Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Am Coll Cardiol. Author manuscript; available in PMC 2011 February 9.
Published in final edited form as:
PMCID: PMC2852685

Compound and Digenic Heterozygosity Contributes to Arrhythmogenic Right Ventricular Cardiomyopathy

Tianhong Xu, PhD,*,1 Zhao Yang, MD, PhD,*,2,3 Matteo Vatta, PhD,3 Alessandra Rampazzo, MD, PhD,4 Giorgia Beffagna, PhD,3,4 Kalliopi Pillichou, PhD,4 Steven E. Scherer, PhD,1 Jeffrey Saffitz, MD, PhD,5 Joshua Kravitz,3 Wojciech Zareba, MD,6 Gian Antonio Danieli, PhD,4 Alessandra Lorenzon, PhD,4 Andrea Nava, MD,8 Barbara Bauce, MD, PhD,8 Gaetano Thiene, MD,9 Cristina Basso, MD, PhD,9 Hugh Calkins, MD,7 Kathy Gear, RN,10 Frank Marcus, MD,10 and Jeffrey A. Towbin, MD11, for the Multidisciplinary Study of Right Ventricular Dysplasia Investigators



To define the genetic basis of arrhythmogenic right ventricular cardiomyopathy.


Arrhythmogenic right ventricular cardiomyopathy (ARVC), characterized by right ventricular fibrofatty replacement and arrhythmias, causes sudden death. Autosomal dominant Inheritance, reduced penetrance, and 7 desmosome-encoding causative genes are known. The basis of low penetrance is poorly understood.


ARVC probands and family members were enrolled, blood obtained, lymphoblastoid cell lines immortalized, DNA extracted, PCR amplification of desmosome-encoding genes performed, PCR products sequenced and diseased tissue samples studied for intercellular junction protein distribution using confocal immunofluorescence microscopy and antibodies against key proteins.


We identified 21 variants in plakophilin-2 (PKP2) in 38 of 198 probands (19%), including missense, nonsense, splice site, and deletion/insertion mutations. Pedigrees showed wide intra-familial variability (severe early-onset disease to asymptomatic individuals). In 9/38 probands, PKP2 variants were identified that were encoded in trans (compound heterozygosity). The 38 probands hosting PKP2 variants were screened for other desmosomal genes mutations; second variants (digenic heterozygosity) were identified in 16/38 subjects with PKP2 variants (42%) including desmoplakin (DSP, n=6), desmoglein-2 (DSG2, n=5), plakophilin-4 (PKP4, n=1), and desmocollin-2 (DSC2, n=1). Heterozygous mutations in non-PKP 2desmosomal genes occurred in 14/198 subjects (7%), including DSP (n=4), DSG2 (n=5), DSC2 (n=3), and junctional plakoglobin (JUP, n=2). All variants occurred in conserved regions; none were identified in 700 ethnic-matched controls.

Immunohistochemical analysis demonstrated abnormalities of protein architecture.


These data suggest that the genetic basis of ARVC includes reduced penetrance with compound and digenic heterozygosity. Disturbed junctional cytoarchitecture in subjects with desmosomal mutations confirms that ARVC is a disease of the desmosome and cell junction.

Keywords: Arrhythmias, Cardiomyopathies, Desmosomes, Intercalated Disks, Genetic Mutations


Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC) has been defined as a primary right ventricular cardiomyopathy initially described by Marcus, Fontaine and colleagues in the early 1980's.1 It is characterized by fibrofatty infiltration of the right ventricular (RV) myocardium and clinically presents with ventricular arrhythmias, heart failure, syncope, and sudden death.1,2 The left ventricle (LV) may also be affected.3 In familial cases of ARVC, autosomal dominant inheritance with reduced penetrance has been reported and is believed to account for approximately 30% of cases.4 In the remaining sporadic cases, the etiology may be an acquired cause such as myocarditis5,6 or an unidentified inherited disorder. To date, multiple genetic loci and 7 genes including desmoplakin (DSP),7 plakophilin-2 (PKP2),8 desmoglein-2 (DSG2),9 desmocollin-2 (DSC2), 10 transforming growth factor β3 (TGF β3),11 ryanodine receptor 2 (RYR2)12, and Transmenbrane Protein-43 (TMEM43) 13 have been identified in ARVC patients. In addition, two complex cardiocutaneous disorders with autosomal recessive inheritance in which cardiomyopathy is associated with woolly hair and palmoplantar keratoderma have been reported. These include Naxos disease with ARVC14 and Carvajal syndrome associated with an LV cardiomyopathy.15 The genes identified for these disorders include homozygous mutations in junctional plakoglobin (JUP) in Naxos disease,16 as well as homozygous mutations in DSP in Carvajal syndrome.17 The most common gene variants identified in ARVC is PKP2, initially reported by Gerull and colleagues to be mutated in ~25% of patients with autosomal dominant inherited disease.8 In the analysis of PKP2, an essential armadillo-repeat protein of the cardiac desmosome, they identified heterozygous mutations in 32 of 120 unrelated individuals with ARVC. 8 Other investigations have confirmed these findings.18-21 The majority of genes identified to date causing ARVC encode desmosomal proteins. Within cardiomyocytes, two types of cell adhesion junctions are responsible for intercellular adhesion: the desmosomes and fascia adherens junctions.22 These cell adhesion junctions are both located at the cardiomyocyte intercalated disk and both contain intracellular proteins that link the cytoplasmic domains of cadherins to components of the cytoskeleton. In cardiomyocytes, a variety of proteins interact to form functional cell-cell junctions. DSGs and DSCs are connected to the desmin cytoskeleton by DSP, JUP (γ-catenin), and PKP2. These latter proteins, JUP and PKP2, are members of the armadillo family of nuclear and junctional proteins.23, 24 PKP-2 interacts with DSP, DSG and intermediate filament proteins at sites within its N-terminus. DSP and PKP2 are located only in desmosomes, whereas JUP participates as a linker in both desmosomes and adherens junctions. The adherens junctions are located at the ends of sarcomeres and are linked to sarcomeric actin through intracellular linker proteins, most notably members of the catenin family including JUP, β-catenin, αcatenin and p120 catenin.

