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Lack of association between polymorphisms of eight candidate genes and idiopathic dilated cardiomyopathy
Journal of the American College of Cardiology © 1999 by the American College of Cardiology Published by Elsevier Science Inc.
Vol. 35, No. 1, 2000 ISSN 0735-1097/00/$20.00 PII S0735-1097(99)00522-7
Lack of Association Between Polymorphisms of Eight Candidate Genes and Idiopathic Dilated Cardiomyopathy The CARDIGENE Study Laurence Tiret, PHD,* Christine Mallet, MSC,† Odette Poirier, PHD,* Viviane Nicaud, MA,* Alain Millaire, MD, PHD,‡ Jean-Brieuc Bouhour, MD,§ Ge´rard Roize`s, PHD,㛳 Michel Desnos, MD,¶ Richard Dorent, MD,# Ketty Schwartz, PHD,†† Franc¸ois Cambien, MD,* Michel Komajda, MD,** FOR CARDIGENE GROUP Paris, Lille, Nantes, and Montpellier, France OBJECTIVES
THE
The study investigated the potential role of eight candidate genes in the susceptibility to idiopathic dilated cardiomyopathy (IDC).
BACKGROUND Idiopathic dilated cardiomyopathy has a familial origin in 20% to 25% of cases, and several genetic loci have been identified in rare monogenic forms of the disease. These findings led to the hypothesis that genetic factors might also be involved in sporadic forms of the disease. In complex diseases that do not exhibit a clear pattern of familial aggregation, the candidate gene approach is a strategy widely used to identify susceptibility genes. All genes coding for proteins involved in biochemical or physiological abnormalities of cardiac function are potential candidates for IDC. METHODS
We studied 433 patients with IDC and 401 gender- and age-matched controls. Polymorphisms investigated were the I/D polymorphism of the angiotensin I-converting enzyme (ACE) gene, the T174M and M235T polymorphisms of the angiotensinogen (AGT) gene, the A-153G and A⫹39C polymorphisms of the angiotensin-II type 1 receptor (AGTR1) gene, the T-344C polymorphism of the aldosterone synthase (CYP11B2) gene, the G-308A polymorphism of the tumor necrosis factor-alpha (TNF) gene, the R25P polymorphism of the transforming growth factor beta1 (TGFB1) gene, the G⫹11/in23T polymorphism of the endothelial nitric oxide synthase (NOS3) gene and the C-1563T polymorphism of the brain natriuretic peptide (BNP) gene.
RESULTS
None of the polymorphisms were significantly associated with the risk or the severity of the disease.
CONCLUSIONS We did not find evidence for an involvement of any of the 10 investigated polymorphisms in the susceptibility to IDC. (J Am Coll Cardiol 2000;35:29 –35) © 1999 by the American College of Cardiology
Dilated cardiomyopathy (DCM) is a myocardial disease characterized by impaired systolic function and dilation of the left or both ventricles (1). Despite recent advances in medical and surgical therapies, it remains an important cause of mortality and morbidity and is a leading indication for heart transplantation. The etiology of DCM is idiopathic in about half the patients. Family studies using From the *INSERM U525, Paris; †INSERM SC7, Paris; ‡CHR de Lille; §CHR de Nantes; 㛳INSERM U249, Montpellier; ¶Hoˆpital Boucicaut, Paris; #Service de Chirurgie Cardiaque and **Service de Cardiologie, Groupe Hospitalier Pitie´Salpeˆtrie`re, Paris; ††INSERM U523, Paris, France. The CARDIGENE study was supported by grants from Parke Davis France, the “De´le´gation a` la Recherche Clinique AP-HP” (EMUL and PHRC) and INSERM. Manuscript received February 19, 1999; revised manuscript received July 29, 1999, accepted October 5, 1999.
echocardiography to identify individuals with presymptomatic disease revealed that 20% to 25% of idiopathic dilated cardiomyopathy (IDC) had a familial origin, a proportion that had been considerably underestimated previously (2,3). This finding raised the hypothesis that gene defects might play an important role in the disease pathogenesis. Indeed, several genetic loci responsible for rare autosomal dominant or X-linked forms of DCM have been subsequently localized by linkage analysis in large pedigrees (review in [4]). The recognition of familial DCM led to the notion that even in sporadic cases, genetic factors might contribute to the disease susceptibility. In complex diseases that do not exhibit a clear pattern of familial aggregation, the candidate gene approach, which tests the role of known genes selected for their potential implication in the pathophysiological
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Tiret et al. Dilated Cardiomyopathy and Candidate Genes
Abbreviations ACE AGT AGTR1 BNP CYP11B2 DCM IDC NOS3 TGFB1 TNF
and Acronyms ⫽ angiotensin I-converting enzyme ⫽ angiotensinogen ⫽ angiotensin-II type 1 receptor ⫽ brain natriuretic peptide ⫽ aldosterone synthase ⫽ dilated cardiomyopathy ⫽ idiopathic dilated cardiomyopathy ⫽ endothelial nitric oxide synthase ⫽ transforming growth factor beta1 ⫽ tumor necrosis factor-alpha
process, is a strategy widely used (5,6). In DCM, all genes coding for proteins involved in biochemical or physiological abnormalities of cardiac function are potential candidates. The CARDIGENE study is a large case-control study designed to identify genetic factors involved in the predisposition to IDC. This article reports the results of an investigation of eight candidate genes: the angiotensin I-converting enzyme (ACE), the angiotensin-II type 1 receptor (AGTR1), the angiotensinogen (AGT), the aldosterone synthase (CYP11B2), the tumor necrosis factoralpha (TNF), the transforming growth factor beta1 (TGFB1), the endothelial nitric oxide synthase (NOS3) and the brain natriuretic peptide (BNP) genes.
