Novel Desmin Mutation p.Glu401Asp Impairs Filament Formation, Disrupts Cell Membrane Integrity, and Causes Severe Arrhythmogenic Left Ventricular Cardiomyopathy/Dysplasia

Introduction

Editorial, see p 1611

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited disorder of genetic origin that causes malignant ventricular arrhythmias leading to sudden cardiac death (SCD).^1–4 Inherited ARVC/D (iARVC/D) is one of the most prevalent causes of SCD in young people, especially in athletes.⁵ Left ventricular (LV) involvement is common and appears to imply a poorer prognosis. Mutations in genes encoding for desmosomal proteins (plakophilin-2 [PKP2], desmoplakin [DSP], desmoglein-2 [DSG2], desmocollin-2 [DSC2], and plakoglobin [JUP]) play a key role in the pathogenesis of this disease.^6–10 With the increasing use of the next-generation sequencing techniques in cardiovascular genetics, nondesmosomal and ion channels gene mutations¹¹ have also been associated with iARVC/D. Two prominent examples are the mutations p.S358L in TMEM43 and p.Arg14del in PLN,¹² encoding the transmembrane protein Luma localized in the nuclear membrane^13,14 and a regulator of the sarcoplasmic reticulum Ca²⁺-(SERCA2a) pump in cardiac muscle, respectively.

Some cytoskeletal proteins, such as filamin C¹⁵ or desmin, are closely related to the development of cardiomyopathy. Desmin, encoded by the gene DES, is a structural intermediate filament present in the cytoskeleton of the leiomyocytes, rhabdomyocytes, and cardiomyocytes, and it is associated with different cellular structures, such as desmosomes, Z-bands, costameres, mitochondria, and the nuclei. Its function is related to the maintenance of the structural integrity of the cardiomyocyte.¹⁶ More than 70% of the described pathogenic DES mutations exhibit cardiac involvement and can be associated with any form of cardiomyopathy, the most common being dilated cardiomyopathy, followed by restrictive, hypertrophic, ARVC/D, and their combinations.¹⁷ It usually presents specific conduction system disturbance and, in addition, evidence of skeletal myopathy. The molecular pathogenic mechanisms causing the ARVC/D-related DES mutation phenotype are unknown, and preexisting data in this regard are rare.

In this study, we demonstrate the pathogenicity of the novel DES mutation p.Glu401Asp in a large family with an inherited LV dominant arrhythmogenic cardiomyopathy/dysplasia and a high incidence of adverse clinical events, such as SCD, major heart failure, and ventricular arrhythmia. We also identify the cellular, histological, and molecular mechanisms leading to the appearance of the iARVC/D phenotype.

Methods

The data, analytic methods, and study materials will not be made available to other researchers for purposes of reproducing the results or replicating the procedure.

Study Population and Clinical Variables

The study population included all available members of a large Spanish family of white origin, composed of 83 individuals spread over 6 generations. Sixty-six family members were available for evaluation (51.6% male; mean age, 43.3±19.3 years old). The study protocol was approved by the local ethics committee (University of Granada) on March 2016. All patients included in the study signed an informed consent form.

Clinical Study

All subjects underwent an initial clinical evaluation that included a clinical history with particular emphasis on the presence of adverse events such as death, heart failure death, SCD, atrioventricular block, sustained ventricular arrhythmia, syncope, heart transplantation, or cardiac device implantation. In addition, all subjects underwent a 12-lead ECG, a transthoracic echocardiogram, a creatine phosphokinase blood test, and a neuromuscular examination to investigate involvement of the skeletal muscle. The mutation carriers underwent 24 hours of ambulatory ECG monitoring (Holter) and a cardiac magnetic resonance (CMR) imaging. The CMR imaging was performed in all participants who accepted the procedure and had no contraindications (cardiac device implant and claustrophobia). The ECG data were analyzed to obtain the PR, QRS, and QT intervals in milliseconds, and data on voltage, conduction disturbance, and the presence of pathological Q waves. In the 24-hour ambulatory ECG, we analyzed the presence of nonsustained ventricular tachycardia, atrioventricular block, and ventricular ectopy morphology and density. The evaluation of LV diameter and function was performed according to current guidelines,¹⁸ including LV end-diastolic and end-systolic diameter and volume, LV ejection fraction using the Simpson biplane method, right ventricular outflow and inflow tract diameter, and right ventricular systolic function, as well. Diagnosis of the index patient was based on modified Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia Task Force criteria.¹⁹ All clinical tests were interpreted by cardiologists specialized in advanced cardiac imaging and electrophysiology. Data collection was limited by the lack of both electro- and echocardiographic information on deceased and precardiac transplant subjects in the corresponding cases. Furthermore, the statistical analysis did not include clinical data or complementary tests of children <13 years of age.

