Desmin mutations as a cause of right ventricular heart failure affect the intercalated disks

Desmin mutations as a cause of right ventricular heart failure affect the intercalated disks Ellen Otten, MD,* Angeliki Asimaki, PhD,† Alexander Maass, MD, PhD,‡ Irene M. van Langen, MD, PhD,*¶ Allard van der Wal, MD, PhD,§ Nicolaas de Jonge, MD, PhD,储 Maarten P. van den Berg, MD, PhD,‡ Jeffrey E. Saffitz, MD, PhD,† Arthur A.M. Wilde, MD, PhD,**†† Jan D.H. Jongbloed, PhD,* J. Peter van Tintelen, MD, PhD,* From the *Department of Genetics, University Medical Center Groningen, University of Groningen, the Netherlands, † Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston Massachusetts, ‡ Department of Cardiology, Thoraxcenter, University Medical Center Groningen, University of Groningen, ¶Department of Clinical Genetics and §Department of Pathology, Academic Medical Center, Amsterdam, 储Department of Cardiology, University Medical Center Utrecht, **Heart Failure Research Center, Department of Cardiology, Academic Medical Center, Amsterdam, and ††Interuniversity Cardiology Institute of the Netherlands, Utrecht, the Netherlands. BACKGROUND Mutations in the gene encoding desmin (DES), an intermediate filament protein, underlie a heterogeneous phenotype, which is referred to as desmin-related myopathy (DRM). Right ventricular involvement including an arrhythmogenic right ventricular cardiomyopathy (ARVC)(-like) phenotype has occasionally been described in DES mutation-carrying patients. OBJECTIVE To determine the effects of a DES missense mutation on the structure of different intercalated disk proteins, to evaluate right ventricular involvement in DES mutation carriers, and to establish the role of DES mutations in ARVC(-like) phenotypes. METHODS We evaluated the clinical phenotype in two families carrying two different DES mutations. One family was diagnosed with DRM, with an ARVC(-like) phenotype in one patient, while the other family presented with a severe biventricular cardiomyopathy. Additional immunohistochemistry of desmosomal proteins was performed in myocardial tissue from two patients of the last family. The DES gene was screened for mutations in 50 ARVC(-like) patients. RESULTS Except for two different DES mutations (p.N342D and p.R454W) in two families with DRM and severe biventricular cardiomyopathy, respectively, we did not find additional DES mutations in ARVC(-like) patients. In addition to desmin aggregates, immunohistochemistry demonstrated a decreased amount of desmoplakin and plakophilin-2 at the intercalated disk in p.R454W mutation carriers.

Introduction Desmin-related myopathy (DRM, MIM no. 601419) is generally inherited in an autosomal dominant fashion and is a subgroup of myofibrillar myopathies in which skeletal myopathy is often comAddress reprint requests and correspondence: J. Peter van Tintelen, M.D., Ph.D., Department of Genetics, University Medical Center Groningen, University of Groningen, P.O. Box 30001, 9700 RB Groningen, the Netherlands. E-mail address: [email protected]. (Received August 25, 2009; accepted April 20, 2010.)

CONCLUSIONS We confirmed that either an ARVC-like phenotype or a severe cardiomyopathy with right ventricular involvement are possible, yet infrequent, cardiac phenotypes in DRM. Moreover, we demonstrated that the DES mutation p.R454W affects the localization of desmoplakin and plakophilin-2 at the intercalated disk, suggesting a link between desmosomal cardiomyopathies (mainly affecting the right ventricle) and cardiomyopathies caused by DES mutations. KEYWORDS Cardiomyopathy; Intercalated disk; Genes; Genetics; Immunohistochemistry; Right ventricle; Desmin ABBREVIATIONS ARVC ⫽ arrhythmogenic right ventricular cardiomyopathy; AVB ⫽ atrioventricular block; CM ⫽ cardiomyopathy; CRYAB ⫽ alpha B crystallin; DCM ⫽ dilated cardiomyopathy; DES ⫽ desmin; DSC2 ⫽ desmocollin 2; DSG2 ⫽ desmoglein 2; DGGE ⫽ denaturing gradient gel electrophoresis; DHPLC ⫽ denaturing high-performance liquid chromatography; DRM ⫽ desminrelated myopathy; DSP ⫽ desmoplakin; ECG ⫽ electrocardiogram; HCM ⫽ hypertrophic cardiomyopathy; JUP ⫽ junction plakoglobin; LMNA ⫽ lamin A/C; LV ⫽ left ventricle, ventricular; PCR ⫽ polymerase chain reaction; PKP2 ⫽ plakophilin-2; PVC ⫽ premature ventricular contraction; RCM ⫽ restrictive cardiomyopathy; RV ⫽ right ventricle, ventricular (Heart Rhythm 2010;7:1058 –1064) © 2010 Heart Rhythm Society. All rights reserved.

