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BMC Medical Genetics BioMed Central Research article Open Access Arrhythmogenic right ventricular cardiomyopathy type 6 (ARVC6): support for the locus assignment, narrowing of the critical region and mutation screening of three candidate genes Luzuko O Matolweni†1,2, Soraya Bardien†2,5, George Rebello2, Ekow Oppon2, Miroslav Munclinger3,6, Rajkumar Ramesar2, Hugh Watkins4 and Bongani M Mayosi*1 Address: 1The Cardiac Clinic, Department of Medicine, Groote Schuur Hospital and University of Cape Town, Cape Town, South Africa, 2MRC/ UCT Research Unit for Human Genetics, Division of Human Genetics, Department of Clinical Laboratory Sciences, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa, 3Sub-department of Cardiology, Department of Medicine, University of Kwa-Zulu Natal, Durban, South Africa, 4Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK, 5Centre for Human Genetics Research and Education, Faculty of Health Sciences, University of Stellenbosch, Tygerberg, Cape Town, South Africa and 6Department of Medicine and Cardiology, Mafraq Hospital, Abu Dhabi, United Arab Emirates Email: Luzuko O Matolweni - [email protected]; Soraya Bardien - [email protected]; George Rebello - [email protected]; Ekow Oppon - [email protected]; Miroslav Munclinger - [email protected]; Rajkumar Ramesar - [email protected]; Hugh Watkins - [email protected]; Bongani M Mayosi* - [email protected] * Corresponding author †Equal contributors Published: 28 March 2006 Received: 24 September 2005 Accepted: 28 March 2006 BMC Medical Genetics2006, 7:29 doi:10.1186/1471-2350-7-29 This article is available from: http://www.biomedcentral.com/1471-2350/7/29 © 2006Matolweni et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heritable disorder characterized by progressive degeneration of right ventricular myocardium, arrhythmias and an increased risk of sudden death at a young age. By linkage analysis, ARVC type 6 was previously mapped to a 10.6 cM region on chromosome 10p12-p14 in a large North American kindred. To date, the genetic defect that causes ARVC6 has not been identified. Methods: We identified a South African family of 13 members with ARVC segregating as an autosomal dominant disorder. The diagnosis of ARVC was based on international diagnostic criteria. All available family members were genotyped with microsatellite markers at six known ARVC loci, and positional candidate gene screening was performed. Results: Genetic linkage and haplotype analysis provided lod scores that are highly suggestive of linkage to the ARVC6 locus on chromosome 10p12-p14, and the narrowing of the critical region to ~2.9 Mb. Two positional candidate genes (ITG8 and FRMD4A) were screened in which defects could possibly disrupt cell- cell adhesion. A non-positional candidate gene with apoptosis inducing properties, LAMR1P6 (laminin receptor 1 pseudogene 6) was also screened. Direct sequencing of DNA from affected individuals failed to detect disease-causing mutations in the exonic sequences of the three genes investigated. Conclusion: The narrowing of the ARVC6 critical region may facilitate progress towards the identification of the gene that is involved in ARVC. Identification of the causative genes for ARVC will contribute to the understanding of the pathogenesis and management of this poorly understood condition. Page 1 of 10 (page number not for citation purposes) BMC Medical Genetics 2006, 7:29 http://www.biomedcentral.com/1471-2350/7/29 Background It is of interest that the discovery of the plakophilin-2 gene Arrhythmogenic right ventricular cardiomyopathy on chromosome 12p11 as a cause of ARVC has resulted in (ARVC, OMIM: 604401) is characterized by progressive the re-assignment of the disease locus in one small Euro- degeneration and fibro-fatty replacement of right ven- pean family. Originally, this family was thought to be tricular myocardium, causing ventricular tachyarrhyth- linked to chromosome 2q32.1-q33.3 using a cumulative mias and resulting in sudden death, often during exercise. lod score together with two other small families [15]. In Italy, where the condition has been studied extensively, Individually, these families did not produce statistically ARVC accounts for 20% of all sudden deaths in individu- significant lod scores. Subsequently, a frame shift muta- als under 35 years and 22% of sudden deaths in athletes tion was identified in the plakophilin-2 gene in the Euro- [1]. Familial occurrence is reported in 30–50% of individ- pean family, which shows that the original linkage uals