In this study, we analyzed probands and family members for ARVC using a standardized clinical protocol either developed as part of the North American ARVD Registry25 or using the standard Task Force criteria (Table 1).26 All individuals were screened for mutations in all of genes encoding proteins involved in desmosomal function, even if a variant were already identified in any of these desmosome-encoding genes. We report identification of multiple mutations in these genes, including autosomal dominant heterozygous mutations in 26% of subjects (52/198), including 38 in PKP2 and 14 in other desmosome-encoding genes.

Table 1
Task Force Diagnostic Criteria for ARVC.26

Additionally, compound heterozygous mutations and digenic mutations were identified in 42% of the subjects (16/38) in whom PKP2 mutations were identified. In addition, we demonstrate that many PKP2 mutations have low penetrance and in many cases may not be the primary cause of the disease, contradicting the previously reported contention that PKP2 is the major ARVC causing gene, accounting for the cause of disease in 25% of ARVC patients.


Patient Evaluation

After informed consent, probands were evaluated by noninvasive and invasive studies including physical examination and history/family history, chest radiography, 12-lead electrocardiogram (ECG), echocardiography and cardiac magnetic resonance imaging (cMRI).

In most cases, the clinical evaluation followed the protocol of the NIH-funded North American ARVD Registry,25 which included invasive studies including cardiac catheterization, ventricular angiography, and endomyocardial biopsy. In these subjects, all studies (noninvasive and invasive testing) were analyzed by Core Laboratories. Family members were evaluated using the noninvasive studies only (ECG, cMRI, echocardiogram, chest X-ray, and physical examination with history/family history). In the subjects in whom genetic studies were performed but who declined enrollment in the Registry, or international subjects not eligible to enroll in the Registry, the Task Force diagnostic criteria (Table 1) were used. 26 Diagnostic criteria previously described by McKenna et al. were used to determine affectation status.26

After informed consent, blood for DNA extraction and lymphoblastoid cell line immortalization was obtained, as approved by the Baylor College of Medicine Institutional Review Board (IRB).

DNA Sequencing Analysis

Genomic DNA samples of the 143 U.S. and 55 Italian ARVC index cases (n=198) were obtained from blood samples and immortalized lymphoblastoid cell lines as previously described27 and amplified by PCR using primers designed to amplify the coding exons of desmosome-encoding genes PKP2, DSP, JUP, DSC2, DSG2, plakophilin-4 (PKP4), and the intermediate filament-encoding gene desmin (DES). Other non-desmosomal genes were excluded from this analysis. PCR products from the U.S. cohort were sequenced using Big Dye Terminator version 3.1 chemistry (Applied Biosystems; ABI) and analyzed using an ABI 3730 DNA sequencer (Applied Biosystems; ABI). In addition, the 55 Italian ARVC index cases were screened for mutations by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. DHPLC analysis was performed with the use of WAVE Nucleic Acid Fragment Analysis System 3500HT with DNASep HT cartridge (Transgenomic Ltd NE). Temperatures for sample analysis were selected with the use of WAVEMAKER software. In all 198 subjects, therefore, we analyzed the entire coding sequence and the surrounding intronic sequences of DES and of the desmosomal genes including PKP2, DSP, DSC2, DSG2, JUP, DSC2, DSG2 and PKP4.

Cloning and Sequencing of PKP2 cDNA

Total RNA was isolated from lymphoblastoid cell lines using an RNeasy Mini Kit (Qiagen, Stanford, CA) and subjected to random hexamer-primed cDNA synthesis using Superscript II (Invitrogen). PKP2 cDNA was amplified by PCR with oligonucleotides specific for the cDNA sequence of PKP2 (5′-CCAGCTGAGTACGGCTACATC-3′; 5′-TCAGTCTTTAAGGGAGTGGT-3′), cloned into TA-vectors (Topo TA Cloning Kit; Invitrogen, Carlsbad, CA) and then introduced into TOP10 cells using a One Shot Chemical Transformation kit (Invitrogen). For each patient sample, plasmid DNA was isolated and the insert was sequenced using the same oligonucleotides.