bined with cardiac involvement. Skeletal muscle weakness usually manifests in the second to fourth decade of life with slowly progressive, painless distal weakness and atrophy of the lower extremities. With disease progression, the upper extremities, the proximal leg, and the trunk muscles may also become affected.1–3 Up to 70% of patients show cardiac involvement, which may precede, coincide with, or succeed skeletal muscle weakness.4 Desmin is the main intermediate filament protein in mature skeletal and heart muscle cells. It forms a scaffold

1547-5271/$ -see front matter © 2010 Heart Rhythm Society. All rights reserved.

doi:10.1016/j.hrthm.2010.04.023

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around the Z-disk and interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton, the intercalated disk, nuclei, and other cytoplasmic components.1,5 Desmin is encoded by a single gene (DES), which is located on chromosome 2q35. In approximately one-third of DRM patients, mutations have been found in the DES gene or the gene encoding alpha-B-crystallin (CRYAB).2 The majority of the respective DES mutations are found in exons encoding the alpha-helical rod domain, with a significant clustering of mutations in exon 6, which encodes the C-terminal part of the alpha-helical 2B segment. The phenotypes of desminopathies are influenced by interactions (or changes in interactions) between desmin and other cell components and the activity of chaperones that are capable of compensating for the detrimental effect of mutant desmin.1 The increased susceptibility of muscle fibers to physical strain, owing to a lack of normal desmin, is believed to play a key role in the pathogenesis of DRM, although its exact pathophysiological mechanism is not yet fully understood.1 We recently described right ventricular (RV) involvement including an arrhythmogenic right ventricular cardiomyopathy (ARVC) phenocopy and abnormalities at the level of the intercalated disk in patients carrying a missense mutation in the head domain (p.Ser13Phe) of the DES protein.6 Therefore we hypothesized that DES mutations can also demonstrate an effect at the level of the myocardial intercalated disk. Here we describe two Dutch families with a DES mutation and the results of DES screening in a series of ARVC and ARVC-like patients. In one of these families with a DES mutation, we studied the effects of the mutation at the level of the intercalated disk.

Methods Patients The index patient of family A was referred to our department for genetic confirmation of DRM. The second family (B) was referred because of an early presenting, familial cardiomyopathy (CM; Figures 1A and 1B, respectively). In addition, 20 patients who fulfilled the generally accepted

1059 1994 clinical task force criteria for ARVC7 and 30 patients with probable ARVC were evaluated for mutations in the DES gene. The majority of these additional patients have been described elsewhere.8,9 They were all at least evaluated by physical examination, 12-lead electrocardiogram (ECG), 24-hour Holter monitoring, exercise testing, and two-dimensional transthoracic echocardiography. Probable ARVC was defined as having either one major and one minor criterion or three minor criteria.

Genetic analyses Genomic DNA was isolated from blood samples using standardized procedures. DNA from 300 chromosomes from ethnically matched, healthy individuals was used as controls. Written informed consent was obtained from the index patients and their relatives, who were analyzed according to our hospital’s medical ethics committees. Primers for polymerase chain reaction (PCR) amplification of the DES gene were designed to encompass the coding exons as well as adjacent intronic sequences as described elsewhere.10 The genomic sequences used to design these primers were obtained from sequences in the GenBank database; accession number NC_000002.10, region 219991343 to 219999705. Amplifications were performed following a standard PCR protocol, and PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) and/or direct sequencing.11 PCR fragments showing an aberrant DGGE pattern were sequenced as described elsewhere.12 Moreover, the known ARVC genes encoding desmoplakin (DSP), plakophilin-2 (PKP2), desmocollin 2 (DSC2), desmoglein 2 (DSG2), and plakoglobin (JUP) were screened in both patients from family B by direct sequencing. The genomic sequences used to design the respective primers were obtained from sequences in the GenBank database: accession number NC_000006.10, region 7541870 to 7586946 (DSP); accession number NC_000012.10, region 32943680 to 33049780 (PKP2); accession number, NC_000018.8 region 28645942 to 28682388 (DSC2); accession number NC_000018.8, region 29078027 to 29128814 (DSG2); accession number NC_000017.9, region 39910859 to 39942964 (JUP). Primer sequences and PCR conditions are available upon request.

