Original Article

Variant on 9p is associated with left ventricular mass: results from two cohorts of essential hypertensive individuals

Cristina Mennia,b,j, Lucia Boffia, Francesca Cesanaa, Chiara Viviani Anselmic, Stefano Navaa, Francesca Bertolad, Anna M. Di Blasioe, Roberta Roncaratic,f, Valentina Trimarcog, Marina Marinoh, Bruno Trimarcoh, Guido Grassia, Cristina Giannattasioa,i, and Giuseppe Manciaa

INTRODUCTION Objectives: It is well known that among hypertensive patients, an increased left ventricular mass (LVM) is a ncreased left ventricular mass (LVM) is a strong and powerful predictor of cardiovascular morbidity and independent risk factor for cardiovascular morbidity mortality. However, the mechanisms underlying LVM I and mortality [1,2]. Even though demographic in hypertension are not completely understood, as characteristics, such as sex and BMI, and hemodynamic the absolute value of blood pressure and other risk variables, such as blood pressure (BP) values, volume load factors associated do not predict alone a definite and contractile efficiency, are known to be associated with LVM progression. Recently, the 9p21 chromosomal LVM, nearly half of the LVM variability remains unexplained region has been consistently associated with coronary [3]. LVM heritability is found to be between 0.17 and 0.59, heart disease. thus suggesting that LVM has a clear genetic component Methods and results: We examined the association of [4–8]. Candidate studies have shown a potential role 384 single nucleotide polymorphisms (SNPs) in the short of genetic polymorphisms located in angiotensin-convert- arm of with LVM in 821 hypertensive ing enzyme [9] and aldosterone synthase (CYP11B2) [10], individuals from northern Italy. We identified a SNP insulin-like growth factor [11], neuropeptide Y [12], guanine (rs894379) in the intronic region of the centlein, nucleotide-binding 3 [13], endothelial nitric oxide centrosomal protein (CNTLN) on chromosome synthase [14] and peroxisome proliferator-activated recep- 9p22, whose minor allele G is associated with an tor-a (PPARa) [15] genes. Genome-wide linkage and increased LVM. We performed a follow-up validation association studies (GWASs) have shown an association analysis for the top SNP in 1038 hypertensive individuals between LVM, assessed especially with ECG criteria, and from southern Italy. We then combined the results and several loci located in different (3, 5, 6, 7, 10, found a nominal association for rs894379 (b ¼ 2.46, 12, 15, 17 and 19) [16–18]. Particularly, in a whole genome P ¼ 0.0026). linkage study of hypertensive families, three regions (10q23.1, 12q14.1 and 17p13.3) were found to approach Conclusion: We describe a new variant associated with suggestive evidence of linkage for particular measures of echocardiography LVM. This result, though it needs to be further investigated, may improve our understanding of the genetic determination of this prognostically relevant trait. Journal of Hypertension 2012, 30:2144–2150 aDepartment of Clinical Medicine and Prevention, San Gerardo Hospital, University of Keywords: association, chromosome 9p21, genetics, left Milano-Bicocca, Milan, Italy, bBritish Heart Foundation, Glasgow Cardiovascular ventricular mass Research Centre, University of Glasgow, Glasgow, UK, cUnit of Genetics and Car- diovascular Research Institute, Istituto Ricovero Cura Carattere Scientifico ‘Multi- Abbreviations: BP, blood pressure; BSA, body surface medica’, Sesto San Giovanni, dConsortium for Human Molecular Genetics, University of Milano-Bicocca, eIstituto Auxologico Italiano IRCCS, fMilan Unit, Biomedical and area; CNTLN, centlein centrosomal protein; ELN, elastin; Genetic Research Institute (IRGB), National Research Council of Italy, Milan, ET-A, endothelin receptor type A; FC-ECG, cardiac gDepartment of Neuroscience, hDepartment of Clinical Medicine and Cardiovascular frequency ECG; FDR, false discovery rate; GWAS, genome- and Immunological Sciences, Federico II University Hospital, Naples, iCardiology IV, Cardiothoracovascular Department A. De Gasperis, Niguarda Hospital, Milan, Italy wide association study; LVEDD, left ventricular end-diastolic and jDepartment of Twin Research and Genetic Epidemiology, King’s College London, diameter; LVM, left ventricular mass; LVMI, LVM index; London, UK LVH, left ventricular hypertrophy; MAF, minor allele Correspondence to Professor Giuseppe Mancia, Department of Clinical Medicine and frequency; MMP9, matrix metallopeptidase 9; SNP, single Prevention, San Gerardo Hospital, University of Milano-Bicocca, Via Pergolesi 33, 20052 Monza, Italy. Tel: +39 039 233 3357; fax: +39 039 322 274; e-mail: nucleotide polymorphism [email protected] Received 4 July 2012 Accepted 13 July 2012 J Hypertens 30:2144–2150 ß 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins. DOI:10.1097/HJH.0b013e3283581f7e