When available, formalin-fixed or snap-frozen cardiac tissues from affected patients were studied for the distribution of intercellular junction proteins using confocal immunofluorescence microscopy, as previously described28,29 Antigens were exposed in paraffin-embedded sections using microwave antigen-recovery techniques28,29 and then stained with commercial rabbit polyclonal antibodies against connexin 43 (Cx43 Invitrogen (Zymed), the C-terminal domain of DSP (Serotec, Raleigh, NC), a conserved sequence in the N-cadherins (Sigma-Aldrich, St. Louis MO), and DES (ScyTek Laboratories, Logan, UT), as well as mouse monoclonal antibodies against JUP (Sigma-Aldrich), DSC 2/3 (Invitrogen (Zymed)), PKP-2 (Biodesign International, Saco, ME), α-catenin (Invitrogen (Zymed)), β-catenin (Invitrogen (Zymed)), and the C-terminus of JUP (Research Diagnostics Inc., Concord, MA). The sections were then stained with appropriate secondary antibodies conjugated with CY3 and visualized by confocal microscopy as described previously.28,29 Patient samples were stained simultaneously with myocardial sections prepared from three age-matched controls with no clinical or autopsy evidence of heart disease. The amount of immunoreactive signal for each protein at the intercalated disks was evaluated in a blinded fashion and scored as being strongly present, weakly present or absent.



After informed consent, 198 probands (143 North American, 55 Italian) meeting clinical criteria for the diagnosis of ARVC using the Task Force criteria (Table 1) and/or ARVD Registry criteria were enrolled in the genetic analysis study. All subjects were screened for mutations in all desmosome-encoding genes, with complete sequencing of the entire gene performed in all cases. 21 variants in PKP2 were identified in 38 of the 198 probands (19%), a frequency similar to previous reports.8, 18-21 The variants identified included 8 missense, 4 nonsense, 2 splice site mutations, and 8 deletion/insertion mutations; the deletion/insertion variants each predict a protein frame shift (Table 2). Several variants were identified in multiple probands, including nonsense and frame-shift variants (Table 2). All of the missense substitutions resulted in changes at residues that are conserved between species (Supplemental Figure 1). Among the variants identified, the splice site substitution detected in intron 10, and the 2509delA in exon 13 have been previously reported.8 The remaining variants are novel. Despite the fact that several of these variants were identified in multiple individuals, analysis of 700 ethnically matched control individuals (1,400 chromosomes) identified only one of these variants (A372P) in the control population (1 in 700).

Table 2
PKP2 gene variants identified in patients with ARVC

Analysis of the family pedigrees shows that there is significantly reduced penetrance of these PKP2 variants, similar to that reported by Gerull et al. (Figure 1A and B).8 However, in 9 of the probands with PKP2 variants, we identified 2 distinct PKP2 variants (Table 3, patients 5, 7, 8, 11, 12, 14, 15, 16 and 17), consistent with compound heterozygosity. One of these probands (patient 5, Table 3; Figure 1C) was a member of a family with a history of ARVC in his generation (Figure 1C). DNA was available from the living affected subject (patient 6, Table 3; Figure 1C) and unaffected siblings of the proband, as well as the phenotypically normal parents, paternal uncle and paternal grandparents. All family members were clinically evaluated using the Task Force criteria (Table 1). Only the clinically affected brother carried both variants (Figure 1C). The parents were carriers of individual PKP2 variants and the clinically unaffected (not meeting Task Force criteria) sibling hosted only the variant transmitted by his father and paternal grandfather (Figure 1C). RT-PCR of PKP2 mRNA was performed using samples obtained from five other affected probands and sequencing revealed that in each case the variants were encoded in trans. Thus, these variants were either inherited independently from the phenotypically normal patients or were de novo. As noted in Table 3, disparities between the clinical phenotypes and genetic findings appear somewhat common. Notably, all subjects had RV involvement, several had biventricular disease, and all had associated arrhythmias. Importantly, not all subjects met full Task Force Criteria, thereby pointing to imperfections in these criteria and necessitating modifications of these criteria (Marcus et al, Circulation, in press) 30.

Figure 1Figure 1
Pedigrees of three families with PKP2 variants and incomplete penetrance
Table 3
Clinical Demographics of ARVC Subjects with Desmosomal Gene Mutations. Note that most, but not all subjects with mutations meet full Task Force Diagnostic Criteria. All subjects have right ventricular involvement and arrhythmias and several subjects also ...

These findings led us to consider the possibility that ARVC may be due to another form of “compound heterozygosity” with mutations/genetic variants in two desmosomal protein encoding genes required for clinical disease, called “digenic heterozygosity”.


