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American Society of Echocardiography,10 the fol- lowing parameters were obtained, each as an aver- age of at least three measurements: left ventricular.
Journal of Human Hypertension (2000) 14, 47–49  2000 Macmillan Publishers Ltd. All rights reserved 0950-9240/00 $15.00 www.nature.com/jhh

ORIGINAL ARTICLE

Lack of association between ACE gene polymorphism and left ventricular hypertrophy in essential hypertension E Gomez-Angelats1, A de la Sierra1, M Enjuto2, C Sierra1, J Oriola3, A Francino2, JC Pare´2, E Poch4 and A Coca1 Departments of 1Internal Medicine, 2Cardiology, 3Hormonology and 4Nephrology, Hospital Clı´nic, Barcelona, Spain

The possible association between the insertion/deletion (I/D) polymorphism of the angiotensin I converting enzyme (ACE) gene and left ventricular hypertrophy (LVH) was investigated in a group of essential hypertensive patients. Seventy-one essential hypertensive patients (35 men and 36 women), aged 51 ⴞ 1 years, were genotyped by PCR for the I/D polymorphism of the ACE gene. Cardiac morphology and function were assessed by means of M-mode echocardiography. The relative frequencies of the three genotypes, DD, DI, and

II, were respectively: 24%, 55%, and 21%. Mean values of left ventricular mass index were 145, 144, and 150 g/m2 for DD, DI, and II genotypes, without significant differences among them (P ⴝ 0.82). Likewise, the prevalence of LVH (76%, 64%, and 87%) was not significantly different among the three genotypes (P ⴝ 0.23). We conclude that the ACE gene I/D polymorphism is not associated with LVH in essential hypertension. Journal of Human Hypertension (2000) 14, 47–49

Keywords: genetics; angiotensin I converting enzyme gene; left ventricular hypertrophy

Introduction Left ventricular hypertrophy (LVH) occurs as a pathophysiological adaptation to high blood pressure in essential hypertension. Nevertheless, the correlation between the severity of high blood pressure and left ventricular mass is poor, suggesting that in addition to haemodynamic overload, other different factors may contribute to the development of LVH in essential hypertension.1 It has been demonstrated that the renin-angiotensin system has an independent direct role in modulating cardiac growth and remodelling through blood pressure regulation. Moreover, angiotensin II is generated locally by myocardial cells, acting as a paracrine regulatory system and its expression is upregulated in the presence of stretch or mechanical overload. An insertion/deletion (I/D) polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene has been recently associated with multiple cardiovascular disorders. Subjects homozygous for the D allele (DD genotype) have higher circulating and cardiac ACE levels. It has been suggested that essential hypertensives with this genotype may have an increased myocardial angiotensin II formation, leading to a greater degree of LVH.2 Despite those data, contradictory findings have been

Correspondence: Dr Alejandro de la Sierra, Hypertension Unit, Department of Internal Medicine, Hospital Cli´nic, 170-Villarroel, 08036-Barcelona, Spain Received 6 August 1999; revised and accepted 10 September 1999

reported about the ACE gene polymorphism and LVH in both normal3–6 and hypertensive populations.7–9 The aim of the present study was to evaluate the possible association between the I/D polymorphism of the ACE gene and the presence of LVH in a group of essential hypertensive patients.

Materials and methods Patient selection Seventy-one never treated white essential hypertensive out-patients of both sexes (35 males, 36 females) aged between 18 and 75 (51 ± 1) were studied at the Hypertension Unit, Hospital Clinic, Barcelona, Spain. None of these had renal impairment (serum creatinine ⬎1.5 mg/dl), cardiac failure or evidence of coronary heart disease. Likewise, patients with severe concomitant pathological condition, daily alcohol intake ⬎80 g for men and ⬎60 g for women, pregnant women, or those taking contraceptive pills were excluded. Blood pressure measurements All patients underwent a 24-h ambulatory blood pressure monitoring (ABPM), using a non-invasive oscillometric device (Spacelabs 90207, Spacelabs Inc, Redmond, WA, USA). Blood pressure was registered automatically at 20-min intervals during the whole period of 24 h.

ACE gene polymorphism and LVH E Gomez-Angelats et al

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Echocardiographic studies Two-dimensional controlled, M-mode echocardiograms were recorded in each patient in the partial left decubitus position after a rest of at least 10 min. All traced echocardiograms were read by two trained physicians. According to the criteria of the American Society of Echocardiography,10 the following parameters were obtained, each as an average of at least three measurements: left ventricular end-diastolic diameter (LVEDD), interventricular septum thickness (IST), posterior wall thickness (PWT), left atrial diameter (LAD), and ejection fraction (EF). Left ventricular mass was calculated using the Penn convention criteria11 and divided by the body surface area to obtain the left ventricular mass index (LVMI) in grams per square meter (g/m2). LVH was diagnosed if LVMI exceeded 110 g/m2 in women and 130 g/m2 in men.12 The relative wall thickness ratio (WTR) was obtained by the standardised formula: 2 × PWT/LVEDD. Determination of ACE genotype DNA was extracted from peripheral blood leukocytes and the ACE I/D genotype was determined by PCR as previously published.13 Subjects were classified as II, DD or heterozygote for insertion/deletion (DI). Mistyping of DI heterozygotes as DD due to preferential amplification of the D allele and inefficiency in amplification of the I allele has been described.14 Therefore, all samples found to be DD were reconfirmed in duplicated, as described by Shanmugam et al.14 Statistical analysis Values are expressed as mean ± s.e.m. Clinical and echocardiographic differences between the three genotypes were analysed using one-way ANOVA, with Bonferroni correction for multiple comparisons, or by Chi-square test, for qualitative variables. A P value of less than 0.05 was considered statistically significant.