2144 www.jhypertension.com Volume 30 Number 11 November 2012 Chromosome 9p and left ventricular mass left ventricular hypertrophy (LVH) [16]. A GWAS on Hypertension was defined as SBP of at least 140 mmHg Koreans reported a significant correlation between the and DBP of at least 90 mmHg or as the reported use of ryanodine receptor 1 (RYR1) (skeletal) gene on chromo- antihypertensive drugs. some 19 and LVH, suggesting a possible role of intracellular From venous blood sample, levels of fasting serum calcium homeostasis [17]. Finally, a large meta-analysis glucose, serum total cholesterol, high-density lipoprotein identified loci associated with left ventricular structure on (HDL), low-density lipoprotein (LDL) cholesterol and the solute carrier family 35, member F1 (SLC35F1), chromo- serum triglycerides were determined. Height and weight some 6 open reading frame 203 (C6orf203) and phospho- were obtained to calculate the patient BMI and waist lamban (PLN) genes [18]. However, the physiopathologic circumference was assessed halfway between the lower link between genes and LVM remains unclear and these ribs and the iliac crest. results explain only a small part of LVM variance. Two-dimensional-guided M-mode echocardiograms In 2007 McPherson et al. [19] identified a 58-kilobase were performed using a dedicated ultrasound machine interval on chromosome 9p21 that has been consistently (SONOS 5500; Philips Healthcare, Andover, Massachusetts, associated with coronary heart disease in six independent USA with an ultrasound transducer of 2.5MHz.) samples (more than 23 000 participants) from four white LVM was calculated using Deveraux formula – LVM populations. Since then, many studies confirmed the (g) ¼ 0.8 1.04 {[left ventricular end-diastolic diameter association between 9p21 and the onset of coronary heart (LVEDD) (cm) þ interventricular septum þ posterior wall disease, but to date there are scanty data on cardiovascular thickness (cm)]3LVEDD3 (cm)} þ 0.6 – from an echo- phenotypes possibly affected by genes located in that cardiographic measurement of left ventricular septum, left same region. ventricular diameter and posterior wall thickness. The Given the continuous relationship between LVM index LVM values were normalized for BSA to obtain LVMI. We (LVMI) [LVM indexed for body surface area (BSA)] and the calculated BSA using the DuBois and DuBois formula [BSA risk of cardiovascular events [2], in this article, we examined (m2) ¼ 0.007184 height (cm)0.725 weigh (kg)0.425]. The the effects of single nucleotide polymorphisms (SNPs) in intraoperator variability in terms of coefficient of variation the short arm of chromosome 9 on LVM. Indeed, cardio- of the mean of two measurements is less than 3% in our vascular risk increases with increasing LVM, and decreases laboratory. LVH was diagnosed by LVMI of at least 125 g/m2 with regression of LVH in response to antihypertensive for men and at least 110 g/m2 in women. All patients were of treatment [20]. In our study we assessed LVM with white origin. the echocardiographic method in accurately phenotyped hypertensive individuals using an independent cohort to Validation cohort validate results. The identification of genes that predispose To validate our result, we selected 1038 patients from hypertensive individuals to abnormal left ventricular struc- the Campania Salute Project [21]. This included essential tural parameters may aid the early diagnosis and develop- hypertensive individuals from the Campania region, Italy, ment of more specific targeted preventive measures. undergoing regular check-up. Data relevant to the present study include demographic/anthropometric and lifestyle METHODS variables, drug therapy, BP measurements and echo- Ethical considerations cardiography. BP was measured by standard aneroid All studies were approved by institutional ethics review sphygmomanometer after 5-min rest in the supine position. committees at the relevant organizations. All participants Three BP measurements were obtained with the patient provided informed written consent. in the sitting position at 2-min intervals. The average of these measurements was used for the analysis. Essential Discovery cohort hypertension was defined with the same criteria used in the From September 2006 to October 2008, we enrolled discovery cohort. 827 consecutive outpatients aged 18–80 years old, followed Two-dimensional-guided M-mode echocardiograms by the Hypertension Centre of S. Gerardo Hospital, Monza, were performed using a dedicated ultrasound machine Italy, affected by essential hypertension, and in apparently (SONOS 5500; Philips) with an ultrasound transducer of adequate BP control. Those with secondary hypertension, 2.5 MHz. LVM and LVMI were calculated as described chronic renal disease, chronic pulmonary disease, sub- above. Intraoperator and interassay variability were 5 and stance abuse and history of cancer were excluded. 6%, respectively. The medical visit took place in the morning and it was a comprehensive medical history and physical examination. Single nucleotide polymorphism selection and Clinic BP was measured twice by a trained physician by genotyping mercury sphygmomanometer with the patient in the sitting We selected 384 tagSNP from HapMap Phase II (http:// position for at least 5 min and with the arm placed at heart www.hapmap.org) using the linkage disequilibrium level. The first and fifth phase of Korotkoff sounds were tagSNP selection approach [22]. SNPs belonged to the short taken as SBP and DBP values while the cuff was deflated arm of chromosome 9 or to three potentially functional at a rate of 2 mmHg/s. We then measured BP twice with a genes [the matrix metallopeptidase 9 (MMP9) gene on semi-automatic device. Four clinic BP measurements were chromosome 20, the elastin (ELN) gene on chromosome taken during the initial visit. The average of the two manual 7 and the endothelin receptor type A (ET-A) gene on and two semi-automatic BP readings was recorded. chromosome 4] [19,23–25]. Selection criteria included the