METHODS Ethics approval for the study was obtained from the relevant committees, and each subject gave written informed consent for the study. Patients. Patients aged 18 to 65 years (n ⫽ 433) were recruited between 1994 and 1996 from 10 hospitals (see list in Appendix). The diagnosis of IDC was based on impaired left ventricular systolic function (ejection fraction ⱕ40% on ventriculography, radionucleotide angiography or echocardiography) and left ventricular dilation (end-diastolic volume ⬎140 ml/m2 on ventriculography or end-diastolic diameter ⬎34 mm/m2 on echocardiography), confirmed over a six-month period. For transplanted patients, these criteria had to be fulfilled before heart transplantation. Coronary angiography had to be performed in all subjects aged ⬎35 years, and patients with ⱖ50% obstruction of one or more coronary arteries were excluded. Patients with myocardial infarction, sustained systemic arterial hypertension, insulin-dependent diabetes, intrinsic valvular disease, congenital cardiopathy, documented myocarditis, specific primary or secondary heart muscle disease were also excluded. All patients had to be born in France, and their parents had to be born in France or neighboring countries. The mean left ventricular ejection fraction was 23.3 ⫾ 7.6% (n ⫽ 433), the mean end-diastolic volume was 194 ⫾ 37 ml/m2 on ventriculography (n ⫽ 182) and the mean end-diastolic diameter was 40.1 ⫾ 5.1 mm/m2 (n ⫽ 371) on
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echocardiography. The mean duration between diagnosis and inclusion was 7.1 ⫾ 5.3 years. Of the 433 patients, 229 (52.9%) had heart transplantation. Family history of DCM, defined as definite or possible cardiomyopathy, or sudden death ⱕ35 years, in first- or second-degree relatives, was reported in 25% of patients. Controls. Controls (n ⫽ 401) were randomly sampled from the French population surveys carried out in Lille (northern France), Strasbourg (eastern France) and Toulouse (southern France) within the framework of the WHO MONICA (MONItoring in CArdiovascular diseases) project (7). A stratification was used to match the approximate age distributions in cases and controls (age at diagnosis in cases and current age in controls). Controls with clinical history of cardiovascular disease or insulindependent diabetes were excluded. Genotyping. Genomic DNA was prepared from white blood cells by phenol extraction. Polymorphisms investigated were an insertion (I)/deletion (D) polymorphism in intron 16 of the ACE gene (8); two nucleotide substitutions resulting in amino acid changes at codons 174 (Thr/Met) and 235 (Met/Thr) of the AGT gene (9); two nucleotide substitutions A-153G and A⫹39C (previously named A⫹1166C) in the AGTR1 gene (10); a nucleotide substitution T-344C in the CYP11B2 gene (11); a nucleotide substitution G-308A in the TNF gene (12); a nucleotide substitution resulting in an amino acid change at codon 25 (Arg/Pro) of the TGFB1 gene (13); a G/T nucleotide substitution in intron 23 of the NOS3 gene (14); and a nucleotide substitution C-1563T in the BNP gene (unpublished). Statistical analysis. Data were analyzed using the SAS statistical software (SAS Institute, Cary, North Carolina). Allele frequencies were estimated by gene counting. HardyWeinberg equilibrium was tested by chi-square analysis. Genotype and allele frequencies were compared between cases and controls by chi-square analysis. Genotypic odds ratios (OR) for disease, assuming a dominant or a recessive model, were computed. Association between polymorphisms and case/control status was also tested by means of logistic regression analysis controlling for age, gender (matching criteria) and different covariates. A p value ⬍0.05 was considered statistically significant.
RESULTS Characteristics of cases and controls are presented in Table 1. The mean age at diagnosis was 44.6 years in male and 48.4 years in female cases. Compared to controls, cases were shorter by approximately 1 cm (p ⬍ 0.05), had a higher alcohol and tobacco consumption (p ⬍ 0.001) and a higher prevalence of non–insulin-dependent diabetes mellitus (p ⬍ 0.05). Transplanted patients were younger, leaner and had a lower alcohol consumption than did nontransplanted patients, and they were characterized by a more severe left
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Table 1. Characteristics of Cases and Controls Men
Age (yr)* Height (cm) Weight (kg) BMI (kg/m2) Alcohol (g/day) Smokers (%) NIDDM (%)
Women
p Value
Cases (n ⴝ 345)
Controls (n ⴝ 329)
Cases (n ⴝ 88)
Controls (n ⴝ 72)
Cases vs. controls (Both genders pooled)
44.6 (0.5) 173.1 (0.4) 76.6 (0.7) 25.5 (0.2) 78.1 (3.1) 55.1 4.4
45.2 (0.5) 174.3 (0.4) 77.6 (0.7) 25.5 (0.2) 26.7 (3.2) 27.1 1.5
48.4 (1.0) 161.7 (0.7) 60.3 (1.4) 23.1 (0.4) 9.0 (6.2) 25.0 4.6
48.9 (1.1) 162.2 (0.8) 62.8 (1.5) 23.9 (0.5) 12.2 (6.8) 19.4 2.8
0.35 0.02 0.15 0.64 ⬍ 0.001 0.001 0.03
*Age at diagnosis for cases and current age for controls. Data are expressed as mean (SE) or %. BMI ⫽ body mass index (weight/height2); NIDDM ⫽ non–insulin-dependent diabetes mellitus.
ventricular dysfunction and a lower ejection fraction (data not shown). There was no deviation from Hardy-Weinberg equilibrium for any of the polymorphisms considered. None of the polymorphisms exhibited significant difference of genotype or allele frequencies between cases and controls (Table 2).