with ARVC, suggesting that genetic factors may play findings to chromosome 2 in this family were erroneous an aetiological role in a significant proportion of affected [12]. people [2]. At least 11 loci have potentially been identi- fied so far: ARVC1 (14q23-q24), ARVC2 (1q42-q43), Therefore, independent corroboration of genetic linkage ARVC3 (14q12-q22), ARVC4 (2q32.1-q32.3), ARVC5 findings, particularly in rare conditions such as ARVC, is (3p23), ARVC6 (10p12-p14), chromosome 10q22.3, essential to facilitate progress towards the identification of ARVD8 (6p24), ARVC9 (12p11), Naxos disease (17q21), causal genetic mutations. We report on the linkage analy- and ARVC with anterior polar cataract [3,4]. To date five sis in a South African family with ARVC, which provided candidate genes have been identified, i.e., ryanodine support for previous chromosomal localization of receptor (RyR2 on 1q42), plakoglobin (JUP on 17q21), ARVC6, the narrowing of the critical region, and exclusion desmoplakin (DSM on 6p24), plakophilin-2 (PKP2 on of disease-causing mutations in the coding regions of two 12p11), and transforming growth factor β3 gene (TGFβ3 positional and one non-positional candidate genes. on 14q24.3) [4,5]. Recently, a serendipitous mouse model of ARVC was discovered in which a functional lam- Methods inin 1 receptor (lamr1) retroposon interacted with hetero- Family and clinical evaluation chromatin protein 1 (HP1) to cause degeneration of A family with ARVC was investigated at Wentworth Hos- cardiomyocytes in the right ventricle of the heart [6]. pital, Durban and Groote Schuur Hospital, Cape Town, South Africa. Family members gave informed consent to Several lines of evidence suggest that impairment of cell- participate in the study, which was approved by the local to-cell adhesion leading to accelerated apoptosis of myo- ethics committee. The protocol included: 12-lead electro- cardial cells may be the underlying pathogenic mecha- cardiogram (ECG), signal averaged ECG, 24-hour ambu- nism in ARVC. According to this hypothesis, disruption of latory ECG, and 2D echocardiogram. Additional cellular junctional cohesion leads to cell death and repair procedures (i.e., cardiac magnetic resonance imaging, by fibrofatty replacement [7]. Initial support for this right ventricular angiogram, and endomyocardial biopsy) hypothesis was provided by the observation of abnormal- were performed when clinically indicated. The diagnosis ities of myocyte intercalated discs on electron microscopy in the index case was based on criteria proposed by McK- [8,9]. Subsequently, the involvement of desmosomal pro- enna et al [16]: i.e., affection status was established when teins (i.e., plakoglobin, desmoplakin and plakophilin-2) two major criteria, or one major criterion plus two minor in different ARVC clinical phenotypes has been reported criteria, or four minor criteria were fulfilled. The diagnosis [7,10-12]. ARVC2, which has features that are more typi- of ARVC in the other family members was based on the cal of a catecholamine-dependent ventricular tachycardia criteria for familial ARVC proposed by Hamid et al [2]; syndrome than ARVC, is associated with a mutation in i.e., the presence of one or more minor criteria in the con- ryanodine receptor-2 [13]. Mutations in the regulatory text of familial ARVC indicate an affected status. regions of the TGFβ3 have been found in association with one of the three families linked to the ARVC2 locus, and Molecular genetic studies in one isolated case of ARVC [5]. The role of TGFβ3 muta- Peripheral blood samples were collected from family tions remain to be confirmed as a cause of ARVC because members in EDTA-coated tubes and genomic DNA was of the absence of mutations in the other two families that prepared from nucleated blood cells, according to the salt- have been linked to this locus [4]. The difficulty in identi- ing out method. PCR-based methods were used for geno- fying the genetic mutations underlying ARVC2, which was typing and sequencing. All PCR amplifications were initially mapped more than 10 years ago [14], underscore carried out on a GeneAmp 9700 thermocycler (Applied the challenge of cloning causal genes in rare disorders Biosystems). Each 50 µl reaction contained 200 ng of with small families, such as ARVC. genomic DNA, 200 µM dNTPs, 50 mM KCL, 10 mM Tris (pH 8.4), 10 pM of each primer and 1 unit of BIOTAQ DNA polymerase (BIOLINE). The concentration of mag- Page 2 of 10 (page number not for citation purposes) BMC Medical Genetics 2006, 7:29 http://www.biomedcentral.com/1471-2350/7/29