Immunofluorescence

Figure 1 Pedigrees of families A and B. Closed symbols depict the patients carrying a DES mutation, showing CM and skeletal myopathy in family A and showing a malignant CM in family B. The half-closed symbols depict the family members showing skeletal myopathy in family A and severe CM in family B, of which no DNA was available to confirm the diagnosis. The arrow indicates the index patient.

Endomyocardial biopsy samples from the left ventricular (LV) septal wall, obtained from two patients of family B, were immunostained using methods that were validated in a previous study.13 Primary antibodies included polyclonal rabbit anti-connexin-43 (Zymed, San Fransisco, CA, USA), monoclonal mouse anti-plakoglobin (Sigma, St. Louis), polyclonal rabbit anti-desmoplakin (Serotec, Raleigh, NC, USA), monoclonal mouse anti N-cadherin (Sigma), monoclonal mouse anti-plakophilin 2 (Fitzgerald, Acton, MA, USA), polyclonal rabbit anti-desmin (AbCam, Cambridge, MA, USA), and polyclonal rabbit anti-desmin (Serotec) antibodies. Immunostained preparations were analyzed by laser-scanning confocal microscopy (Sarastro Model 2000,

1060 Molecular Dynamics, Sunnyvale, CA, USA) as described elsewhere.13 As in a previous study, we did not quantify immunostaining signals.14

Results Patients Family A The index patient of family A (Figure 1A) was referred to our department for genetic screening because of signs of a generalized skeletal muscular disease with cardiac involvement. He presented at the age of 36 years with loss of strength of his leg musculature. In addition, he had experienced palpitations ever since he was a child. At the age of 39 years, he was diagnosed with atrial fibrillation, and on ECG 2 years later, extensive pathology was seen, including first-degree atrioventricular block (AVB), sinus arrests, premature ventricular contractions (PVCs), negative T waves in multiple leads, and an epsilon wave (Figure 2). At the age of 42 years, he collapsed and had to be resuscitated: an implantable cardioverter-defibrillator was implanted because of repeating ventricular tachycardias. His ECG demonstrated multiple pathologies, and over 1,000 PVCs were present on 24-hour Holter monitoring. Echocardiography revealed RV dilatation with hypokinesia and akinesia. This confirmed a diagnosis of ARVC. A skeletal muscle biopsy at the age of 44 years demonstrated signs of a desminopathy. Four years later, echocardiography revealed a dilated LV with a slightly diminished global systolic function, a severely impaired diastolic function, and a poorly functioning RV. The weakness of his leg musculature was progressive, and his hand and diaphragmatic musculatures were also involved some years later. Family history showed that his brother also presented with a progressive loss of strength of his leg musculature, at the age of 41 years. In a skeletal muscle biopsy at the age of 43 years, significant accumulation of desmin-positive ma-

Heart Rhythm, Vol 7, No 8, August 2010 terial was seen. Cardiac evaluation at the age of 46 years showed a first-degree AVB and a slightly diminished LV function, compatible with incipient CM. The father of these two brothers was also said to have had a progressive loss of strength of his leg muscles, which eventually made him wheelchair dependent. He died at the age of 59 years from pneumonia. He had no cardiac complaints but had never been cardiologically evaluated. Family B The female index patient of family B (Figure 1B) presented at the age of 9 years with a total AVB and slow ventricular escape rhythm. At the age of 18 years, she had complaints of tiredness and a year later a DDD pacemaker had been implanted. A slightly increased diameter of the left atrium and an increased RV diameter were observed. Furthermore, the LV ejection fraction (EF) progressively decreased. An endomyocardial biopsy from the LV septal wall at the age of 21 years showed slightly hypertrophic cardiac muscle cells with extensive fibrosis and focal infiltrations of inflammatory cells (lymphocytes and macrophages). At the time of biopsy, progression of RV dilatation was seen with still normal cardiac function. At the age of 27 years, she was suffering from heart failure and progressive dyspnea. She died at age 28 from heart failure. No neurological evaluation was ever performed, although she had never complained about muscle weakness. Her brother underwent cardiological screening at the age of 17 years. He also had a total AVB, for which a DDDR pacemaker was implanted 2 years later. Cardiological investigation showed dilatation of the left and right atria and a normal systolic LV function (EF 57%). At age 19 years, an endomyocardial biopsy from the LV septal wall showed findings identical to those in his sister. At that time, the LVEF was still normal but later on deteriorated (35% at the age of 26 years). From the age of 24, he had atrial fibrilla-