No significant association was observed when considering either a dominant or a recessive model for each polymorphism (Fig. 1). We further investigated whether polymorphisms were associated with the severity of disease assessed by ejection fraction (⬍/ⱖ24%), left ventricular dilation (end-diastolic diameter ⬍/ⱖ40 mm/m2) or heart transplan-
Table 2. Genotype and Allele Frequencies of Candidate Gene Polymorphisms in Cases and Controls Genotypes
ACE I/D Cases Controls AGT T174M Cases Controls AGT M235T Cases Controls AGTR1 A-153G Cases Controls AGTR1 A⫹39C Cases Controls CYP11B2 T-344C Cases Controls TNF G-308A Cases Controls TGFB1 R25P Cases Controls NOS3 G⫹11/in23T Cases Controls BNP C-1563T Cases Controls
n (%)
n (%)
n (%)
Allele Frequency
II 94 (22.3) 71 (18.4) TT 333 (77.8) 295 (74.1) MM 157 (36.7) 131 (32.9) AA 296 (69.5) 267 (67.4) AA 223 (52.6) 209 (52.5) TT 132 (31.0) 114 (29.0) GG 322 (75.2) 288 (72.7) RR 360 (85.3) 344 (86.2) GG 186 (43.4) 177 (44.9) CC 401 (95.3) 379 (95.7)
ID 200 (47.4) 190 (49.1) TM 88 (20.6) 93 (23.4) MT 200 (46.7) 195 (49.0) AG 121 (28.4) 111 (28.0) AC 161 (38.0) 152 (38.2) TC 219 (51.4) 195 (49.6) GA 100 (23.4) 95 (24.0) RP 58 (13.7) 52 (13.0) GT 189 (44.1) 169 (42.9) CT 18 (4.3) 17 (4.3)
DD 128 (30.3) 126 (32.6) MM 7 (1.6) 10 (2.5) TT 71 (16.6) 72 (18.1) GG 9 (2.1) 18 (4.6) CC 40 (9.4) 37 (9.3) CC 75 (17.6) 84 (21.4) AA 6 (1.4) 13 (3.3) PP 4 (1.0) 3 (0.8) TT 54 (12.6) 48 (12.2) TT 2 (0.5) 0 (0.0)
f (D) 0.54 0.57 f (M) 0.12 0.14 f (T) 0.40 0.43 f (G) 0.16 0.19 f (C) 0.28 0.28 f (C) 0.43 0.46 f (A) 0.13 0.15 f (P) 0.08 0.07 f (T) 0.35 0.34 f (T) 0.03 0.02
Note: No significant difference existed between cases and controls.
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Figure 1. Genotypic odds ratios for DCM and 95% confidence intervals, assuming a recessive (left) or dominant (right) genetic model. For all polymorphisms, the major allele was taken as the reference allele, except for ACE I/D where I was the reference allele. For TGFB1 R25P and BNP C-1563T, the recessive model was not considered because of the low frequency of the minor allele.
tation. No significant difference was found between subgroups of disease severity. The genotype and allele frequencies did not differ between patients with and without a familial history of DCM. Because alcohol intake is a major etiologic factor for DCM, we investigated whether alcohol might modulate the effect of polymorphisms on the risk of DCM, but we did not find any evidence for alcohol– genotype interaction. Finally, for each polymorphism, we performed multivariate logistic regression analysis controlling for age, gender, height, weight, diabetes, alcohol intake, smoking status and genotype (assuming either a recessive or a dominant model), but genotypic ORs were little modified by adjustment and remained nonsignificant.
DISCUSSION Selection of the candidate genes and of the polymorphisms examined in the present study was based on pathophysiological considerations and/or results previously reported in published data. Genes of the renin-angiotensin-aldosterone system. The renin-angiotensin-aldosterone system may be involved in the susceptibility to DCM because of its action on cellular hypertrophy, cell proliferation and cardiac remodeling. The major biologically active product of the renin-angiotensin system is angiotensin II, which is liberated from AGT by the sequential action of renin and ACE. In adult humans, the effects of angiotensin II are mainly mediated by the AGTR1, a G-protein-coupled receptor expressed by many cell types, in particular cardiomyocytes. Each component of this system constitutes then a candidate for DCM. The ACE I/D polymorphism was the first genetic factor reported to be associated with nonfamilial IDC (15). However, the association seemed mainly due to a unexpected low frequency of the DD genotype in controls rather than to an
excess in cases, and this finding failed to be confirmed in later studies (16 –19). In contrast, the DD genotype was found associated with poorer survival and reduced left ventricular performance in idiopathic heart failure and IDC (17,20). Angiotensinogen (AGT) is the precursor of angiotensin-I and its concentration is rate-limiting for the generation of angiotensin-II. Ever since the initial finding of an association between the M235T and T174M polymorphisms of the AGT gene and essential hypertension (9), the implication of the AGT locus in the pathogenesis of hypertension and the regulation of blood pressure has been confirmed in a large number of studies. A possible involvement of these two polymorphisms in the susceptibility to IDC was investigated in a Japanese study, which failed to detect any association with the disease itself or its severity (19). The AGTR1 has been shown to be selectively downregulated in failing left ventricle from patients with endstage heart failure due to IDC, suggesting that the failing human heart is exposed to increased concentrations of angiotensin-II (21). Genetic variation at the AGTR1 locus might then influence susceptibility to cardiomyopathy or progression of the disease, although this possibility has not yet been examined. The selection of the two polymorphisms investigated in the present study (A-153G and A⫹39C) was based on the fact that they had been previously suggested to be involved in susceptibility to myocardial infarction (10,22) and essential hypertension (23). Aldosterone, whose production is regulated primarily by the renin–angiotensin system, may have indirect effects on cardiac structure and function through its role in blood pressure regulation (24). In addition, aldosterone has direct effects on the heart in animal studies, including induction of myocardial hypertrophy and fibrosis (25,26). The CYP11B2 T-344C polymorphism, which is associated with plasma