Journal of Hypertension www.jhypertension.com 2145 Menni et al. following: SNPs with validation data, successful predictive Association analysis was done using Plink [26]. We genotyping scores for Illumina GoldenGate (Illumina Inc., performed linear regression using the additive model San Diego, California, USA) assays, a minor allele frequency and adjusting for covariates determined by stepwise (MAF) of at least 0.05 and a pairwise linkage disequilibrium regression. The covariates used for multivariate adjustments threshold (r2 0.80) for whites. were age, sex and SBP. A further refinement was made by selecting SNPs We also explored the association of the top hits with LVH belonging to coding, intronic and 50-untranslated and 30- using logistic regression and adjusting for age, sex and SBP. untranslated regions with similar proportions. Because testing multiple SNPs could lead to false- On the 9p region, 335 SNP were selected in almost positive associations, we estimated the false discovery rate 200 different genes within 100-kilobase distance. The (FDR) and used an FDR threshold of 0.1, indicating that cyclin-dependent kinase inhibitor 2A and cyclin-dependent we should expect only 10% of the declared discoveries kinase inhibitor 2B regions on chromosome 9 were below this threshold to be false [27]. studied with a denser coverage (30 SNPs selected with an average distance of 15 kilobase) given their potential Validation analysis importance [19]. The remaining 19 SNPs belonged to For validation, we selected the top SNP and we tested it for the MMP9 (six SNPs), ELN (seven SNPs) and ET-A (six association using linear regression, with adjustment for SNPs) genes and were within a distance of about 6 kilobase the same covariates (age, sex, SBP). from one another. The full list of SNPs selected, along Finally, we combined the results using the inverse with the gene they fall into and their position on the variance fixed-effect meta-analysis. gene, is presented in Table S1 (http://links.lww.com/ HJH/A196). RESULTS Genomic DNA was extracted from 300 ml of fresh peripheral blood using Wizard Genomic DNA Purification The demographic characteristics of the discovery and vali- kit (Promega, Madison, Wisconsin, USA) and then dation cohort are presented in Tables 1 and 2, respectively. resuspended in 70 ml of pure water. DNA aliquots were stocked at 808C. Genotyping was performed on the on Discovery cohort Illumina BeadXpress Reader platform (Illumina Inc.). Males accounted for 57% of the total cohort, they were Genotypes were called using the Illumina GenomeStudio younger than females, had higher BMI, DBP, total chole- Software (Illumina, Inc.). To validate Illumina results, sterol, triglycerides, glucose, creatinine and LVMI. They 100 samples were screened further for six of the 384 SNPs had significantly lower heart rate (cardiac frequency on the ABI Prism 3130 Avant Automatic Sequencer (Applied ECG, FC-ECG), HDL and LDL cholesterol. Eighteen percent Biosystems). of men and 16% of females were current smokers and 81% In validation analysis, genotyping was performed men and 78% females were on antihypertensive treatment. with Taqman predesigned SNP genotyping assays (Applied Of the 384 autosomal SNPs selected, after quality control, Biosystems). 31 were excluded because of poor clustering, two were excluded because of MAF less than 0.01, three were Statistical analysis excluded because of deviation from Hardy–Weinberg Quality checking included manual review of all cluster equilibrium and 348 SNPs were included in the final plots, genotype frequency, call rate and deviation from analysis. Genotyping was successful in 99.5% of Hardy–Weinberg equilibrium. For Hardy–Weinberg equi- individuals. librium, a P value threshold of 5 10–4 was used. Analysis Single-marker association analysis showed the most of variance and the x2-test were used to compare continu- significant association for rs894379 on the centlein, ous and categorical variables, respectively, between sexes. centrosomal protein (CNTLN) gene on chromosome 9