In all 198 probands, the desmosome-encoding genes DSP, DSC2, DSG2, JUP, DSC2,DSG2, PKP4, and DES were sequenced in addition to PKP2 sequencing. This sequencing identified 13 variants in second desmosomal genes in 13 subjects with PKP2 variants, including DSP in 6 cases, DSG2 in 5 cases, PKP4 in 1 subject, and DSC2 in 1 subject (Table 3). None of these variants were identified in at least 700 ethnic-matched controls (>1,400 chromosomes). Thus, of the 38 probands with PKP2 variants, compound or digenic heterozygosity was identified in 16 (42%), including 6 probands with 3 variants: in two of these cases, one of the PKP2 variants was the A372P polymorphism identified in the control population. Five probands had additional family members available for clinical and genetic evaluation including the following probands: patient 1 with the Q90R DSP variant (Table 3), the DSP W207X variant (patient 2, Table 3), the DSG2 V56M variant (patient 14, Table 3), the DSG2 R146H variant (patient 15, Table 3) and the DSP R1255K variant (patient 16, Table 3). The DSP Q90R variant was a de novo substitution and therefore could be pathogenic alone or in combination with the PKP2 variant (Figure 1A). In patient 12, a 4609C>T DSP substitution (R1537C) was also identified, a variant previously reported as a single nucleotide polymorphism ( Whether this variant becomes clinically relevant in combination with the PKP2 variants and is needed for the development of clinical features is speculation until animal models are completed. In the family of Patient 2 (Figure 2A), the PKP2 I531S variant was identified in three of the four siblings available for study. All family members were clinically evaluated using the Task Force criteria (Table 1) and one of the three siblings was clinically unaffected (and also has twin carrier daughters) while the other two siblings were clinically affected. Only the proband hosted the second variant, DSP W207X. In this case, we propose three potential interpretations of these data. Since the proband had the most severe phenotype (sudden death at age 25 years and severe ARVC on autopsy), it is possible that the DSP W207X mutation was required to manifest severe clinical disease. Secondly, this mutation may have no involvement in the development of disease and another, as yet identified mutation, is carried by the affected siblings but not by the unaffected sibling. Thirdly, the I531S mutation may be disease-causing alone with extremely reduced penetrance, as has been described in other forms of cardiomyopathy, and requires other factors, such as acquired agents, to manifest and contribute to clinical expression. In this regard, it should be noted that the proband developed mononucleosis two years prior to death.

Figure 2Figure 2
Pedigree of two U.S. families with compound desmosomal mutations (digenic heterozygosity)

As previously noted, Patient 14 hosted a DSG2 V56M variant; in addition, however, this subject also had two additional PKP2 variants (Figure 2B). Based upon evaluation of the pedigree, it is difficult to be certain as to the role of each of these variants; however, it appears that the combination of PKP2 S140F and DSG2 V56M, which was only detected in the proband, led to a more severe clinical phenotype (Figure 2B).

Patient 15, a 51 year old male, was diagnosed with a severe form of ARVC at the age of 39 years after an episode of ventricular fibrillation. He was found to host three different variants (PKP2 S209R, PKP2 T816fsX825 and DSG2 R146H) (Figure 3A). The PKP2 T816fsX825 was inherited from his mother (II,2). Since his father's DNA was not available, it was not possible to establish if the two missense variations (PKP2 S209R and DSG2 R146H) were inherited from the father or are de novo mutations. His mother and maternal aunt, who both carried the PKP2 frameshift variant, were clinically unaffected by Task Force criteria and asymptomatic.

Figure 3Figure 3
Pedigree of two Italian families with compound desmosomal mutations

Patient 16, a 40 year old male, was diagnosed with a severe form of ARVC after an episode of ventricular fibrillation that occurred during a sports activity. After resuscitation, he received an implantable cardioverter defibrillator (ICD). His family history included a male cousin who died suddenly at 30 years of age while hiking. No autopsy was performed. The proband (II,1) inherited the DSP missense mutation and PKP2 stop mutation from his mother (I,2), and the missense PKP2 mutation from the father (I,1) (Figure 3B). At least one mutation was also detected in the proband's sons. In this family, all mutation carriers, with the exception of the proband, were completely asymptomatic and clinically unaffected by Task Force criteria.

Overall, there was more VF and exercise-induced VT in those subjects having compound or digenic heterozygosity compared to those subjects with single heterozygous PKP2 mutations. In addition, the age of onset of symptomatic ARVC, most commonly ventricular arrhythmias or syncope, was earlier in those subjects with multiple genetic variants in PKP2 or in PKP2 plus other desmosomal genes (mean age 31.5 years) compared to those subjects with single heterozygous gene mutations (mean age 39.5 years). Therefore, it appears that compound and digenic heterozygosity leads to more clinically apparent and severe ARVC with an earlier age of onset than those hosting heterozygous mutations in these genes.


In 14 of the 198 subjects analyzed (7%), single heterozygous mutations were identified in desmosome-encoding genes other than PKP2. The heterozygous mutations identified included 4 variants in DSP, 5 variants in DSG2, 3 variants in DSC2, and 2 variants in JUP (Table 4). All variants were identified in conserved regions and none of these variants were identified in 700 ethnic-matched controls (1,400 chromosomes).

Table 4
Heterozygous mutations identified in Desmosomal genes other than PKP2


Myocardial autopsy samples were available from two individuals with PKP2 variants, including one patient (PG) with a single heterozygous PKP2 W528X nonsense mutation (no second variant has been identified to date) and a second patient (M686) with digenic heterozygosity including the PKP2 I531S variant as well as a DSP W207X mutation. These myocardial samples were sectioned and stained for a variety of junctional proteins, including PKP2, DSP, JUP, N-cadherin, and DSC2/3, as well as for Cx43 and DES (Figure 4). Staining of samples from patient PG was very weak or absent for each of these proteins except for DSP, which was normal. In contrast, staining of samples from patient M686 was essentially normal for each antibody, except for Cx43, which was absent (Figure 4), and DSP, which appeared to stain weakly. These data suggest that the DSP nonsense mutation in patient M686 does not effect DSP localization and that these variants do not grossly affect the desmosomal junctions. In contrast, both the desmosomal and cadherin junctions appear to be affected in patient PG, with loss of staining for N-cadherin and JUP, in addition to PKP2. Signal intensities were comparable in samples obtained from both left ventricle and right ventricle and in areas relatively unaffected by fibrofatty replacement.