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aldosterone levels (27), has been shown in a small study to strongly affect left ventricular size and mass in young adults free of clinical heart disease (28). The TNF gene. The hypertrophic response to myocardial failure may be considered as a generalized inflammatory response (29). In this line of evidence, several proinflammatory cytokines have been shown to be increased in the myocardium of subjects with heart failure, among which one of the most important by its powerful biological effects is TNF. Levels of this protein are elevated in the circulation of patients with congestive heart failure (30 –32) and an increased expression has been observed in the failing human heart (33–35). Moreover, in transgenic mice, a cardiac overexpression of TNF produces heart failure and DCM (36,37). All these findings concur to suggest that TNF may be an important mediator of cardiac inflammation evolving to DCM. The G-308A polymorphism was selected because it had been shown to affect transcriptional activity (38). The TGFB1 gene. The TGFB1 is another cytokine that might play an important role in the immune modulation of cardiac function. It is a known regulator of cardiac fibroblasts and myocyte function. Its release by activated macrophages stimulates cardiac fibrosis and influences cardiac remodeling, contributing to impairment of myocardial contractility (39,40). The nonsynonymous TGFB1 R25P polymorphism, which affects TGFB1 production in vitro (41), has been found associated with several diseases, in particular hypertension (13,42). The NOS3 gene. Nitric oxide is a powerful vasoactive molecule produced from L-arginine by three distinct nitric oxide synthase enzymes. Two constitutively present enzymes are found in neuronal and endothelial cells, respectively, and the third one is inducible in many cell types after immunological or inflammatory stimuli. The endothelial isoform NOS3 is also constitutively expressed in cardiac myocytes (43). Enhanced basal production of nitric oxide has been reported in patients with heart failure (44 – 46) while increased activity of inducible nitric oxide synthase has been found in cardiac tissue from patients with DCM (34,47– 49). An explanation for these findings might be that an excessive production of nitric oxide in cardiomyocytes, when exerted over long periods, might lead to a depression of myocardial contractility, an important feature of DCM. A family study recently demonstrated genetic linkage between the level of plasma metabolites of nitric oxide and markers located in the region of the NOS3 gene (50). This result suggests that sequence variation in the NOS3 gene might influence nitric oxide production, and thereby affect the risk and/or the progression of DCM. The BNP gene. Brain natriuretic peptide is mainly produced by the ventricles (51) and its secretion has been found increased in the failing heart in proportion to the severity of left-ventricular dysfunction (52). Its expression has been observed primarily in myocytes in the interstitial fibrous area
Tiret et al. Dilated Cardiomyopathy and Candidate Genes
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in DCM (53). Plasma levels of BNP are raised in patients with heart failure (51,54 –56), left-ventricular dysfunction (57,58), DCM (59) and have a prognostic role in mortality of patients with congestive heart failure (60). Power of the study. The CARDIGENE study, including more than 400 patients with DCM, is the largest study conducted so far on this topic. Assuming an additive or a dominant genetic model, the sample size provided a ⱖ80% power to detect the effect of any polymorphism having a frequency ⱖ0.1 and associated with an OR for disease ⱖ1.6. Under a recessive model, polymorphisms with a frequency ⱖ0.25 and associated with an OR ⱖ2 could be detected. The power of the study was then adequate to detect associations of modest magnitude. Moreover, patients were selected on strict criteria of disease severity, and both patients and controls had to fulfill conditions of ethnic homogeneity. In control subjects, there was no difference of allele frequencies among the three MONICA regions despite their geographical distance (northern, eastern and southern France), making unlikely a risk of stratification of the population for the polymorphisms considered. Several reasons might explain the negative results. The first one is that the investigated genes, despite being strong candidates a priori, do not play any significant role in the pathogenesis of DCM. A second reason might be that the polymorphisms selected in each gene were not appropriate and that there exist other unmeasured polymorphisms of these genes whose effect on disease could not be detected through linkage disequilibrium with the polymorphisms studied. However, this explanation is rather unlikely, given the strong linkage disequilibrium generally observed within candidate genes. A third explanation might be related to the heterogeneity of patients with respect to progression of the disease. In actuality, the sample included both new and old patients, some of them being followed up for more than 10 years. If the same genetic factor contributed to both the susceptibility to disease and its mortality, then mixing new cases and long-term survivors might mask the genetic effect. This question would have to be addressed in longitudinal studies.
APPENDIX CARDIGENE Project Management Group: M. Desnos, Service de Cardiologie, Hoˆpital Boucicaut, Paris; R. Dorent, Service de Chirurgie Cardiaque, Groupe Hospitalier Pitie´Salpeˆtrie`re, Paris; M. Komajda, Service de Cardiologie, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Paris; G. Roize`s, INSERM U249, Montpellier; K. Schwartz, INSERM U523, Paris; L. Tiret, INSERM U525, Paris, France. CARDIGENE recruitment centers: CHU Ambroise Pare´, Boulogne Billancourt (O. Dubourg); CHU Henri Mondor, Cre´teil (F. Paillart); CHR de Lille (A. Millaire); Hoˆpital Louis Pradel, Lyon (X. Andre´-Foue¨t, J. Beaune, P. Touboul); CHR de Nancy (Y. Juillie`re); CHR de Nantes (J-B. Bouhour, N. Chedru); CHU Pitie´-Salpeˆtrie`re, Paris
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(R. Dorent, M. Komajda); Hoˆpital Boucicaut, Paris (M. Desnos); Hoˆpital Bichat, Paris (M-C. Aumont); CHR de Strasbourg (A. Sacrez). Acknowledgments We thank Dominique Arveiler (MONICA Strasbourg), Philippe Amouyel (MONICA Lille) and Jean-Bernard Ruidavets (MONICA Toulouse) for providing the control subjects, and Christiane Souriau for DNA extraction. Reprint requests and correspondence: Dr. Laurence Tiret, INSERM U525, 17 rue du Fer a` Moulin, 75005 Paris, France. E-mail: [email protected].