Results The distribution of the three ACE genotypes among this group of essential hypertensive patients was: 15 II (21%), 39 DI (55%) and 17 DD (24%). The frequencies of the I and D alleles were 0.49 and 0.51, respectively, and they did not deviate from Hardy– Weinberg equilibrium. Age, gender distribution, weight and body mass index did not differ among essential hypertensive patients classified by ACE genotype (Table 1). Twenty-four hour systolic and diastolic blood pressures tended to be higher in DD patients (154/94 mm Hg) than in DI (150/94 mm Hg) or II essential hypertensives (148/91 mm Hg), although these differences were not statistically significant. As also shown in Table 1, left ventricular parameters were almost identical in the three genotype groups. In fact, there were no differences in left ventricular diastolic diameter, interventricular septum Journal of Human Hypertension

Table 1 Clinical and echocardiographic parameters in essential hypertensive patients classified by angiotension-converting enzyme gene polymorphism DD (n = 17) Clinical data Age (years) Sex (men/women) Weight (kg) Body mass index (kg/m2) 24-h systolic BP (mm Hg) 24-h diastolic BP (mm Hg)

DI (n = 39)

II (n = 15)

53 ± 2 9/8

50 ± 1 18/21

54 ± 2 8/7

75 ± 3 28 ± 1

79 ± 2 29 ± 1

74 ± 3 28 ± 1

154 ± 4

150 ± 2

148 ± 3

94 ± 2

94 ± 1

91 ± 2

Echocardiographic parameters LVEDD (mm) 50.6 ± 1.4 50.1 ± 0.8 IST (mm) 11.4 ± 0.3 12.0 ± 0.2 PWT (mm) 11.0 ± 0.3 11.0 ± 0.2 LAD (mm) 35.3 ± 1.1 36.6 ± 0.6 EF (%) 67.1 ± 1.5 68.8 ± 1.3 144.5 ± 8.6 143.5 ± 5.6 LVMI (g/m2) WTR 0.44 ± 0.01 0.45 ± 0.01

51.4 ± 1.1 11.6 ± 0.5 11.1 ± 0.4 36.2 ± 1.1 64.8 ± 1.6 149.9 ± 7.4 0.43 ± 0.02

LVEDD, left ventricular end diastolic diameter; IST, interventricular septum thickness; PWT, posterior wall thickness; LAD, left atrial diameter; EF, ejection fraction; LVMI, left ventricular mass index; WTR, wall thickness ratio.

thickness, posterior wall thickness, left atrial diameter, ejection fraction or relative wall thickness ratio. Mean values of LVMI in the three genotype groups were: 144.5 g/m2 in DD, 143.5 g/m2 in DI, and 149.9 g/m2 in II (P = 0.82). Likewise, the number of patients with echocardiographic criteria of LVH in each group was: 13 (76%) in DD, 25 (64%) in DI, and 13 (87%) in II patients (P = 0.23).

Discussion The present study shows a lack of association between ACE gene I/D polymorphism and LVH in a group of 71 never treated essential hypertensive patients. Left ventricular mass and geometry are determined by hereditary, neurohumoral, and haemodynamic factors. The identification of hereditary factors is important since LVH is a predictor of an adverse cardiovascular outcome. Several studies have emphasized the role of the renin-angiotensin system by showing that I/D polymorphism of the ACE gene is associated with some myocardial diseases, including hypertrophic cardiomyopathy.3,15 It has been demonstrated that the DD genotype of the ACE gene is accompanied by high plasma ACE activity, and it is believed that the resulting higher local concentrations of angiotensin II could promote inappropriate vascular wall thickening and myocardial hypertrophy.2 Nevertheless, contradictory findings have been reported about the association of ACE gene polymorphism and LVH in both normotensive3–6 and hypertensive populations.7–9 Schunkert et al3 first reported in 1994 a higher prevalence of the DD genotype in subjects with electrocardiographic evidence of LVH. However, three

