

199.2 ⫾ 45.9 .0001

39.5 ⫾ 12.3 127.6 ⫾ 45.1 129.8 ⫾ 48.1 55.7 ⫾ 71.1 25.7 ⫾ 20.4

40.4 ⫾ 9.0 154.15 ⫾ 25.5 137.8 ⫾ 34.9 106.4 ⫾ 72.9 33.8 ⫾ 19

.0001 .0001 .0001 .02

.54 .0001 .11 .0001 .008

Data are mean ⫾ SD. Comparisons are performed by ␹2 test for categoric variables. Plasma triglycerides and lipoprotein(a) were logarithmically transformed (log10) before analysis, but untransformed values are shown in the Table. Positive family history is defined as the presence of a first-degree relative with hypertension. HDL ⫽ high-density lipoprotein.

in the presence of the insertion. Agarose gel electrophoresis enabled us to identify the DD, ID, or II genotype in each sample, according to the size of the amplified bands. For the I allele a 490-bp band was

observed and for the D allele a 190-bp band was observed. To avoid mistyping of the ID genotype, an additional PCR was performed in samples classified as DD. None of the samples were found to be initially mistyped. The distribution of the DD, ID, and II ACE genotypes was 65, 77, and 23 in hypertensive patients, and 42, 82, and 19 in normotensive patients (␹2 ⫽ 3.9, P ⫽ .14), respectively. The estimated frequency of the insertion allele was 0.37 in hypertensives and 0.42 in normotensives. The allele frequencies did not statistically deviate from the Hardy-Weinberg equilibrium. We also investigated the frequencies of ACE genotypes in age groups younger and older than 60 years. ACE genotype distribution was age related and showed significant differences among groups (Table 2). Odds ratios were calculated as measures of the association of the ACE genotype with hypertension and its different subgroups based on risk factors and potential confounders (Table 3). To rule out ACE treatment effects, we confined analysis of the association between serum ACE activity and ACE genotypes to persons who had not received ACE inhibitor treatment (n ⫽ 105). ACE activity was significantly higher in hypertensives (P ⫽ .008). In both normotensive and hypertensive subjects, ACE activity was highest in the DD genotype, intermediate in the ID genotype, and lowest in the II genotype (Table 4). However, the within-group difference was not significant. ACE levels were similar among men and women in both normotensive and hypertensive groups. The difference of ACE activity by gender between normotensives and hypertensives was statistically significant in men (23 ⫾ 19 U/L v 36 ⫾ 19 U/L, P ⫽ .007) but not in women (28 ⫾ 21 U/L v 32 ⫾ 19 U/L, P ⫽ .25). The linear relationship among the measured covariates was examined with correlation analysis restricted to the hypertensive subjects not being on ACE inhibitor treatment. A modest relationship between ACE activity and both SBP and DBP was detected (r ⫽ 0.25, P ⫽ .03 and r ⫽ 0.27, P ⫽ .02, respectively). Multivariate analyses were carried out to assess the potential independence among factors such as ACE activity, BMI, age, and gender (Table 5). DISCUSSION

FIGURE 1. PCR products from amplification of the polymorphic region in intron 16 of the ACE gene. Lane ID contains the products from an ID heterozygote with both the 490- and 190-bp fragments; lane II, the 490-bp product from a II homozygote; lane DD, the 190-bp product from a DD homozygote; lane M, markers.

The recent data accumulated on the relationship of the ACE gene to its physiologic concomitants and blood pressure have created a paradox. Despite the evidence that the renin-angiotensin system (RAS) is an important determinant of blood pressure, and the complementary findings on the benefit of ACE inhibition, a direct relationship between I/D ACE polymorphism and hypertension has been difficult to demonstrate.7,10 However, in the recent studies17,18 on large popula-

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TABLE 2. FREQUENCY OF INSERTION/DELETION (I/D) POLYMORPHISM OF THE ACE GENE IN NORMOTENSIVES AND HYPERTENSIVES CATEGORIZED BY AGE Genotype

All normotensives All hypertensives Hypertensives by age group ⱖ60 y ⬍60 y

No.

II

ID

DD

␹

P

II ⴙ ID

DD

␹2

P

143 165

19 23

82 77

42 65

— 3.9

— 0.14

101 100

42 65

— 3.4

— 0.06

41 124

7 16

23 54

11 54

0.4 6.2

0.82 0.04

30 70

11 54

0.1 5.8

0.75 0.02

2

The ␹2 and P values shown are obtained by comparing data for each category of hypertensives with values for all normotensives used as controls.