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Vol. 35, No. 1, 2000 ISSN 0735-1097/00/$20.00 PII S0735-1097(99)00522-7
Lack of Association Between Polymorphisms of Eight Candidate Genes and Idiopathic Dilated Cardiomyopathy The CARDIGENE Study Laurence Tiret, PHD,* Christine Mallet, MSC,† Odette Poirier, PHD,* Viviane Nicaud, MA,* Alain Millaire, MD, PHD,‡ Jean-Brieuc Bouhour, MD,§ Ge´rard Roize`s, PHD,㛳 Michel Desnos, MD,¶ Richard Dorent, MD,# Ketty Schwartz, PHD,†† Franc¸ois Cambien, MD,* Michel Komajda, MD,** FOR CARDIGENE GROUP Paris, Lille, Nantes, and Montpellier, France OBJECTIVES
THE
The study investigated the potential role of eight candidate genes in the susceptibility to idiopathic dilated cardiomyopathy (IDC).
BACKGROUND Idiopathic dilated cardiomyopathy has a familial origin in 20% to 25% of cases, and several genetic loci have been identified in rare monogenic forms of the disease. These findings led to the hypothesis that genetic factors might also be involved in sporadic forms of the disease. In complex diseases that do not exhibit a clear pattern of familial aggregation, the candidate gene approach is a strategy widely used to identify susceptibility genes. All genes coding for proteins involved in biochemical or physiological abnormalities of cardiac function are potential candidates for IDC. METHODS
We studied 433 patients with IDC and 401 gender- and age-matched controls. Polymorphisms investigated were the I/D polymorphism of the angiotensin I-converting enzyme (ACE) gene, the T174M and M235T polymorphisms of the angiotensinogen (AGT) gene, the A-153G and A⫹39C polymorphisms of the angiotensin-II type 1 receptor (AGTR1) gene, the T-344C polymorphism of the aldosterone synthase (CYP11B2) gene, the G-308A polymorphism of the tumor necrosis factor-alpha (TNF) gene, the R25P polymorphism of the transforming growth factor beta1 (TGFB1) gene, the G⫹11/in23T polymorphism of the endothelial nitric oxide synthase (NOS3) gene and the C-1563T polymorphism of the brain natriuretic peptide (BNP) gene.
RESULTS
None of the polymorphisms were significantly associated with the risk or the severity of the disease.
CONCLUSIONS We did not find evidence for an involvement of any of the 10 investigated polymorphisms in the susceptibility to IDC. (J Am Coll Cardiol 2000;35:29 –35) © 1999 by the American College of Cardiology
Dilated cardiomyopathy (DCM) is a myocardial disease characterized by impaired systolic function and dilation of the left or both ventricles (1). Despite recent advances in medical and surgical therapies, it remains an important cause of mortality and morbidity and is a leading indication for heart transplantation. The etiology of DCM is idiopathic in about half the patients. Family studies using From the *INSERM U525, Paris; †INSERM SC7, Paris; ‡CHR de Lille; §CHR de Nantes; 㛳INSERM U249, Montpellier; ¶Hoˆpital Boucicaut, Paris; #Service de Chirurgie Cardiaque and **Service de Cardiologie, Groupe Hospitalier Pitie´Salpeˆtrie`re, Paris; ††INSERM U523, Paris, France. The CARDIGENE study was supported by grants from Parke Davis France, the “De´le´gation a` la Recherche Clinique AP-HP” (EMUL and PHRC) and INSERM. Manuscript received February 19, 1999; revised manuscript received July 29, 1999, accepted October 5, 1999.
echocardiography to identify individuals with presymptomatic disease revealed that 20% to 25% of idiopathic dilated cardiomyopathy (IDC) had a familial origin, a proportion that had been considerably underestimated previously (2,3). This finding raised the hypothesis that gene defects might play an important role in the disease pathogenesis. Indeed, several genetic loci responsible for rare autosomal dominant or X-linked forms of DCM have been subsequently localized by linkage analysis in large pedigrees (review in [4]). The recognition of familial DCM led to the notion that even in sporadic cases, genetic factors might contribute to the disease susceptibility. In complex diseases that do not exhibit a clear pattern of familial aggregation, the candidate gene approach, which tests the role of known genes selected for their potential implication in the pathophysiological
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Tiret et al. Dilated Cardiomyopathy and Candidate Genes
Abbreviations ACE AGT AGTR1 BNP CYP11B2 DCM IDC NOS3 TGFB1 TNF
and Acronyms ⫽ angiotensin I-converting enzyme ⫽ angiotensinogen ⫽ angiotensin-II type 1 receptor ⫽ brain natriuretic peptide ⫽ aldosterone synthase ⫽ dilated cardiomyopathy ⫽ idiopathic dilated cardiomyopathy ⫽ endothelial nitric oxide synthase ⫽ transforming growth factor beta1 ⫽ tumor necrosis factor-alpha
process, is a strategy widely used (5,6). In DCM, all genes coding for proteins involved in biochemical or physiological abnormalities of cardiac function are potential candidates. The CARDIGENE study is a large case-control study designed to identify genetic factors involved in the predisposition to IDC. This article reports the results of an investigation of eight candidate genes: the angiotensin I-converting enzyme (ACE), the angiotensin-II type 1 receptor (AGTR1), the angiotensinogen (AGT), the aldosterone synthase (CYP11B2), the tumor necrosis factoralpha (TNF), the transforming growth factor beta1 (TGFB1), the endothelial nitric oxide synthase (NOS3) and the brain natriuretic peptide (BNP) genes.