Figure 4
Immunostaining of myocardial sections from a control heart and from the hearts of ARVC patients PG (PKP2 W538X) and M686 (PKP2 I531S and DSP W207X)


Arrhythmogenic right ventricular cardiomyopathy (ARVC) has emerged as a significant cause of sudden death, heart failure and the need for heart transplantation over the past decade.2, 31The incidence was initially thought to be particularly high in the Veneto region of Italy and in other parts of Europe, but low elsewhere.2 More recently, the disease has been increasingly recognized throughout the world.3,32,33 It is believed to be inherited in a moderate percentage of cases, with autosomal dominant inheritance predominating.4 Multiple genes, mostly those encoding desmosomal proteins, have been identified as causative in ARVC, with PKP2 reported to be responsible for approximately 25% of all cases.8, 18-21 In the work presented herein, however, interpretation of these data are questioned and the concepts of low penetrance, as well as compound and digenic heterozygosity are proposed as potential determinants of the clinical presentation in subjects carrying mutations and in their family members. In addition, identification of novel heterozygous mutations in other desmosome-encoding genes, including novel mutations in JUP, further supports the notion that the “final common pathway” for ARVC is the cell-cell junctions. 34

In this report, we demonstrate that, while variants in PKP2 are relatively common (identified in 38/198 or 19% of probands), harboring one PKP2 variant may not by itself be sufficient to determine overt clinical disease. In at least 16/38 (42%) cases, concomitant causes such as either a “second hit” in the same gene (compound heterozygosity) or in a second desmosome-encoding gene (digenic heterozygosity) or an acquired disruption of these proteins or environmental factors, has been shown to be required for the overt clinical phenotype to develop or for modification of disease severity. Hence, while PKP2 “mutations” are relatively common, a second variant in PKP2 or in DSP, DSC2, DSG2, PKP4, JUP or other interacting junction protein-encoding gene appears to be important for the disease and its clinical consequences to be manifest. Other “second hit” genes or interactors are likely to be discovered in the future. In addition, a variety of heterozygous mutations in all of the known desmosome-encoding genes were identified to cause ARVC and its associated clinical signs and symptoms.

Interestingly, the subjects harboring more than one variant in PKP2 and/or PKP2 plus other desmosome-encoding genes appear to have earlier onset of disease and more clinical severity than those individuals harboring heterozygous mutations alone. Interpretation of these data, however, is confounded by the small number of large pedigrees available for analysis. In order to track segregation of the genotype with disease, large families with clinical ARVC are required but, in our study, small families and sporadic cases were predominantly seen. However, in the families in which multiple individuals were enrolled and genetically screened, a single genetic variant commonly did not lead to overt clinical disease using the Task Force criteria (Table 1) or the stringent diagnostic evaluation used by the ARVD Registry. It will be interesting to determine if the carriers previously phenotyped as unaffected will be found to have any signs of early ARVC upon detailed examination (MRI, echocardiogram, etc) or whether later development of clinical signs due to an otherwise concealed pathological process will occur over time. These genetic findings should encourage physicians caring for patients with ARVC to screen as many families as possible, irrespective of clinical presentation. Clearly, longitudinal follow-up of patients and carrier family members will be critical in determining the influence of these gene variants and, more importantly, will be critical in providing excellent preventive care. As noted, we have only identified second variants in about 40% of the probands in whom PKP2 variants were identified. What about the remainder? Firstly, many genes that encode proteins that either directly or indirectly contributes to the function and integrity of cell adhesion junctions remain to be screened, such as the genes encoding α- or β-catenin. Mutations in proteins of the myocyte cytoarchitecture which are linked to these proteins, such as actin, α-actinin-2, metavinculin, or Z-disk proteins could be important and will also be evaluated. Secondly, some mutations could result in dominant negative proteins giving rise to autosomal dominant inheritance, the mode of inheritance widely held as the most common in patients suffering from ARVC. In addition to the compound heterozygous and digenic mutations noted, we also identified apparent isolated heterozygous mutations in desmosome-encoding genes in another 7% of subjects. We would speculate that other relevant genes have yet to be discovered. An interesting observation from these studies is that nonsense or frame-shift mutations alone in PKP2 often do not result in clinical disease in these families. These data would suggest that haploinsufficiency for PKP2 is not critically important or even sufficient or that compensatory mechanisms occur. Only when the remaining copy of PKP2 is mutated or there is a mutation in another gene, does overt clinical disease develop. It also suggests that the description of these genetic variants as “disease-causing mutations” may be inaccurate and that defining affectation status by genetic analysis alone may not be entirely appropriate. For instance, on face value, the I531S variant would appear to be a polymorphism. However, the detection of this variant in 5 of the 143 probands (from different regions of the U.S.), but in none of the 700 controls, would suggest that this variant is linked to disease. On the other hand, nonsense mutations in PKP2 would be expected to result in overt clinical disease but, in many cases, does not. For this reason, we would caution potential “fee-for-service” laboratories offering “clinical testing” for ARVC from definitive statements regarding cause-and effect relationships, particularly in subjects in whom PKP2 variants are identified, especially if the remaining genes are not screened. In this circumstance, not only is the affected subject at risk to have a mistaken causative gene assigned, but “at-risk” family members may be either mistakenly diagnosed with a causative mutation or, more concerning, be given a negative result. In the latter case, this could lead to discharge from follow-up despite actually carrying a disease causing mutation in another gene not analyzed. Thia could lead to tragic outcomes. Grossman et al.35 ablated the PKP2 gene in mice, causing lethal alteration in heart morphogenesis at mid-gestation characterization by reduced trabeculations, disarrayed cytoskeleton, rupture of the cardiac wall, and hemopericardium. In the absence of PKP2, the cytoskeletal linker protein desmoplakin dissociates from the junctional plaques that connect cardiomyocytes, resulting in reduced architectural stability of intercalated disks. Constant mechanical stress, as seen in the contracting heart, is likely to be deleterious to the weakened intercellular junctions, leading to disruption, dilation and dysfunction. However, heterozygous mice were healthy and fertile. This is consistent with our notion that haploinsufficiency for PKP2 is not sufficient to cause disease. The right ventricle, based on its geometry, architecture, and role in cardiac function, dilates to a variety of volume and pressure abnormalities and would likely develop structural and functional disease earlier than the left ventricle. This was clearly shown in the DSP transgenic mouse model of Yang et al, who demonstrated early right ventricular dilation with later left ventricular dilation, as well as intercalated disk disruption and loss of desmosomes.29 In addition, further support is provided by the fact that the left ventricle develops late-onset disease in some patients with ARVC.3, 36 In addition to the genetic mutations, mechanical stress and stretch forces on the disturbed desmosome and intercalated disk, likely plays a role in disease development and severity.34,37