Figure 2 ECG from the index patient of family A, suggestive of ARVC. This ECG shows a first-degree AV block (1), sinusarrests (2), ventricular extrasystoles (3), and negative T-waves in multiple leads and an epsilon wave in lead V1 (4).

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tion, dizziness, and hypotension due to forward failure. He died at age 27. The father of both patients was diagnosed with CM of unknown origin at the age of 19 years and a total AVB at the age of 20 years. One year later, a pacemaker was implanted. He suffered from progressive fatigue and dyspnea and died at the age of 31 years from recurrent thromboembolisms with widespread hemorrhagic lung infarctions in addition to progressive congestive heart failure. Postmortem examination revealed a severely enlarged and dilated heart with extensive circumferential fibrosis and little remaining myocardial tissue.

Genetic analyses Analysis of the DES gene showed a c.1024A⬎G mutation in index patient A, which results in an asparagineto-aspartate substitution at position 342 in exon 6 (p.Asn342Asp). This mutation was not found in 300 control chromosomes. In brother and sister B, sequence analysis demonstrated a c.1360C⬎T mutation in exon 8 of the DES gene. This results in an arginine-to-tryptophan substitution at position 454 (p.Arg454Trp; Figure 3). The mutation was absent in the unaffected mother and was therefore presumed to be inherited from the clinically affected father. The mutations identified are considered to be pathogenic because they result in a significant change in polarity of the respective residues; the affected amino acid residues are highly conserved among a variety of species and are located in highly conserved regions of the DES protein. Moreover, both mutations are situated in a functionally important domain of the DES protein and have been described before in patients with (cardio-)myopathies.15,16 In both siblings from family B, mutations in the DSP, PKP2, DSC2, DSG2, and JUP genes

1061 were excluded. In addition, mutations in the LMNA gene were excluded in both families (data not shown). Screening of the DES gene in 20 ARVC patients and in 30 probable ARVC patients without mutations in the ARVC-related PKP2, DSC2, and DSG2 genes did not reveal any additional mutations.

Immunofluorescence To examine the effects of the DES missense mutation on the distribution of desmin and intercalated disk proteins and on the composition of the corresponding cellular structures, immunohistochemistry was performed on the myocardial samples from the brother and sister in family B. Myocardial specimens from two age-matched individuals without clinical history or pathological evidence of heart disease were subjected to the same staining protocol and used as controls. Immunostaining using anti-desmin antibodies in control myocardium gave a characteristic striated pattern, reflecting the association of the intermediate filament protein with Z-band areas, while pronounced deposition was observed at the intercalated disks. Conversely, desmin distribution was severely disrupted in the diseased myocardial samples of these two patients. No signal was observed at the intercalated disks, and instead desmin formed large cytoplasmic and perinuclear blobs (Figure 4). When the results of immunostaining of diseased and control myocardium using anti N-cadherin and anti-plakoglobin specific antibodies were compared, the incorporation of comparable levels of these proteins into the intercalated disk was observed (Figure 4). In contrast, a marked decrease in the amount of immunoreactive signal at intercalated disks, corresponding to the desmosomal proteins desmoplakin and plakophilin 2, and the major gap junction protein in cardiac ventricles,

Figure 3 DNA sequences showing nucleotide substitutions and amino acid sequences displaying residue conservation in different species. A: c.1024A⬎G; p.Asn342Asp; B: c.1360C⬎T; p.Arg454Trp.