METHODS Ethics approval for the study was obtained from the relevant committees, and each subject gave written informed consent for the study. Patients. Patients aged 18 to 65 years (n ⫽ 433) were recruited between 1994 and 1996 from 10 hospitals (see list in Appendix). The diagnosis of IDC was based on impaired left ventricular systolic function (ejection fraction ⱕ40% on ventriculography, radionucleotide angiography or echocardiography) and left ventricular dilation (end-diastolic volume ⬎140 ml/m2 on ventriculography or end-diastolic diameter ⬎34 mm/m2 on echocardiography), confirmed over a six-month period. For transplanted patients, these criteria had to be fulfilled before heart transplantation. Coronary angiography had to be performed in all subjects aged ⬎35 years, and patients with ⱖ50% obstruction of one or more coronary arteries were excluded. Patients with myocardial infarction, sustained systemic arterial hypertension, insulin-dependent diabetes, intrinsic valvular disease, congenital cardiopathy, documented myocarditis, specific primary or secondary heart muscle disease were also excluded. All patients had to be born in France, and their parents had to be born in France or neighboring countries. The mean left ventricular ejection fraction was 23.3 ⫾ 7.6% (n ⫽ 433), the mean end-diastolic volume was 194 ⫾ 37 ml/m2 on ventriculography (n ⫽ 182) and the mean end-diastolic diameter was 40.1 ⫾ 5.1 mm/m2 (n ⫽ 371) on
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echocardiography. The mean duration between diagnosis and inclusion was 7.1 ⫾ 5.3 years. Of the 433 patients, 229 (52.9%) had heart transplantation. Family history of DCM, defined as definite or possible cardiomyopathy, or sudden death ⱕ35 years, in first- or second-degree relatives, was reported in 25% of patients. Controls. Controls (n ⫽ 401) were randomly sampled from the French population surveys carried out in Lille (northern France), Strasbourg (eastern France) and Toulouse (southern France) within the framework of the WHO MONICA (MONItoring in CArdiovascular diseases) project (7). A stratification was used to match the approximate age distributions in cases and controls (age at diagnosis in cases and current age in controls). Controls with clinical history of cardiovascular disease or insulindependent diabetes were excluded. Genotyping. Genomic DNA was prepared from white blood cells by phenol extraction. Polymorphisms investigated were an insertion (I)/deletion (D) polymorphism in intron 16 of the ACE gene (8); two nucleotide substitutions resulting in amino acid changes at codons 174 (Thr/Met) and 235 (Met/Thr) of the AGT gene (9); two nucleotide substitutions A-153G and A⫹39C (previously named A⫹1166C) in the AGTR1 gene (10); a nucleotide substitution T-344C in the CYP11B2 gene (11); a nucleotide substitution G-308A in the TNF gene (12); a nucleotide substitution resulting in an amino acid change at codon 25 (Arg/Pro) of the TGFB1 gene (13); a G/T nucleotide substitution in intron 23 of the NOS3 gene (14); and a nucleotide substitution C-1563T in the BNP gene (unpublished). Statistical analysis. Data were analyzed using the SAS statistical software (SAS Institute, Cary, North Carolina). Allele frequencies were estimated by gene counting. HardyWeinberg equilibrium was tested by chi-square analysis. Genotype and allele frequencies were compared between cases and controls by chi-square analysis. Genotypic odds ratios (OR) for disease, assuming a dominant or a recessive model, were computed. Association between polymorphisms and case/control status was also tested by means of logistic regression analysis controlling for age, gender (matching criteria) and different covariates. A p value ⬍0.05 was considered statistically significant.
RESULTS Characteristics of cases and controls are presented in Table 1. The mean age at diagnosis was 44.6 years in male and 48.4 years in female cases. Compared to controls, cases were shorter by approximately 1 cm (p ⬍ 0.05), had a higher alcohol and tobacco consumption (p ⬍ 0.001) and a higher prevalence of non–insulin-dependent diabetes mellitus (p ⬍ 0.05). Transplanted patients were younger, leaner and had a lower alcohol consumption than did nontransplanted patients, and they were characterized by a more severe left
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Table 1. Characteristics of Cases and Controls Men
Age (yr)* Height (cm) Weight (kg) BMI (kg/m2) Alcohol (g/day) Smokers (%) NIDDM (%)
Women
p Value
Cases (n ⴝ 345)
Controls (n ⴝ 329)
Cases (n ⴝ 88)
Controls (n ⴝ 72)
Cases vs. controls (Both genders pooled)
44.6 (0.5) 173.1 (0.4) 76.6 (0.7) 25.5 (0.2) 78.1 (3.1) 55.1 4.4
45.2 (0.5) 174.3 (0.4) 77.6 (0.7) 25.5 (0.2) 26.7 (3.2) 27.1 1.5
48.4 (1.0) 161.7 (0.7) 60.3 (1.4) 23.1 (0.4) 9.0 (6.2) 25.0 4.6
48.9 (1.1) 162.2 (0.8) 62.8 (1.5) 23.9 (0.5) 12.2 (6.8) 19.4 2.8
0.35 0.02 0.15 0.64 ⬍ 0.001 0.001 0.03
*Age at diagnosis for cases and current age for controls. Data are expressed as mean (SE) or %. BMI ⫽ body mass index (weight/height2); NIDDM ⫽ non–insulin-dependent diabetes mellitus.
ventricular dysfunction and a lower ejection fraction (data not shown). There was no deviation from Hardy-Weinberg equilibrium for any of the polymorphisms considered. None of the polymorphisms exhibited significant difference of genotype or allele frequencies between cases and controls (Table 2).
