European Journal of Human Genetics (2003) 11, 659–664 & 2003 Nature Publishing Group All rights reserved 1018-4813/03 $25.00 www.nature.com/ejhg ARTICLE Polymorphisms of genes of the cardiac calcineurin pathway and cardiac hypertrophy Odette Poirier1, Viviane Nicaud1, Theresa McDonagh2, Henry J Dargie2, Michel Desnos3, Richard Dorent4,Ge´rard Roize`s5, Ketty Schwartz6, Laurence Tiret1, Michel Komajda7 and Franc¸ois Cambien*,1 1INSERM U525, Epidemiologic and Molecular Genetics of Cardiovascular Diseases, Paris, France; 2Department of Cardiology and MRC Clinical Research Initiative in Heart Failure, Western Infirmary and University of Glasgow, UK; 3Hoˆpital Boucicaut, Paris, France; 4Service de Chirurgie Cardiaque, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Paris, France; 5CNRS UPR 1142, Montpellier, France; 6INSERM U523, Paris, France; 7Service de Cardiologie, Groupe Hospitalier Pitie´-Salpeˆtrie`re, Paris, France The study investigated the role of genetic polymorphisms in four genes of the calcineurin pathway on cardiac hypertrophy and dilated cardiomyopathy. The cardiac calcineurin pathway has been suggested to play a role in the development of cardiac hypertrophy in response to a number of physiological and pathological stimuli. Calcineurin, a heterodimeric protein composed of a catalytic and a regulatory subunit, activates the nuclear factor NFATC4 which after translocation to the nucleus associates with the transcription factor GATA4 to activate several cardiac genes involved in hypertrophic response. We have screened the genes encoding the four major components of the heart calcineurin pathway in 95 individuals and identified 27 polymorphisms. These polymorphisms were investigated in 400 selected subjects obtained from a population-based study (LOVE) in relation to echocardiographic parameters. A Gly/Ala substitution at position 160 of the NFATC4 protein (G160A) was associated with left ventricular mass and wall thickness (P ¼ 0.02 and 0.006, respectively, GA+AA vs GG), the minor allele (Ala) being associated with lower mean values of these parameters. The other polymorphisms identified by the gene screen were not associated with cardiac phenotypes. For the G160A polymorphism in NFATC4, genotype frequencies were compared between patients with dilated cardiomyopathy and controls obtained from the CARDIGENE Study. Allele A carriers were less frequent in the patient than in the control group (P ¼ 0.04). Although the strength of the associations was rather weak, these observations raise the hypothesis that the G160A polymorphism of the NFATC4 gene plays a role in the development of human cardiac hypertrophy. European Journal of Human Genetics (2003) 11, 659–664. doi:10.1038/sj.ejhg.5201023 Keywords: calcineurin; cardiac hypertrophy; genetic polymorphisms; association study Introduction including hypertension, myocardial infarction and endo- Hypertrophic growth is an adaptative response of the heart crine disorders. to a variety of physiological and pathological stimuli, The hypertrophic response of cardiac myocytes to hemodynamic overload, mechanical stress or humoral 1 *Correspondence: Dr F Cambien, INSERM U525, Epidemiologic and factors is mediated by different signaling pathways. Molecular Genetics of Cardiovascular Diseases, Faculte´ de Me´decine Experimental evidence suggests that calcium handling Pitie´-Salpeˆtrie`re, 91 Boulevard de l’Hoˆpital, 75634 Paris cedex 13, France. may play an important role in cardiac hypertrophy.2 In Tel: +33 1 40 77 96 39; Fax: +33 1 40 77 97 28; response to growth stimuli, cardiomyocytes increase their E-mail: [email protected] 2+ Received 5 December 2002; revised 17 March 2003; accepted 1 April 2003 cytosolic Ca level, which in turn activates calcineurin, a Genes of calcineurin pathway and cardiac hypertrophy O Poirier et al 660 Ca2+/calmodulin-dependent protein phosphatase. Calci- on left ventricule telediastolic dimension. Ejection fraction neurin is a heterodimeric protein composed of a catalytic (EF) was calculated by the biplane disc summation method subunit, calcineurin A, also known as protein phosphatase (Simpson’s rule). DNA was not available for 39% of the 3, catalytic subunit and a regulatory subunit, calcineurin B, subjects. For the LOVE Study, 400 subjects (180 men, 200 also known as protein phosphatase 3, regulatory women, mean age: 45713 years, not taking cardiovascular subunit. Whereas calcineurin B is encoded by a single gene drugs and whose body mass index (BMI) was o28 kg/m2), (PPP3R1; OMIM #601302), calcineurin A subunits were selected in order to study the variation of LVM, WT can be coded by three different genes, respectively, PPP3CA and EF according to polymorphisms of candidate genes. (isoform alpha; OMIM #114105), PPP3CB (isoform beta; OMIM #114106) and PPP3CC (isoform gamma; The CARDIGENE Study 3 4 OMIM #114107). Molkentin et al reported that calcineur- This study has been already described.7 Briefly, 433 patients in plays a central role in the development of cardiac with dilated cardiomyopathy (DCM), aged 18–65 years, hypertrophy through activation by dephosphorylation of were recruited between 1994 and 1996 from 10 hospitals in the nucleic factor of activated T cells (NFATC4). After France. The diagnosis was based on an ejection fraction activation, NFATC4 translocates to the nucleus where it p40 and left ventricular dilation (end-diastolic volume associates with the transcription factor GATA-binding 4140 ml/m2 on ventriculography or end-diastolic dia- protein 4 (GATA4), to induce several cardiac genes meter 434 mm/m2 on echocardiography) confirmed over involved in hypertrophic response. Given the potential a 6-month period. All subjects had to be born in France and importance of the calcineurin pathway in cardiac hyper- their parents had to be born in France or neighboring trophy in humans, we designed this study to investigate countries. Controls (n ¼ 433) free of hypertension were the genetic variability of its major components PPP3CA, recruited in Centres for Preventive Medicine all over PPP3CB, NFATC4, GATA4 and its possible association with metropolitan France and were matched for age, sex and echocardiographic indexes of cardiac hypertrophy in a region of birth. Their parents had to be born in France and subsample of subjects participating in the Glasgow Heart their four grandparents had to be born in Europe. Each Scan Study (LOVE Study). In addition, the NFATC4/G160A subject gave written informed consent. polymorphism was further investigated in the CARDIGENE Study to assess its possible association with dilated Identification of polymorphisms myocardiopathy. Genomic DNA was prepared from leukocytes. Polymorph- isms discovery was performed in a sample of 95 indivi- duals. In addition, in order to increase the power of Methods detecting relatively rare variants that are associated with The LOVE Study cardiac hypertrophy, these individuals were selected from The LOVE Study is a substudy of the Glasgow Heart Scan among individuals in the Glasgow Heart Scan Study whose Study. This study has been previously described:5 1600 age-adjusted LVM (estimated by regression analysis) was in subjects (51% women) aged 25–74 years (mean age: 51714 the upper decile of the sex-specific distribution. years) from North Glasgow (UK), who had participated in DNA was amplified by polymerase chain reaction (PCR). the Third Glasgow MONICA Risk Factors Survey in 1992,6 All the primers used were designed to generate overlapping were randomly selected. Participants underwent a standard fragments of o300 bp spanning each exon of the four echocardiography. Left ventricule mass (LVM) was indexed genes as well as their promoter and intronic sequences on body surface area and wall thickness (WT) was indexed flanking exons (Table 1). The detection of sequence Table 1 Description of the explored genes Length explored (bp) OMIM Flanking OMIM gene accession Chromosome Number of intronic Analyzed-SSCP name number location exons 50 region 50UTR Exons region 30UTR 30 region fragments PPP3CA 114105 4p21–q24 14 324 148 1566 1735 543 0 16 PPP3CB 114106 10q21–q22 14 1091 117 1521 1919 214 0 17 GATA4 600576 8p23.1–p22 6 0 240 1324 949 199 0 16 NFATC4 602699 14q11–2 10 544 141 2703 1213 53 94 18 OMIM, online Mendelian Inheritance in Man (site available at: http://www3.ncbi.nlm.nih.gov/Omim/); PPP3CA, protein phosphatase 3, catalytic subunit, alpha isoform; PPP3CB, protein phosphatase 3, catalytic subunit, beta isoform; GATA4, GATA-binding protein 4; NFATC4, nuclear factor of activated T cells, cytoplasmic, 4; UTR, untranslated region of the exon. European Journal of Human Genetics Genes of calcineurin pathway and cardiac hypertrophy O Poirier et al 661 variants was performed using the single-strand conforma- Table 1. Among the observed variants, six were located in tion polymorphism (SSCP) analysis followed by direct the PPP3CA gene, two in the PPP3CB gene, twelve in the sequencing of PCR fragments from patients with variable GATA4 gene, and seven in the NFATC4 gene (Table 2). All electrophoretic patterns. polymorphisms except three (for technical reasons) were genotyped in the LOVE Study. Complete description of Genotyping the polymorphisms, linkage disequilibrium coefficients For the À83 repeat in the PPP3CA gene, genotypes were and haplotype frequencies for each gene can be found on deduced after an 8% polyacrylamide denaturing gel our web site (http://genecanvas.idf.inserm.fr). All observed electrophoresis (PAGE) of a radiolabeled PCR fragment. genotype distributions were