TABLE 1. Demographic characteristics of the discovery cohort overall and by sex Variable All Males Females P value

N (%) 821 464 (57) 357 (43) Age (years) 53.92 (13.72) 52.98 (13.46) 55.14 (13.98) 0.0248 BMI (kg/m2) 26.76 (4.19) 27.38 (3.63) 25.94 (4.70) <0.0001 SBP (mmHg) 142.40 (18.34) 142.93 (18.07) 141.7 (18.68) 0.345 DBP (mmHg) 86.78 (10.55) 87.64 (10.68) 85.65 (10.28) 0.0077 FC-ECG (beats/min) 66.42 (10.74) 65.28 (10.93) 67.90 (10.32) 0.0006 Total cholesterol (mg/dl) 197.03 (35.19) 192.43 (33.84) 202.98 (36.05) <0.0001 HDL cholesterol (mg/dl) 53.46 (13.62) 48.97 (11.6) 59.31 (13.86) <0.0001 LDL cholesterol (mg/dl) 118.72 (31.87) 117.16 (31.47) 120.74 (32.32) 0.1521 Triglycerides (mg/dl) 121.66 (77.96) 129.51 (81.75) 111.39 (71.54) 0.0015 Glucose (mg/dl) 89.99 (23.27) 92.66 (25.99) 86.52 (18.63) 0.0004 Creatinine (mg/dl) 0.86 (0.22) 0.96 (0.21) 0.75 (0.16) <0.0001 LVMI (g/m2) 111.52 (32.71) 119.91 (32.25) 100.58 (30.01) <0.0001

FC-ECG, cardiac frequency ECG; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LVMI, left ventricular mass index. Data are presented as means (SD).

2146 www.jhypertension.com Volume 30 Number 11 November 2012 Chromosome 9p and left ventricular mass

TABLE 2. Demographic characteristics of the validation cohort overall and by sex Variable All Males Females P value

N (%) 1038 600 (58) 438 (42) Age (years) 50.6 (9.98) 49.9 (10.17) 51.5 (9.64) 0.0112 BMI (kg/m2) 27.61 (4.15) 27.82 (3.65) 27.31 (4.75) 0.0508 SBP (mmHg) 153.3 (15.63) 152.39 (15.81) 154.60 (15.32) 0.0258 DBP (mmHg) 98.16 (7.56) 98.31 (7.82) 97.96 (7.19) 0.4585 FC-ECG (beats/min) 72.89 (12.02) 72.16 (12.10) 73.88 (11.84) 0.0235 Total cholesterol (mg/dl) 206.54 (38.96) 203.95 (39.67) 210.13 (37.70) 0.0206 HDL cholesterol (mg/dl) 51.03 (13.13) 46.90 (11.21) 56.86 (13.45) <0.0001 LDL cholesterol (mg/dl) 128.32 (34.92) 127.25 (34.80) 129.84 (35.12) 0.3736 Triglycerides (mg/dl) 134.32 (70.55) 144.73 (74.03) 119.93 (62.73) <0.0001 Glucose (mg/dl) 96.93 (20.19) 97.87 (21.21) 95.58 (18.58) 0.1042 Creatinine (mg/dl) 0.96 (0.20) 1.03 (0.18) 0.84 (0.16) <0.0001 LVMI (g/m2) 115.29 (22.23) 120.34 (23.48) 108 (18.21) <0.0001

FC-ECG, cardiac frequency ECG; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LVMI, left ventricular mass index. Data are presented as means (SD).