No significant association was observed when considering either a dominant or a recessive model for each polymorphism (Fig. 1). We further investigated whether polymorphisms were associated with the severity of disease assessed by ejection fraction (⬍/ⱖ24%), left ventricular dilation (end-diastolic diameter ⬍/ⱖ40 mm/m2) or heart transplan-
Table 2. Genotype and Allele Frequencies of Candidate Gene Polymorphisms in Cases and Controls Genotypes
ACE I/D Cases Controls AGT T174M Cases Controls AGT M235T Cases Controls AGTR1 A-153G Cases Controls AGTR1 A⫹39C Cases Controls CYP11B2 T-344C Cases Controls TNF G-308A Cases Controls TGFB1 R25P Cases Controls NOS3 G⫹11/in23T Cases Controls BNP C-1563T Cases Controls
n (%)
n (%)
n (%)
Allele Frequency
II 94 (22.3) 71 (18.4) TT 333 (77.8) 295 (74.1) MM 157 (36.7) 131 (32.9) AA 296 (69.5) 267 (67.4) AA 223 (52.6) 209 (52.5) TT 132 (31.0) 114 (29.0) GG 322 (75.2) 288 (72.7) RR 360 (85.3) 344 (86.2) GG 186 (43.4) 177 (44.9) CC 401 (95.3) 379 (95.7)
ID 200 (47.4) 190 (49.1) TM 88 (20.6) 93 (23.4) MT 200 (46.7) 195 (49.0) AG 121 (28.4) 111 (28.0) AC 161 (38.0) 152 (38.2) TC 219 (51.4) 195 (49.6) GA 100 (23.4) 95 (24.0) RP 58 (13.7) 52 (13.0) GT 189 (44.1) 169 (42.9) CT 18 (4.3) 17 (4.3)
DD 128 (30.3) 126 (32.6) MM 7 (1.6) 10 (2.5) TT 71 (16.6) 72 (18.1) GG 9 (2.1) 18 (4.6) CC 40 (9.4) 37 (9.3) CC 75 (17.6) 84 (21.4) AA 6 (1.4) 13 (3.3) PP 4 (1.0) 3 (0.8) TT 54 (12.6) 48 (12.2) TT 2 (0.5) 0 (0.0)
f (D) 0.54 0.57 f (M) 0.12 0.14 f (T) 0.40 0.43 f (G) 0.16 0.19 f (C) 0.28 0.28 f (C) 0.43 0.46 f (A) 0.13 0.15 f (P) 0.08 0.07 f (T) 0.35 0.34 f (T) 0.03 0.02
Note: No significant difference existed between cases and controls.
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Figure 1. Genotypic odds ratios for DCM and 95% confidence intervals, assuming a recessive (left) or dominant (right) genetic model. For all polymorphisms, the major allele was taken as the reference allele, except for ACE I/D where I was the reference allele. For TGFB1 R25P and BNP C-1563T, the recessive model was not considered because of the low frequency of the minor allele.
tation. No significant difference was found between subgroups of disease severity. The genotype and allele frequencies did not differ between patients with and without a familial history of DCM. Because alcohol intake is a major etiologic factor for DCM, we investigated whether alcohol might modulate the effect of polymorphisms on the risk of DCM, but we did not find any evidence for alcohol– genotype interaction. Finally, for each polymorphism, we performed multivariate logistic regression analysis controlling for age, gender, height, weight, diabetes, alcohol intake, smoking status and genotype (assuming either a recessive or a dominant model), but genotypic ORs were little modified by adjustment and remained nonsignificant.
DISCUSSION Selection of the candidate genes and of the polymorphisms examined in the present study was based on pathophysiological considerations and/or results previously reported in published data. Genes of the renin-angiotensin-aldosterone system. The renin-angiotensin-aldosterone system may be involved in the susceptibility to DCM because of its action on cellular hypertrophy, cell proliferation and cardiac remodeling. The major biologically active product of the renin-angiotensin system is angiotensin II, which is liberated from AGT by the sequential action of renin and ACE. In adult humans, the effects of angiotensin II are mainly mediated by the AGTR1, a G-protein-coupled receptor expressed by many cell types, in particular cardiomyocytes. Each component of this system constitutes then a candidate for DCM. The ACE I/D polymorphism was the first genetic factor reported to be associated with nonfamilial IDC (15). However, the association seemed mainly due to a unexpected low frequency of the DD genotype in controls rather than to an
excess in cases, and this finding failed to be confirmed in later studies (16 –19). In contrast, the DD genotype was found associated with poorer survival and reduced left ventricular performance in idiopathic heart failure and IDC (17,20). Angiotensinogen (AGT) is the precursor of angiotensin-I and its concentration is rate-limiting for the generation of angiotensin-II. Ever since the initial finding of an association between the M235T and T174M polymorphisms of the AGT gene and essential hypertension (9), the implication of the AGT locus in the pathogenesis of hypertension and the regulation of blood pressure has been confirmed in a large number of studies. A possible involvement of these two polymorphisms in the susceptibility to IDC was investigated in a Japanese study, which failed to detect any association with the disease itself or its severity (19). The AGTR1 has been shown to be selectively downregulated in failing left ventricle from patients with endstage heart failure due to IDC, suggesting that the failing human heart is exposed to increased concentrations of angiotensin-II (21). Genetic variation at the AGTR1 locus might then influence susceptibility to cardiomyopathy or progression of the disease, although this possibility has not yet been examined. The selection of the two polymorphisms investigated in the present study (A-153G and A⫹39C) was based on the fact that they had been previously suggested to be involved in susceptibility to myocardial infarction (10,22) and essential hypertension (23). Aldosterone, whose production is regulated primarily by the renin–angiotensin system, may have indirect effects on cardiac structure and function through its role in blood pressure regulation (24). In addition, aldosterone has direct effects on the heart in animal studies, including induction of myocardial hypertrophy and fibrosis (25,26). The CYP11B2 T-344C polymorphism, which is associated with plasma
