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Association of ACE I/D polymorphism in Tunisian patients with dilated cardiomyopathy Sinda Mahjoub*a, Sounira Mehri*†a, Rafik Bousaada‡, Fatma Ouarda‡, Amira Zaroui‡, Bechir Zouari§, Rachid Mechmeche‡, Mohamed Hammami† and Saida Ben Arab*

Key words: angiotensinconverting enzyme, polymorphism, dilated cardiomyopathy, association, severity. *

Unité d`Epidémiologie Génétique et Moléculaire, Faculté de Médecine de Tunis, Tunisia †

Laboratoire de Biochimie, U.S.C.R. de Spectrométrie de Masse, la Faculté de Médecine de Monastir, Tunisia ‡

Services des Explorations Fonctionnelles Cardiologiques, Hôpital La Rabta de Tunis, Tunisia §

Service de Médecine Préventive et Communautaire, Faculté de Médecine de Tunis, Tunisia Correspondence to: Pr Saïda Ben Arab, Unité d’Epidémiologie Génétique et Moléculaire Faculté de Médecine de Tunis, 15 Rue Djebel Lakhdar La Rabta, 1007 Tunis,Tunisia Email: Benarab_saida@ yahoo.fr a These two authors contributed equally to this work.

Journal of the ReninAngiotensinAldosterone System (Including other Peptidergic systems) Month 2010 Volume 11 Number 3

Abstract Primary cardiomyopathies are multifactorial diseases. Genetic factors other than the causal mutations in the modified genes affect the phenotypic expression of dilated cardiomyopathy. The aim of this study was to determine the association of angiotensinconverting enzyme I/D polymorphism with the risk of dilated cardiomyopathy in a Tunisian population. A total of 76 patients with dilated cardiomyopathy was compared to 151 ethnically, age- and gender-matched controls. The frequencies of the DD genotype and D allele were significantly higher in patients as compared with controls, and were associated with increased risk of dilated cardiomyopathy (ACE DD versus ID and II: OR = 3.05 (95% CI, 1.58–5.87; p = 0.001)); D versus I: OR = 2 (95% CI: 1.35–2.97; p = 0.001)). No association was found between the combined genotypes (DD+ID) or D allele and left ventricular end diastolic diameter in dilated cardiomyopathy patients with severe and moderate clinical phenotypes. DD genotype and D allele of angiotensin-converting enzyme I/D gene polymorphism are associated with increased risk of dilated cardiomyopathy in a Tunisian population but do not influence the cardiac phenotype severity. Introduction The role of genetic factors in the pathogenesis of primary cardiomyopathies (CMs) has received increasing attention during the past 15 years. Primary CMs are a heterogeneous group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that usually (but not invariably) exhibit inappropriate ventricular hypertrophy or dilatation and are due to a variety of causes that are frequently genetic.1 Hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM) and restrictive cardiomyopathy are the major types of CM. Dilated forms of CM are characterized by ventricular chamber enlargement and

systolic dysfunction with normal left ventricular (LV) wall thickness; usually diagnosis is made with two-dimensional echocardiography. DCM leads to progressive heart failure and a decline in LV contractile function, ventricular and supraventricular arrhythmias, conduction system abnormalities, thromboembolism, and sudden or heart failure-related death. Several studies suggest that DCM occurs as a result of mutations in genes encoding a variety of proteins, including sarcomeric and structural proteins,2 and may result from activation of several different independent mechanisms. However, clinical evaluations of families with DCM gene mutations often reveal the absence of disease in individuals carrying disease-causing mutations. These data suggest that more knowledge of the cellular and molecular events triggered by DCM mutations is needed to understand the pathways by which the human heart remodels and ultimately fails.3 Human association studies have evaluated candidate molecules as genetic modifiers of DCM. The phenotype is also highly heterogeneous, in terms of functional severity, degree of cardiac dilation, and impairment of systolic function, and also because the myocardial disease may be isolated, or associated with cardiac conduction or muscular dystrophy.4 Collectively, these data strongly suggest that modifying factors, both genetic and/or environmental, play an important role in explaining the phenotypic diversity seen in DCM. Several genes including those encoding the components of the renin–angiotensin–aldosterone system (RAAS) have emerged as the potential modifiers.5 The angiotensin-converting enzyme (ACE) gene is localized on chromosome 17q23 and is characterized by a major insertion/deletion (I/D) polymorphism consisting of the presence or absence of a 287-base-pair Alu repeat sequence within intron 16.6 ACE is an ectoenzyme found on the external surface of the endothelial and epithelial