(P ¼ 0.0000174; FDR ¼ 0.06) (Table 3). Figure 1 shows (see Table 2). Twenty-four percent of males and 34% of association results for the genotyped SNPs within females were current smokers and, 82% males and females 400 kilobase of rs894379, along with recombination rates. were on antihypertensive treatment. The log10 P values (y-axis) of the SNPs are shown When comparing the overall demographic characteristic according to their chromosomal positions (x-axis). The of the two study populations, we found that individuals top SNP (rs894379) is labeled. The extent of linkage dis- in the discovery cohort were significantly older, had lower equilibrium with rs894379 is reflected by the color intensity BMI, BP, hear rate, total and LDL cholesterol, triglycerides, of each symbol. As it emerges from the graph, there is creatinine and LVMI. They had significantly higher HDL no linkage disequilibrium between rs894379 and all the cholesterol. near genotyped (r2 < 0.2). Genetic recombination rates We genotyped the top SNP (rs894379) and we (cM/Mb), estimated using HapMap Caucasian European performed association analysis assuming an additive samples, are shown with a light blue line. Physical positions model of inheritance and correcting for age, sex and are based on build 36 (NCBI) of the (http:// SBP. SNP rs894379 was no longer significant (b-coefficient www.ncbi.nlm.nih.gov/). Also shown are the relative for LVMI ¼ 1.16; P ¼ 0.2). However, the b-coefficient was positions of genes mapping to the region of association. in the same direction as before, thus suggesting that, Other SNPs, which showed nominal association, were because of the Winner’s curse (i.e. the actual genetic effect rs7873813 (P ¼ 0.000287), rs7866746 (P ¼ 0.000723) and is typically smaller than its estimate), the problem was rs603085 (P ¼ 0.000862), but these did not remain signifi- power. We, therefore, combined the results using inverse cant after multiple testing correction. variance fixed-effect meta-analysis and we obtained a We further investigated the association of the main nominal association (b ¼ 2.4599, P ¼ 0.0026. See Figure 2). hit (rs894379) with LVH and we found a similar trend of association as the one observed with LVMI (OR ¼ 1.41, DISCUSSION SE ¼ 0.17, P ¼ 0.004). The genotype counts for rs894379 according to LVH status are reported in Table 4. In an accurately phenotyped cohort of essential hyper- tensive individuals from northern Italy, we identified SNP Validation cohort rs894379 located in the intronic region of the CNTLN gene In the validation cohort, males accounted for 58%, on chromosome 9p22, whose minor allele is associated they were significantly younger, had a lower SBP, heart with an increased LVM. We took this further and tried rate (FC-ECG) and total and HDL cholesterol. They to validate our result in 1038 hypertensive patients from had significantly higher triglycerides, creatinine and LVMI the Campania Salute Project. Even though the association between rs894379n and LVM had a nonsignificant P value in the validation cohort, we found the same direction for the b-coefficient, thus suggesting that, because of the Winner’s TABLE 3. Linear regression for left ventricular mass index and curse effect, the problem was power. Hence, we combined rs894379 adjusting for age, sex and SBP in discovery the results using inverse variance fixed-effect meta-analysis cohort and were able to obtain a nominal association. LVMI b SEM P value SNP rs894379 is located on the CNTLN gene that rs894379 5.61 1.51 1.74E-04 encodes for a centrosomal protein, named centlein, firstly Age 0.91 0.05 <0.0001 described in 2008 [28]. Centlein is a 721 amino acids Sexa 20.60 1.44 <0.0001 protein and possesses coiled-coil domains. Western blot SBP 0.34 0.04 <0.0001 analysis in animal models indicates that centlein is

LVMI, left ventricular mass index. expressed in all tissues, including heart and also in most aThe reference category is male. of cancer cell lines.