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aldosterone levels (27), has been shown in a small study to strongly affect left ventricular size and mass in young adults free of clinical heart disease (28). The TNF gene. The hypertrophic response to myocardial failure may be considered as a generalized inflammatory response (29). In this line of evidence, several proinflammatory cytokines have been shown to be increased in the myocardium of subjects with heart failure, among which one of the most important by its powerful biological effects is TNF. Levels of this protein are elevated in the circulation of patients with congestive heart failure (30 –32) and an increased expression has been observed in the failing human heart (33–35). Moreover, in transgenic mice, a cardiac overexpression of TNF produces heart failure and DCM (36,37). All these findings concur to suggest that TNF may be an important mediator of cardiac inflammation evolving to DCM. The G-308A polymorphism was selected because it had been shown to affect transcriptional activity (38). The TGFB1 gene. The TGFB1 is another cytokine that might play an important role in the immune modulation of cardiac function. It is a known regulator of cardiac fibroblasts and myocyte function. Its release by activated macrophages stimulates cardiac fibrosis and influences cardiac remodeling, contributing to impairment of myocardial contractility (39,40). The nonsynonymous TGFB1 R25P polymorphism, which affects TGFB1 production in vitro (41), has been found associated with several diseases, in particular hypertension (13,42). The NOS3 gene. Nitric oxide is a powerful vasoactive molecule produced from L-arginine by three distinct nitric oxide synthase enzymes. Two constitutively present enzymes are found in neuronal and endothelial cells, respectively, and the third one is inducible in many cell types after immunological or inflammatory stimuli. The endothelial isoform NOS3 is also constitutively expressed in cardiac myocytes (43). Enhanced basal production of nitric oxide has been reported in patients with heart failure (44 – 46) while increased activity of inducible nitric oxide synthase has been found in cardiac tissue from patients with DCM (34,47– 49). An explanation for these findings might be that an excessive production of nitric oxide in cardiomyocytes, when exerted over long periods, might lead to a depression of myocardial contractility, an important feature of DCM. A family study recently demonstrated genetic linkage between the level of plasma metabolites of nitric oxide and markers located in the region of the NOS3 gene (50). This result suggests that sequence variation in the NOS3 gene might influence nitric oxide production, and thereby affect the risk and/or the progression of DCM. The BNP gene. Brain natriuretic peptide is mainly produced by the ventricles (51) and its secretion has been found increased in the failing heart in proportion to the severity of left-ventricular dysfunction (52). Its expression has been observed primarily in myocytes in the interstitial fibrous area
Tiret et al. Dilated Cardiomyopathy and Candidate Genes
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in DCM (53). Plasma levels of BNP are raised in patients with heart failure (51,54 –56), left-ventricular dysfunction (57,58), DCM (59) and have a prognostic role in mortality of patients with congestive heart failure (60). Power of the study. The CARDIGENE study, including more than 400 patients with DCM, is the largest study conducted so far on this topic. Assuming an additive or a dominant genetic model, the sample size provided a ⱖ80% power to detect the effect of any polymorphism having a frequency ⱖ0.1 and associated with an OR for disease ⱖ1.6. Under a recessive model, polymorphisms with a frequency ⱖ0.25 and associated with an OR ⱖ2 could be detected. The power of the study was then adequate to detect associations of modest magnitude. Moreover, patients were selected on strict criteria of disease severity, and both patients and controls had to fulfill conditions of ethnic homogeneity. In control subjects, there was no difference of allele frequencies among the three MONICA regions despite their geographical distance (northern, eastern and southern France), making unlikely a risk of stratification of the population for the polymorphisms considered. Several reasons might explain the negative results. The first one is that the investigated genes, despite being strong candidates a priori, do not play any significant role in the pathogenesis of DCM. A second reason might be that the polymorphisms selected in each gene were not appropriate and that there exist other unmeasured polymorphisms of these genes whose effect on disease could not be detected through linkage disequilibrium with the polymorphisms studied. However, this explanation is rather unlikely, given the strong linkage disequilibrium generally observed within candidate genes. A third explanation might be related to the heterogeneity of patients with respect to progression of the disease. In actuality, the sample included both new and old patients, some of them being followed up for more than 10 years. If the same genetic factor contributed to both the susceptibility to disease and its mortality, then mixing new cases and long-term survivors might mask the genetic effect. This question would have to be addressed in longitudinal studies.
APPENDIX CARDIGENE Project Management Group: M. Desnos, Service de Cardiologie, Hoˆpital Boucicaut, Paris; R. Dorent, Service de Chirurgie Cardiaque, Groupe Hospitalier Pitie´Salpeˆtrie`re, Paris; M. Komajda, Service de Cardiologie, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Paris; G. Roize`s, INSERM U249, Montpellier; K. Schwartz, INSERM U523, Paris; L. Tiret, INSERM U525, Paris, France. CARDIGENE recruitment centers: CHU Ambroise Pare´, Boulogne Billancourt (O. Dubourg); CHU Henri Mondor, Cre´teil (F. Paillart); CHR de Lille (A. Millaire); Hoˆpital Louis Pradel, Lyon (X. Andre´-Foue¨t, J. Beaune, P. Touboul); CHR de Nancy (Y. Juillie`re); CHR de Nantes (J-B. Bouhour, N. Chedru); CHU Pitie´-Salpeˆtrie`re, Paris
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(R. Dorent, M. Komajda); Hoˆpital Boucicaut, Paris (M. Desnos); Hoˆpital Bichat, Paris (M-C. Aumont); CHR de Strasbourg (A. Sacrez). Acknowledgments We thank Dominique Arveiler (MONICA Strasbourg), Philippe Amouyel (MONICA Lille) and Jean-Bernard Ruidavets (MONICA Toulouse) for providing the control subjects, and Christiane Souriau for DNA extraction. Reprint requests and correspondence: Dr. Laurence Tiret, INSERM U525, 17 rue du Fer a` Moulin, 75005 Paris, France. E-mail: [email protected].
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