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Figure 4 Immunofluorescence images (magnification ⫻40) of myocardium from the p.Arg454Trp mutation-carrying sibs of family B and a control subject. Visualization of desmin with Serotec antibody and Abcam antibody demonstrates a characteristic striated pattern of this protein in control myocardial tissue and a disrupted pattern of desmin distribution in the myocardial tissue of our patients. Signal levels of N-cadherin and plakoglobin at the intercalated disk in myocardium of our patients is comparable with control myocardium. A marked decrease in the amounts of connexin-43, desmoplakin, and PKP2 immunoreactive signals was seen at the intercalated disks in myocardial tissue of our two patients compared with control myocardial tissue.

connexin 43 (Figure 4), was demonstrated by immunostaining using the respective antibodies.

Discussion Desmin is the main intermediate filament protein in mature skeletal and heart muscle cells, providing stability to muscle cells.1,5 In DRM, progressive weakness of skeletal muscles and development of CM or arrhythmias arise to varying degrees, independently of each other, and with broad interand intrafamilial variability.1,2,17 Cardiac phenotypes related to DRM and described earlier are dilated, restrictive, and hypertrophic cardiomyopathy (DCM, RCM, and HCM, respectively).2,4 Recently, we described RV involvement in patients carrying a DES missense mutation (p.Ser13Phe).6 Here we describe two different DES missense mutations in two families (mutations p.N342D and p.R454W in families A and B, respectively). In the first family there is a DRM with an ARVC-like cardiac phenotype. In the other family, a severe biventricular CM phenotype is present. Additional screening of DES in 50 ARVC(-like) patients did not reveal any other mutations, suggesting that DES mutations are an uncommon cause of an ARVC(-like) phenotype. The p.N342D DES mutation in family A, with a predominantly skeletal myopathy phenotype, has been described earlier in two patients from a single family with skeletal myopathy. These patients had normal ECGs, and no CM involvement was reported, in contrast with the predominantly RV cardiac involvement in our index patient.15,18 Expression studies demonstrated the inability of the

p.N342D mutant to form a cellular filamentous network.15,18 In our index patient, RV involvement is followed by LV dilatation. In ARVC, the RV is primarily involved. However, the LV can also be involved, especially in more advanced stages of disease.19,20 Debate is still going on about whether LV involvement in ARVC represents a larger spectrum of the disease or must be seen as a separate CM entity.20 The second mutation (p.R454W), which was found in family B, has been described before in an HCM patient with early disease onset and a cardiac transplantation at age 25.16 This patient also carried a myotilin mutation (p.Q74K), which is considered conditionally pathogenic.16 Importantly, in vivo and in vitro analyses showed an effect of this DES mutation on filament formation.16 In the same study, defects in filament formation and impaired networking properties were shown by viscometry.16 The phenotype of family B apparently resembles the case described above, in terms of a severe CM. DRM is characterized by granulofilamentous aggregates.15 This principal pathognomonic characteristic of DRM was also found in our patients by using immunohistochemistry and laser-scanning confocal microscopy (Figure 4). Previous immunohistochemical studies in a mouse model of DRM also demonstrated marked redistribution of desmin as well as of other intercellular junctional proteins.21 Recently Asimaki et al14 demonstrated that immunoreactive signal for plakoglobin is significantly reduced at intercalated discs of ARVC patients, suggesting that immunohis-

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tochemical analysis of this desmosomal protein may be useful as a diagnostic test for ARVC. To further investigate the effects of the DES tail missense mutation (p.R454W) identified in family B on intercellular junction protein interactions, we analyzed myocardialsamples from the two patients using immunohistochemistry. This revealed that the signal levels of both N-cadherin and plakoglobin at the intercalated disk were comparable to that in control myocardium, while the signal levels of desmoplakin, plakophilin 2, and connexin-43 at junctional sites were markedly reduced in comparison with control myocardium (Figure 4). The presence of plakoglobin in samples from both p.R454W mutation carriers differentiates our patients from those with ARVC described by Asimaki et al.14 In addition to reduced plakoglobin, Asimaki et al also found reduced plakophilin 2 and desmoplakin signals at the intercalated disk in seven of 11 ARVC cases, like the p.R454W DES mutation carriers. As a major component of adherens junctions, whose role is to anchor actin microfilaments, N-cadherin was not anticipated to be affected by a mutation in an intermediate filament protein.22 Plakoglobin is a major component of both adherens junctions and desmosomes.23 At the desmosomal sites, plakoglobin is known to interact with the desmosomal cadherins desmocollin and desmoglein and with desmoplakin.23 It does not, however, directly associate with desmin, which may explain why its distribution might not be affected by a DES mutation. Among its other reported roles, desmoplakin is responsible for binding intermediate filaments and anchoring them at membrane-associated plaques.24 It has been reported that the association between desmoplakin and cytoskeletal elements mediates the correct localization of the plakin protein at the desmosomal complexes.24 Therefore, it is understandable that a mutation that affects the organization of the desmin filament network will affect the distribution of desmoplakin at the junctional sites. In their recent study of desmosomal protein changes in ARVC patients, Asimaki et al14 found decreased signal levels of desmoplakin next to plakoglobin and plakophilin 2 in seven of 11 ARVC patients, of whom four carried mutations in either PKP2 or DSG2. They also found reduced desmoplakin in two DCM patients from a control group. Further studies are warranted to investigate whether this latter finding is a nonspecific finding in idiopathic DCM patients or whether it can be attributed to a mutation related to the desmosomal architecture like the DES mutation in family B. Mutations in the DSP and PKP2 and other ARVC-related genes underlying the reduced signal levels of desmoplakin and plakophilin 2 at the intercalated disk in family B were excluded. In the PKP2 knockout mouse, desmoplakin was virtually absent from all junctions and instead appeared at dense granular aggregates in the cytoplasm. Intermediate filament arrays, normally interspersed between the myofibrils and enriched at intercalated discs, were also displaced in the knockout animals. Extensive swirls of disordered filaments were observed surrounding the desmoplakin aggregates.25