Together, the genetic and functional data provided here should help to define the nature of the genetic and clinical basis of ARVC. This work could significantly impact on clinical genetic screening, as simple single gene analysis (particularly for PKP2) would be inappropriate and the potential for inaccurate interpretation based on single (or even multiple) gene analysis appears to be high. More detailed information regarding the true clinical relevance of PKP2 mutations is needed. Further, the moderate number of mutations in the other desmosome-encoding genes strongly suggests that all genes in this pathway should be screened in all subjects. Identification of heterozygous mutations in JUP, as well as the potential mutation within the PKP4 gene adds to the spectrum of affected genes involving the desmosome. Further studies of PKP4 in subjects with ARVC, as well as functional analysis of models with mutant PKP4 will be needed to clearly state that this is a potential disease-causing gene in ARVC.

The lack of functional studies of the variants described is a limitation of the study. Cellular and animal models incorporating these mutations individually and together could help to resolve these issues to some extent but these approaches may not necessarily recapitulate the human condition. In attempt to better understand the mechanisms involved in the development (or lack of development) of the clinical phenotype associated with these variants, we are in the process of developing these models for study. In addition to the models, the addition of mechanical stretch and stress on the cells and animals could facilitate the development of phenotypic differences from non-stressed models and provide greater insight.

Supplementary Material


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

No conflicts.