Odds Ratio (95% CI)

P

1.56 (0.97 to 2.52)

.065

1.89 (1.07 to 3.32)

.025

1.27 (0.71 to 2.27)

.420

2.03 (1.04 to 3.38)

.036

2.14 (1.01 to 4.59)

.048

1.61 (0.98 to 2.65)

.058

ment model, suggested by Forrester et al,19 seems reasonable to explain these interactions with the exception of no association of ACE activity with BMI. With our data, BMI must be evaluated independently from ACE activity. At the genetic level, a fixed effect, linked to the I/D polymorphism, determines a portion of the variance in serum ACE. At the level of the physiologic intermediate, ACE expression is further influenced by unmeasured environmental factors, and is correlated with blood pressure but not with BMI. The two distant phenotypes, obesity and blood pressure, are related to each other and causal relationship between them is likely to be unidirectional. In addition, our data of different frequencies of DD genotype in hypertensive subgroups by age are in favor of the hypothesis, proposed by Morris et al6 from the observation in Australian whites, that DD genotype in hypertension may associate with risk of premature death. In a recent publication by Kario et al,20 more marked endothelial cell damage observed in elderly hypertensives with DD genotype than in those with II genotype may be another evidence for the explanation of low frequency of ACE D allele in elderly patients with hypertension. Although most researchers have found that no relationship exists between ACE activity and blood pressure, the reported data are not entirely negative.21–24 A French study demonstrated a correlation in a sample of 434 male volunteers (r ⫽ 0.11, P ⫽ .02). 24

1.88 (1.04 to 3.37) 1.85 (1.12 to 3.07) 0.88 (0.40 to 1.92)

.034 .016 .750

TABLE 4. SERUM ACE ACTIVITY (U/L) IN DIFFERENT GENOTYPES OF NORMOTENSIVES AND HYPERTENSIVES

2.02 (1.15 to 3.56)

.014

1.20 (0.67 to 2.14)

.530

tion-based samples, there is also evidence of genetic linkage of the ACE locus with hypertension and with diastolic blood pressure in men but not women. ACE locus exerts greater effects on a subset of subjects with a positive family history of hypertension,18 being another support for our data on sensitivity analysis. From our study, the following relationships emerge. ACE genotype influences ACE activity, although the effect is quantitatively small. ACE activity, on the other hand, has a positive relationship with SBP and DBP, independent of ACE genotype. A three-compart-

TABLE 3. RISK FOR HYPERTENSION ACCORDING TO GENOTYPE WHEN COMPARING CONTROLS WITH CASE-PATIENTS AND OTHER SUBGROUPS Group of Case-Patients

n

All cases 165 Cases with positive family history 84 Cases with negative family history 81 Cases with severe hypertension 48 Cases with positive family history and severe hypertension 34 Cases with BMI ⬎25.1 kg/m2 137 Cases with BMI ⬎25.1 kg/m2 and positive family history 73 Cases ⬍60 years old 124 Cases ⱖ60 years old 41 Cases with hypertension longer than 30 months 81 Cases with hypertension shorter than 30 months 84

Positive family history is defined as the presence of a first-degree relative with hypertension. ACE genotype: 1 ⫽ DD, 2 ⫽ ID ⫹ II. Severe hypertension is defined as systolic hypertension ⬎179 mm Hg, diastolic hypertension ⬎105 mm Hg, or both.

Normotensives Hypertensives P

II

ID

DD

P

20 ⫾ 11 (n ⫽ 14) 28 ⫾ 19 (n ⫽ 11) 0.25

24 ⫾ 19 (n ⫽ 64) 33 ⫾ 19 (n ⫽ 32) 0.04

31 ⫾ 24 (n ⫽ 35) 37 ⫾ 20 (n ⫽ 30) 0.29

0.35 0.12

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TABLE 5. MULTIPLE REGRESSION ANALYSIS OF SBP AND DBP ON ACE ACTIVITY AND COVARIABLES IN HYPERTENSIVE SUBJECTS (n ⴝ 105)* Dependent Variable

Independent Variable

Coefficient

P

SBP

ACE activity BMI Age Gender ACE activity BMI Age Gender

0.33 1.05 0.54 2.71 0.23 0.91 0.02 1.15

0.018 0.076 0.014 0.63 0.004 0.008 0.85 0.73

DBP

* Hypertensive patients receiving the ACE inhibitor treatment were not included.

ACE levels were increased in offspring of parents with high blood pressure.21 Collection of a homogeneous population is improved by selecting appropriate case subjects and even more importantly, appropriate control subjects, by using explicit criteria like a precise definition of hypertension, family history, severity of disease, and age of onset. Sensitivity analysis suggests that a stronger association was detected in homogeneous populations with more specific selection of cases regarding family history and severity of hypertension, whereas the effect vanished with broader and less specific inclusion criteria. In conclusion, we found important association only between ACE I/D polymorphism and more homogeneous group within the heterogeneous essential hypertensives and further studies with a more rigorous design are warranted to conclusively establish an association between ACE activity and essential hypertension in whites. In addition, environmental factors that alter the expression of hypertension-prone genotypes must be clearly characterized to connect variants at the molecular level with the distant phenotype of blood pressure.25 REFERENCES 1.

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