ACE gene polymorphism and LVH E Gomez-Angelats et al

later studies4 –6 failed to confirm this association by assessing LVH by echocardiography. One of these studies, carried out by Lindpaintner et al4 included a total of 2439 subjects from the Framingham population. These authors did not find significant differences in LV mass among the three ACE genotypes. Taking into account the number of subjects included in this study, it seems difficult to assign to ACE gene I/D polymorphism a major role in the development of LVH, at least in the general population. Despite these negative results, more concern has been raised about the possible association between ACE gene polymorphism and LVH in the group of essential hypertensive patients. Three previous studies analysed this issue and yielded to contradictory conclusions. Pontremoli et al7 and Gharavi et al8 found a higher LVMI in DD and DI genotypes, compared with patients wearing the II genotype. However, hypertensives included in Gharavi’s study8 showed an unexpected high frequency of DD genotypes (45%), compared with other studies, including the present one. Moreover, both the unadjusted means for LVMI and the prevalence of LVH were not significantly different among the three genotypes. Finally, it is also important to note that 20% of patients included in the Gharavi study received antihypertensive medication until 2 weeks before echocardiography and this could have influenced the measurement of left ventricular mass. On the contrary, West et al9 did not find any difference in ACE gene polymorphisms in a group of 72 essential hypertensive patients and 44 normotensive subjects classified as having or not LVH. Our results are in agreement with those reported by West et al.9 In this sense, mean values for left ventricular dimensions and thickness, as well as LVMI and the prevalence of LVH were almost identical in the three genotype subgroups. This lack of association found in our study is reinforced by the subgroup analysis of hypertensive patients included in general population studies. In fact, Lindpaintner et al,4 Hamon et al5 and Kupari et al6 did not find any significant difference in LV mass among the three ACE genotypes, when the subset of hypertensives were analysed separately. This study, as other cross-sectional, observational studies, does not completely neglect a possible minor role of ACE on left ventricular growth. It is possible that subjects homozygous for the deletion allele have a greater stimulus for cardiac growth. However, the importance of ACE genotype is obviously surpassed by a great variety of genes and environmental factors influencing left ventricular mass directly or indirectly, through blood pressure or neurohormonal regulation, at variable levels for variable amounts of time. Thus, it seems very difficult the assessment of a gene-environment interaction in an heterogeneous population of hypertensive patients.

In conclusion, we did not find a relationship between ACE gene I/D polymorphism and LV mass in a group of essential hypertensive patients. These results, in agreement with other previously reported, does not support the possibility of a major role of ACE gene I/D polymorphism in the development of LVH in essential hypertension.

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References 1 Levy D et al. Echocardiographycally detected left ventricular hypertrophy: Prevalence and risk factors: The Framingham Heart Study. Ann Intern Med 1988; 108: 7–13. 2 Lindpaintner K, Ganten D. The cardiac renin-angiotensin system: an appraisal of present experimental and clinical evidence. Circ Res 1991; 68: 905–921. 3 Schunkert H et al. Association between a deletion polymorphism of the angiotensin-converting-enzyme gene and left ventricular hypertrophy. N Engl J Med 1994; 330: 1634 –1638. 4 Lindpaintner K et al. Absence of association of genetic linkage between the ACE gene and left ventricular mass. N Engl J Med 1996; 334: 1023–1028. 5 Hamon M et al. Association of ACE and angiotensin II type 1 receptor genotypes with left ventricular function and mass in patients with angiographically normal coronary arteries. Heart 1997; 77: 502–505. 6 Kupari M et al. Left ventricular size, mass, and function in relation to angiotensin-converting enzyme gene polymorphism in humans. Am J Physiol 1994; 267: H1107–H1111. 7 Pontremoli M et al. The deletion polymorphism of the Angiotensin I-converting enzyme gene is associated with target orgen damage in essential hypertension. J Am Soc Nephrol 1996; 7: 2550–2558. 8 Gharavi AG et al. Deletion polymorphism of the ACE gene is independently associated with left ventricular mass and geometric remodelling in systemic hypertension. Am J Cardiol 1996; 77: 1315–1319. 9 West MJ et al. Renin and angiotensin-converting enzyme genotypes in patients with essential hypertension and left ventricular hypertrophy. Clin Exp Pharm Physiol 1994; 21: 207–210. 10 Devereux RB, Reichek N. Echocardiographic determination of left ventricular mass in man: anatomic validation of the method. Circulation 1977; 55: 613–618. 11 Devereux RB et al. Performance of primary and derived M-mode echocardiographic measurements for detection of left ventricular hypertrophy in necropsied subjects and in patients with systemic hypertension, mitral regurgitation and dilated cardiomyopathy. Am J Cardiol 1986; 57: 1388–1393. 12 Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols. A guide to methods and applications. Academic Press Inc: San Diego, 1989, pp 146–168. 13 Ferna´ndez-Llama P et al. Angiotensin converting enzyme gene I/D polymorphism in essential hypertension and nephroangiosclerosis. Kidney Int 1998; 53: 1743–1747. 14 Shanmugam V, Sell KW, Saha BK. Mistipying ACE genotypes. PCR Meth Appl 1993; 3: 120–121. 15 Marian AJ et al. Angiotensin-converting enzyme polymorphism in hypertrophic cardiomyopathy and sudden cardiac death. Lancet 1993; 342: 1085–1086.

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