1063 In addition to desmoplakin, plakophilin 2 is also known to play a role in anchoring the cytoskeleton to the cell membrane. Since no mutations that could explain the protein’s redistribution were found when the PKP2 gene was screened, we may hypothesize that the shift of the desmindesmoplakin complexes to the cytoplasm may affect localization of the armadillo protein. Reduced localization of connexin-43 at intercalated disks has previously been observed in heart samples from patients with Naxos disease and Carvajal syndrome, cardiocutaneous syndromes caused by mutations in JUP and DSP, respectively.26,27 Indeed, it has been suggested that when adhesive junctions are disrupted, gap junctions cannot be formed and maintained normally, which may slow electrical conduction, enhance conduction heterogeneity, and predispose patients to developing reentrant arrhythmias, a common characteristic of a number of CMs.26 –28 In a previous study by Gard et al,21 decreased expression and function of connexin-43 was also observed in a mouse model of DRM. Similarly, in the cases we presented here, disruption of the desmosomal-desmin interactions may explain the disrupted distribution of connexin-43 in the cardiac myocytes. Moreover, in a recent study by Fidler et al,29 immunofluorescence showed decreased amounts of connexin-43 at the intercalated disk in ARVC patients who had mutations in one of the other desmosomal proteins, PKP2. Fidler et al29 suggested there was a remodeling of gap junctions in the myocardium of ARVC patients secondary to PKP2 mutations. Asimaki et al14 recently suggested that a reduced signal of connexin-43 can be seen as a marker of severe heart disease by immunohistochemistry testing of myocardial tissue from patients with different types of cardiac disease. Since it results in a decrease of the signal levels of desmoplakin and plakophilin 2 at this structure, the effect of the p.R454W tail mutation on the intercalated disk in our present study is different from the recently described effect of the p.S13F head domain mutation.2,6 Although both mutations have an effect on the architecture of the intercalated disks, the influence on desmosomal proteins seems to be more explicit in our p.R454W tail mutation, which suggests involvement of this part of desmin in mechanical coupling. Additional pathological and functional studies are needed to confirm our clinical observations of reduced desmoplakin and plakophilin 2 regarding the effects on intercalated disk architecture.

Conclusion So far, the occurrences of the cardiac phenotypes of RCM, DCM, and HCM have all been described in DRM. Here we confirm that an ARVC-like phenotype or a severe CM including RV involvement are also possible, although uncommon, cardiac phenotypes in DRM. More importantly, we report the novel finding that the DES mutation p.R454W affects the localization of the desmosomal proteins desmoplakin and plakophilin 2 at the intercalated disk. This suggests that there is a link between desmosomal CMs that

1064 generally affect the RV and CMs due to desmin mutations, in which there is occasional RV involvement.

Acknowledgments The authors thank Jackie Senior for editing the text and Hennie Bikker, molecular geneticist at the Academic Medical Center Amsterdam, the Netherlands, for performing the DSP mutational analysis. We would also like to thank family A for their contribution to this study.

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