Original articleGenetic testing of candidate genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia
Introduction
Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/ARVD, MIM10790), is a rare cardiac disorder characterized by progressive fibro-fatty replacement of myocardial tissue in the right ventricle (RV). Left ventricle (LV) might be affected also in certain patients [1]. The National Center for Biotechnology Information (NCBI) describes that the disease affects around 5–45 individuals per 10,000. Patients with ARVC/D may present from palpitations to syncope and often also sudden cardiac death (SCD) [2], [3].
ARVC/D is an inherited disease in up to 50% of cases, most of them with an autosomal dominant pattern of transmission even though recessive inheritance has also been described [4]. The diagnosis is based on fulfillment of major and minor criteria suggested by a Task Force Criteria on ARVC/D [5], recently revised [6]. Diagnosis of ARVC/D carries serious implications both for the affected patient and also for family members, especially because SCD can be the first manifestation of the disorder [7].
ARVC/D has been linked to several genes encoding proteins of the intercalar disk or associated proteins. The majority of mutations identified are localized on 5 desmosomal genes, plakophilin-2 (PKP2), desmoplakin (DSP), plakoglobin (JUP), desmoglein-2 (DSG2) and desmocollin-2 (DSC2), being PKP2 the most frequently affected gene [2]. Other 6 nondesmosomal genes have been also suggested as disease-causing: transforming growth factor beta-3 (TGFB3), the cardiac ryanodine receptor (RYR2), the gene transmembrane protein 43 (TMEM43), the tumor protein p63 (TP63) and desmin (DES). Recently, Taylor et al described the association among the disease and titin gene (TTN) [8], the largest protein in mammals. However, debate continues on whether the morphological alterations found in the few ARVC/D families with a TP63 or RYR2 mutation [9], [10], [11], [12] are truly associated with ARVC/D or with other genetic conditions such as catecholaminergic ventricular tachycardia.
All things considered, so far, only about 30%–50% of patients have causal mutations in any of known genes underscoring the genetic heterogeneity of the disease and reflecting that new additional genes will probably be linked with the disease in upcoming years [13].
ARVC/D is currently considered to be a disease of myocytes adhesion caused by defects at intercellular junction level. Myocyte-to-myocyte adhesion is maintained at the intercellular junctions by the desmosomes together with adherens and gap junctions [14]. The desmosomes have a complex structure that includes adhesion molecules of the cadherin family (desmoglein and desmocollin) and proteins of the plakin and catenin families (desmoplakin, plakophilin, and plakoglobin) which link intermediate filaments of the cytoskeleton to the desmosomal cadherinas. The main function of the adherens junctions is anchoring the actin microfilaments [15]. Components of this complex are cadherins, catenins and other catenin-related proteins. One of the most important cadherin is N-cadherin or also named cadherin 2 type 1, encoded by CDHN gene and considered a vital protein in gap junction assembly [16]; it is also critical for maintaining connexin43 (Cx43) function [17], [18]. Adhesive activity is regulated by its cytoplasmatic binding partners, the catenins which mediate linkage of the cadherin to the actin cytoskeleton [19]. Among catenin-related proteins, one key protein is metavinculin, encoded by MVCL gene and expressed in cardiac and smooth muscle [20]. Metavinculin is located at main sites of contractile force transmission in myocytes, anchoring actin filaments to intercalated discs in the human heart [21]. Gap junctions are small intercellular pores that permit the passage of small regulatory molecules and ions through a channel generated by a family of proteins called connexins [22]. In the heart, the primary connexin expressed in human ventricular myocardium is Cx43, protein encoded by GJA1 gene [23], [24], [25].
Genetic studies in cardiomyopathies have shown how mutations in one group of genes can induce different phenotypes in cardiomyopathies [26], [27], [28], [29], [30]. This was first shown in genes encoding for sarcomeric proteins that can be related with dilated, hypertrophic or restrictive cardiomyopathy [31]. Recently, mutations in genes classically linked to ARVC/D have been related to DCM [32], and mutations in genes related to DCM (as titin), have been shown that could cause ARVC/D [8].
To further genetically characterize ARVC/D, we hypothesized that disease-causing mutations could be found in some of these genes not previously studied as possible ARVC/D-associated: ACTC1, CDHN, CTNNA1, GJA1, MVCL, MYL2, and MYL3. All these candidate genes encode key structural proteins for myocyte adhesion and are expressed at the intercalated disk in human heart.
Section snippets
Study sample
Patients were evaluated at dedicated arrhythmia and cardiomyopathy units at two university hospitals from Spain (Hospital Clinic of Barcelona, Barcelona, and Hospital Puerta de Hierro, Madrid). The study was approved by the local ethics committee, followed the Helsinki II declaration and all subjects gave written informed consent before inclusion in the study.
All patients were Caucasian and native of Spain. They were identified after the death of a family member with the disease or after
Patients
Study cohort comprised 14 ARVC/D patients (mean age 30.35 years, range 11–54, 13 males). None of them exhibited mutations in plakophilin-2 (PKP2), desmoplakin (DSP), plakoglobin (JUP), desmoglein-2 (DSG2) and desmocollin-2 (DSC2), transforming growth factor beta-3 (TGFB3), the gene transmembrane protein 43 (TMEM43), the tumor protein p63 (TP63) and desmin (DES). Clinical characteristics of participants are summarized in Tables 1and 2.
Genetic screening
With the analysis of these additional genes, we identified a
Discussion
In the present report we performed the genetic analysis of 7 ARVC/D candidate genes (ACTC1, CDHN, CTNNA1, GJA1, MVCL, MYL2, and MYL3) in a Spanish cohort of 14 ARVC/D patients. Our affected group showed a mean age of 28 years with male prevalence. Both parameters are in concordance with previously published studies of ARVC/D cohorts. Clinical data showed similar affectation in all our patients. Additionally, each one of our patients showed similar number of genetic variations, and most of these
Conclusions
Genetic screening of seven candidate genes involved in structural formation and function of the myocyte intercalar disk did not show any ARVC/D-causing mutations in a Spanish cohort. Further studies of these genes in larger Spanish cohorts and patients of other countries should be necessary to definitively discard all these genes as disease-related.
Acknowledgments
This work was supported by Consejo Superior de Deportes/Ministerio de la Presidencia (003/UPB10/11), CNIC-Translational 2008 (CNIC-03-2008), the Spanish Ministry of Health [PI11/00699 and Red Cooperativa de Insuficiencia Cardiaca (REDINSCOR) RD06/03/0018], Eugenio Rodriguez Pascual Foundation, and Obra Social “la Caixa”. OC has a Sara Borrell post-doctoral grant of Instituto de Salud Carlos III. MA has a pre-doctoral grant of Universitat de Girona.
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