1. Marcus FI, Fontaine GH, Guiraudon G, Frank R, Laurenceau JL, Malergue C, Grosgogeat Y. Right ventricular dysplasia: a report of 24 adult cases. Circulation. 1982;65:384–398. [PubMed]
2. Corrado D, Basso C, Thiene G, McKenna WJ, Davies MJ, Fontaliran F, Nava A, Silvestri F, Blomstrom-Lundqvist C, Wlodarska EK, Fontaine G, Camerini F. Spectrum of clinicopathologic manifestations of arrhythmogenic right ventricular cardiomyopathy/dysplasia: a multicenter study. J. Am. Coll. Cardiol. 1997;30:1512–1520. [PubMed]
3. Horimoto M, Funayama N, Satoh M, Igarashi T, Sekiguchi M. Histologic evidence of left ventricular involvement in arrhythmogenic right ventricular dysplasia. Jpn Circ J. 1989;53:1530–1534. [PubMed]
4. Towbin JA. Molecular genetics of sudden cardiac death. Cardiovasc Pathol. 2001;10:283–295. [PubMed]
5. Bowles NE, Ni J, Marcus F, Towbin JA. The detection of cardiotropic viruses in the myocardium of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy. J.Am. Coll. Cardiol. 2002;39:892–895. [PubMed]
6. Basso C, Thiene G, Corrado D, Angelini A, Nava A, Valente M. Arrhythmogenic right ventricular cardiomyopathy. Dysplasia, dystrophy, or myocarditis? Circulation. 1996;94:983–991. [PubMed]
7. Rampazzo A, Nava A, Malacrida S, Beffagna G, Bauce B, Rossi V, Zimbello R, Simionati B, Basso C, Thiene G, Towbin JA, Danieli GA. Mutation in human desmoplakin domain binding to plakoglobin causes a dominant form of arrhythmogenic right ventricular cardiomyopathy. Am J Hum Genet. 2002;71:1200–1206. [PubMed]
8. Gerull B, Heuser A, Wichter T, Paul M, Basson CT, McDermott DA, Lerman BB, Markowitz SM, Ellinor PT, MacRae CA, Peters S, Grossman KS, Drenckhahn J, Michely B, Sasse-Klaassen S, Birchmeier W, Dietz R, Breithardt G, Schulze-Bahr E, Thierfelder L. Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy. Nat Genet. 2004;36:1162–1164. [PubMed]
9. Pilichou K, Nava A, Basso C, Beffagna G, Bauce B, Lorenzon A, Frigo G, Vettori A, Valente M, Towbin JA, Thiene G, Danieli GA, Rampazzo A. Mutations in desmoglein-2 gene are associated with arrhythmogenic right ventricular cardiomyopathy. Circulation. 2006;113:1171–1179. [PubMed]
10. Syrris P, Ward D, Evans A, Asimaki A, Gandjbakhch E, Sen-Chowdhry S, McKenna WJ. Arrhythmogenic right ventricular dysplasia/cardiomyopathy associated with mutations in the desmosomal gene desmocollin-2. Am J. Hum Genet. 2006;79:978–984. [PubMed]
11. Beffagna G, Occhi G, Nava A, Vitiello A, Ditadi A, Basso C, Bauce B, Carraro G, Thiene G, Towbin JA, Danieli GA, Rampazzo A. Regulatory mutations in transforming growth factor-beta3 gene cause arrhythmogenic right ventricular cardiomyopathy type 1. Cardiovasc Res. 2005;65:366–73. [PubMed]
12. Tiso N, Stephan DA, Nava A, Bagattin A, Devaney JM, Stanchi F, Larderet G, Brahmbhatt B, Brown K, Bauce B, Muriago M, Basso C, Thiene G, Danieli GA, Rampazzo A. Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2) Hum Mol Genet. 2001;10:189–194. [PubMed]
13. Merner ND, Hodgkinson KA, Haywood AF, Connors S, French VM, Drenckhahn JD, Kupprion C, Ramadanova K, Thierfelder L, McKenna W, Gallagher B, Morris-Larkin L, Bassett AS, Parfrey PS, Young TL. Arrhythmogenic right ventricular cardiomyopathy type 5 is a fully penetrant, lethal arrhythmic disorder caused by a missense mutation in the TMEM43 gene. Am J Hum Genet. 2008;82:809–821. [PubMed]
14. Coonar AS, Protonotarios N, Tsatsopoulou A, Needham EW, Houlston RS, Cliff S, Otter MI, Murday VA, Mattu RK, McKenna WJ. Gene for arrhythmogenic right ventricular cardiomyopathy with diffuse nonepidermolytic palmoplantar keratoderma and woolly hair (Naxos disease) maps to 17q21. Circulation. 1998;97:2049–58. 1. [PubMed]
15. Carvajal-Huerta L. Epidermolytic palmoplantar keratoderma with woolly hair and dilated cardiomyopathy. Am. Acad. Dermatol. 1998;39:418–421. [PubMed]
16. McKoy G, Protonotarios N, Crosby A, Tsatsopoulou A, Anastasakis A, Coonar A, Norman M, Baboonian C, Jeffery S, McKenna WJ. Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease) Lancet. 2000;355:2119–24. [PubMed]
17. Norgett EE, Hatsell SJ, Carvajal-Huerta L, Cabezas JC, Common J, Purkis PE, Whittock N, Leigh IM, Stevens HP, Kelsell DP. Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma. Hum Mol Genet. 2000;9:2761–6. [PubMed]
18. Syrris P, Ward D, Asimaki A, Sen-Chowdhry S, Ebrahim HY, Evans A, Hitomi N, Norman M, Pantazis A, Shaw AL, Elliott PM, McKenna WJ. Clinical expression of plakophilin-2 mutations in familial arrhythmogenic right ventricular cardiomyopathy. Circulation. 2006;113:356–364. [PubMed]
19. Van Tintelen JP, Entius MM, Bhuiyan ZA, Jongbloed R, Wiesfeld AC, Wilde AA, van der Smagt J, Boven LG, Mannens MM, van Langen IM, van Hofstra RM, Otterspoor RC, Doevendens PA, Rodriguez LM, van Gelder IC, Hauer RN. Plakophilin-2 mutations are the major determinant of arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circulation. 2006;113:1650–1658. [PubMed]