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cell membranes. It enhances the synthesis of angiotensin II (Ang II), which promotes proliferation, migration and hypertrophy of vascular smooth muscle cells. Ang II also induces the production of proinflammatory cytokines and matrix metalloproteinases.7 Moreover, the increased free radical generation by Ang II contributes to endothelial dysfunction.6 Studies from different populations have shown conflicting data; the results have been inconsistent and inconclusive, with a few studies reporting association8-10 whereas others have shown no association between ACE gene I/D polymorphism and CM.5,11 In the present study, we have examined the prevalence of ACE I/D polymorphism in Tunisian DCM patients and assessed the association of ACE I/D genotypes with clinical phenotype. Materials and methods Patients This study was carried out in 76 unrelated patients, affected by primary DCM of unknown aetiology and recruited from the department of cardiology in Rabta Hospital, Tunisia. All patients with ischaemic, hypertensive, congenital, valvular or pericardial disease were excluded. No person in the DCM group was administered any drugs such as ACE inhibitors or angiotensin II type 1 receptor antagonists (ICE or ARA-2). The average age at diagnosis was 19.9 ± 13.1 years. The mean age of patients with DCM was 18.6 ± 12.6  years. The diagnosis of DCM was made according to criteria provided by the World Health Organization.12 DCM is diagnosed in the presence of (i) fractional shortening less (LV FS) than 25% and/or ejection fraction (LV FE) less than 45%; (ii) left ventricular end diastolic diameter (LVEDD) greater than 69  mm excluding any known cause of myocardial disease. We classified the DCM patients into two groups: patients with LVEDD ≥ 69  mm, with severe clinical phenotype as the first group; and the second group with patients with values of LVD < 69 mm, where the disease is considered to be of moderate clinical phenotype.

Journal of the ReninAngiotensinAldosterone System (Including other Peptidergic systems) Month 2010 Volume 11 Number 3

A total of 151 healthy, age, sex and ethnicitymatched controls without any previous history of cardiovascular disorders were enrolled in the study. The mean age of healthy controls was 19.1 ± 9.8 years. This study was approved by our hospital ethical committee, and informed consent was obtained from all healthy controls and patients before their enrolment.

SAGE Publications 2010 Los Angeles, London, New Delhi and Singapore 188

Table 1 Demographic profile of patients with dilated cardiomyopathy and healthy controls. Parameters

Mean age (years) Sex (male/female) NYHA (I /II) LVEDD, mm LVESD, mm LV FS, % LV EF, % IVST, mm PWT, mm LV mass, g

DCM patients

Healthy controls

(N = 76)

(N = 151)

19.9 ± 13.1 46/30 23/53 68.7 ± 9.84 41.33 ± 8.67 20.63 ± 6.56 37.03 ± 12.7 7.66 ± 1.93 7.55 ± 13.28 298.1 ± 138.7

19.1 ± 9.8 90/61

p-value

0.622 0.965

Values given as means ± SD. DCM, dilated cardiomyopathy; IVST, interventricular septal wall thickness; LV, left ventricular; LVEDD, left ventricular end-diastolic dimension; LVEF, left ventricular ejection fraction; LVESD, left ventricular end-systolic dimension; LVFS, left ventricular fractional shortening; NYHA, New York Heart Association; PWT, posterior wall thickness.

Genomic DNA from patients and controls was extracted from peripheral blood lymphocytes using a standard phenol–chloroform technique. Genotyping of ACE I/D polymorphism was performed using the method of Rigat et al.13 To exclude mistyping of the heterozygotes as DD homozygotes, all the DD genotypes samples were confirmed with an insertion-specific PCR.14 Statistical analysis Differences in clinical measurements were evaluated with the Epi Info 6.04d statistical software of the Centers for Disease Control) (Atlanta, Georgia). Genotype and allele frequencies were compared between patients and controls by chisquare test. To determine whether ACE I/D polymorphism is independently associated with the severity of DCM, binary logistic regression analysis, which allows adjustment for confounding factors, was performed. The strength of an association was expressed by the odds ratios (OR) with a 95% confidence interval (CI) according to Woolf’s method. A p-value