Journal of Hypertension www.jhypertension.com 2147 Menni et al.

Plotted SNPs

10 2 r rs894379 100 0.8 0.6

8 Recombination rate (cM/Mb) 0.4 80 0.2

6 60

4 40

2 20

0 0

CNTLN→ SH3GL2→

17.2 17.4 17.6 17.8 Position on chr9 (Mb) FIGURE 1 Regional association plot for rs894379.

Moreover, centlein is associated with centrosomes in it is one of the closest genes that can, therefore, be involved interphase and mitosis in a microtubules-independent in cell function and survival; however, more studies manner. Evidence exists showing that centlein is located are currently underway to confirm a possible role for this in the mother centriole in G1 phase, suggesting that it may protein in tumor genesis [29]. be involved in mother centriole-related functions, such as A minor finding from this study is that we confirm duplication of centrioles and generation of primary cilia. the lack of association between rs1333049 and LVMI To best of our knowledge, no biomolecular model of as previously reported in white individuals with stable cardiac hypertrophy can contemplate a role of centrosomes coronary artery disease [30]. and, up to now, no GWAS has shown a link between There are many strengths in this study. First, the centrosomes and LVM. discovery cohort is a large sample of accurately phenotyped For this reason, the impact of centlein on cardiac hypertensive patients. This enabled us to account for physiology is unknown, although the protein is expressed several potential confounders (e.g. age, sex, SBP). LVM in cardiac tissue in animal models. was assessed via echocardiographic method, which has a GWASs have the advantage to be hypothesis-free and higher sensibility and specificity than the ECG criteria and this gives the chance to identify new physiopathologic has a stronger prognostic value. Second, we were able pathways; on the contrary, results from GWAS are often to genotype the top SNP in an independent accurately difficult to interpret and to link with the observed phenotyped cohort. Third, we accurately studied the short phenotype. arm of chromosome 9, which has been previously shown to A recent GWAS identified 12 SNPs in 9p22.2 region being have a high biological plausibility in cardiovascular trait. associated with a decreased risk of ovarian cancer, and We selected on that region SNPs that tagged variants in the even if none of them was located in CNTLN intronic region, genes and their flanking 10-kilobase region. There are also some limitations. First, our validation cohort was probably underpowered to replicate rs894379 signal and when we combined the results we reached TABLE 4. Genotype counts for rs894379 according to left nominal association. Second, both the discovery and ventricular hypertrophy status in discovery cohort replication cohorts include hypertensive individuals, LVH so the results may not be applicable to normotensive. Yes No One further limitation is the use of an ultrasound-derived value for LVM. Although echocardiography is known to rs894379 AA 134 235 have much more sensibility and specificity than the ECG AG 148 210 criteria for LVH detection, the accuracy and reproducibility GG 39 47 of M-mode at measuring LVM has several limitations, LVH, left ventricular hypertrophy. such as the not excellent interobserver and intraobserver

2148 www.jhypertension.com Volume 30 Number 11 November 2012 Chromosome 9p and left ventricular mass

Study Beta SE Beta 95%-CI W(fixed)

Discovery 5.61 1.51 5.61 [2.65; 8.57] 29.2% Validation 1.16 0.97 1.16 [–0.74; 3.06] 70.8%

Fixed effect model 2.46 [0.86; 4.06] 100%

02468 Beta FIGURE 2 Forest plots of association with rs894379 and left ventricular mass index.

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Heritability of left in favor of a greater comparability of the two groups. ventricular mass: the Framingham Heart Study. Hypertension 1997; It is also possible that our result is statistical noise, 30:1025–1028. possibly due to indirect association as a result of underlying 8. Sharma P, Middelberg RP, Andrew T, Johnson MR, Christley H, Brown coronary diseases, but every effort has been made to MJ. Heritability of left ventricular mass in a large cohort of twins. minimize measurement noise in both our populations J Hypertens 2006; 24:321–324. 9. Doolan G, Nguyen L, Chung J, Ingles J, Semsarian C. Progression of left and we believe that our result is novel and interesting. ventricular hypertrophy and the angiotensin-converting enzyme gene Further studies are needed to better dissect the mechanistic polymorphism in hypertrophic cardiomyopathy. Int J Cardiol 2004; basis of this association. 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