20. Dalal D, Molin LH, Piccini J, Tichnell C, James C, Bomma C, Prakasa K, Towbin JA, Marcus FI, Spevak PJ, Bluemke DA, Abraham T, Russell SD, Calkins H, Judge DP. Clinical features of arrhythmogenic right ventricular dysplasia/cardiomyopathy associated with mutations in plakophilin-2. Circulation. 2006;113:1641–1649. [PubMed]
21. Dalal D, James C, Devanagondi R, Tichnell C, Tucker A, Prakasa K, Spevak PJ, Bluemke DA, Abraham T, Russell SD, Calkins H, Judge DP. Penetrance of mutations in plakophilin-2 among families with arrhythmogenic right ventricular dysplasia/cardiomyopathy. J. Am. Coll. Cardiol. 2006;48:1416–1424. [PubMed]
22. Garrod DR, Merritt AJ, Nie Z. Desmosomal adhesion: structural basis, molecular mechanism and regulation. Mol Membr Biol. 2002;19:81–94. [PubMed]
23. Peifer M, Berg S, Reynolds AB. A repeating amino acid motif shared by proteins with diverse cellular roles. Cell. 1994;76:789–91. [PubMed]
24. Yin T, Green KJ. Regulation of desmosome assembly and adhesion. Semin Cell Dev Biol. 2004;15:665–77. [PubMed]
25. Marcus F, Towbin JA, Zareba W, Moss A, Calkins H, Brown M, Gear K, ARVD/C Investigators Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C): a multidisciplinary study: design and protocol. Circulation. 2003;107:2975–2978. [PubMed]
26. McKenna WJ, Thiene G, Nava A, Fontaliran F, Blomstrom-Lundqvist C, Fontaine G, Camerini F. Diagnosis of arrhythmogenic right ventricular dysplasia/cardiomyopathy: Task Force of the Working Group Myocardial and Pericardial Disease of the European Society of Cardiology and of the Scientific Council on Cardiomyopathies of the International Society and Federation of Cardiology. Br Heart J. 1994;71:215–218. [PMC free article] [PubMed]
27. Vatta M, Mohapatra B, Jimenez S, Sanchez X, Faulkner G, Perles Z, Sinagra G, Lin JH, Vu TM, Zhou Q, Bowles KR, Di Lenarda A, Schimenti L, Fox M, Chrisco MAA, Murphy RT, McKenna W, Elliott P, Bowles NE, Chen J, Valle G, Towbin JA. Mutations in Cypher/ZASP in patients with dilated cardiomyopathy and left ventricular non-compaction. J. Am. Coll. Cardiol. 2003;42:2014–2027. [PubMed]
28. Kaplan SR, Gard JJ, Carvajal-Huerta L, Ruiz-Cabezas JC, Thiene G, Saffitz JE. Structural and molecular pathology of the heart in Carvajal syndrome. Cardiovasc Pathol. 2004;13:26–32. [PubMed]
29. Yang Z, Bowles NE, Scherer SE, Taylor MD, Kearney DL, Ge S, Nadvoretskiy VV, Defreitas G, Carabello B, Brandon LI, Godsel LM, Green KJ, Saffitz JE, Li H, Danieli GA, Calkins H, Marcus F, Towbin JA. Desmosomal dysfunction due to mutations in desmoplakin causes arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circ Res. 2006;99:646–655. [PubMed]
30. Marcus FI, McKenna WJ, Sherrill D, Basso C, Bauce B, Bluemke DA, Caulkins H, Corrado DA, Cox MGPJ, Daubert J, Fontaine G, Gear K, Hauer RNW, Nava A, Picard MH, Protonotarios N, Saffitz JE, Yoerger Sanborn DM, Steinberg JS, Tandri HM, Thiene G, Towbin JA, Tsatsopoulou A, Wichter T, Zareba W. Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D): Proposed modification of Task Force Criteria. Circulation. 2009 in press. [PMC free article] [PubMed]
31. Thiene G, Nava A, Corrado D, Rossi L, Pennelli N. Right ventricular cardiomyopathy and sudden death in young people. N Engl J Med. 1988;318:129–33. [PubMed]
32. Dalal D, Nasir K, Bomma C, Prakasa K Tandri H, Piccini J, Roquin A, Tichnell C, James C, Russell SD, Judge DP, Abraham T, Spevak PJ, Bluemke DA, Calkins H. Arrhythmogenic right ventricular dysplasia: a United States experience. Circulation. 2005;112:3823–3832. [PubMed]
33. Cho Y, Park T, Shin D, Lee JH, Ryu HM, Jang GL, Lee DY, Park Y, Lee H, Kim H, Shin SC, Heo JH, Kang H, Lee BR, Nah DY, Yang DH, Park HS, Chae SC, Jun JE, Park WH. Clinical manifestations of arrhythmogenic right ventricular cardiomyopathy in Korean patients. Int J Cardiol. 2007;122:137–142. [PubMed]
34. Vatta M, Marcus FI, Towbin JA. Arrhythmogenic right ventricular cardiomyopathy: a “final common pathway that defines clinical phenotype” Eur Heart J. 2007;28:529–530. [PubMed]
35. Grossmann KS, Grund C, Huesken J, Behrend M, Erdmann B, Franke WW, Birchmeier W. Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation. J Cell Biol. 2004;167:149–160. [PMC free article] [PubMed]
36. Takahashi N, Ishida Y, Maeno M, Hirose Y, Kawano S, Fukuoka S, Hayashida K, Kuribayashi S, Hamada S, Yamada N, Takamiya M, Shimomura K, Ohne T. Noninvasive identification of left ventricular involvements in arrhythmogenic right ventricular dysplasia: comparison of 123I-MIB G, 201 TICI, magnetic resonance imaging and ultrafast computed tomography. Ann Nucl Med. 1997;11:233–241. [PubMed]
37. Towbin JA, Bowles NE. Dilated cardiomyopathy: a tale of cytoskeletal proteins and beyond. J Cardiovasc Electrophysiol. 2006;17:919–926. [PubMed]