Genetic Analysis

After obtaining the peripheral blood of the index patient (IV.31 on Figure 1), the genetic analysis was performed by using a next-generation sequencing gene panel containing 149 genes (Figure I in the online-only Data Supplement). This study included an analysis of all coding exons and intron flanking regions of a personalized panel of genes, which had been previously associated with or are regarded as candidates for the development of hereditary cardiovascular diseases. Sample preparation was performed using the Agilent SureSelect Target Enrichment Kit based on the paired-end multiplexed sequencing method according to the manufacturer’s instructions. Low-coverage regions of the relevant genes were resequenced using the Sanger method. Bioinformatic analysis was performed through an in-house–developed pipeline that included software such as NovoAlign, SAMtools, and BCFtools. Information such as frequency in different populations (from sources including exome variant server, 1K genomes, and the Single Nucleotide Polymorphism Database), and various bioinformatic predictions, as well, was added. Pathogenicity of the identified variants was classified according to current guidelines of the American College of Medical Genetics and Genomics.²⁰ Following mutation DES-p.Glu401Asp identification in the index patient, genetic and clinical cascade screening was performed on all available family members.

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Histological Analysis

Cardiac tissue from patient III.21 (Figure 1) carrying DES-p.Glu401Asp and subjected to heart transplantation was used for histological analysis. In addition, normal healthy cardiac tissue obtained from a patient without the mutation was used as control. The tissues were fixed for 24 hours in 10% neutral buffered formaldehyde, washed, dehydrated in ascending concentrations of ethanol, cleared in xylene, and embedded in paraffin using a conventional protocol.²¹ Five-micrometer histological sections were dewaxed and stained with hematoxylin-eosin for morphological evaluation. Stromal collagen fibers were histochemically evaluated by picrosirius staining as described elsewhere.²² To evaluate myofibril organization in the cardiomyocytes, tissue sections were histochemically stained according to the Heidenhain iron hematoxylin method as previously described.²³ The histomorphological pattern of the desmin intermediate filament was evaluated by indirect immunohistochemistry. Desmosomes and intercellular gap junctions localized at the intercalated discs were identified by immunohistochemical staining of desmoplakin, plakoglobin, and connexin-43, respectively. Immunohistochemistry was prepared as previously described^22–24 using positive and negative technical controls. All procedures were performed simultaneously under the same conditions to ensure reproducibility of the results. Technical information about the used antibodies is summarized in Table I in the online-only Data Supplement.

Stem Cell Culture and Cardiomyocyte Differentiation

To evaluate the impact of DES mutation on cell function, adipose-derived mesenchymal stem cells (ADMSCs) were isolated from subcutaneous adipose biopsies obtained from 3 members of the family evaluated in this study: a healthy non-DES mutation carrier (healthy control) and 2 mutation carriers, 1 with mild symptomatology and 1 with a severe clinical condition, were included. The biopsies were mechanically fragmented into small pieces and then digested for 8 hours in a 2 mg/mL solution of Clostridium histolyticum type I collagenase (Gibco BRL Life Technologies) as previously described.²¹ The ADMSCs were harvested by centrifugation and cultured in a basal medium consisting of DMEM supplemented with 10% FBS and antibiotics-antimycotics (Sigma-Aldrich). After obtaining a subconfluent cell culture, the ADMSCs were differentiated ex vivo into the cardiomyocyte lineage, using the Gibco Cardiomyocyte Differentiation Kit (A29212-01, Thermo Fisher Scientific), for 14 days according to the manufacturer’s instructions. The growth pattern and differentiation process of these cells were continuously monitored by phase-contrast microscopy. The human cardiomyocyte phenotype was confirmed using a commercially available Human Cardiomyocyte Immunocytochemistry Kit (A25973, Thermo Fisher Scientific).

Real-Time Quantitative Polymerase Chain Reaction Analyses

Total RNA was extracted from each cell type (differentiated and nondifferentiated cell cultures from each of the 3 family members studied) using a Qiagen RNeasy Mini Kit system (Qiagen). Then, cDNA was synthetized from total RNA with an iScript Advanced cDNA Synthesis Kit (Bio-Rad Laboratories) using 1 µg of each RNA. Expression quantification of a set of genes with important roles in cardiac differentiation and function was analyzed using real-time quantitative polymerase chain reaction (PCR). Thus, a 96-well plate containing oligonucleotides for the genes and controls selected was designed as a PrimePCR custom assay. The selection of these genes was based on 2 previously described gene expression panels for cardiac diseases: Cardiac Arrhythmia Next-Generation Sequencing Multi-Gene Panel and Bio-Rad Arrhythmias Cardiac H96 Panel. For each PCR reaction, 2 µL of cDNA, 8 µL of H₂O, and 10 µL of SsoAdvanced Universal SYBR Green Supermix were added, and a PCR protocol consisting of 40 amplification cycles with an annealing temperature of 60°C was applied using a Bio-Rad CFX Connect-96 instrument. Results were corrected according to the efficiency of the reverse transcription reaction and normalized to the GAPDH housekeeping gene expression using Bio-Rad CFX Manager 3.1 software. Each sample was analyzed in triplicate (n=18). Relative gene expression was calculated for each gene using the ΔΔCq value (CFX Manager 3.1 software, Bio-Rad) by comparing each differentiated cell culture with its nondifferentiated control samples. By using this approach, we obtained the fold-change expression increase or decrease of every gene in the 3 sample types analyzed as average differentiated cell expression divided by average nondifferentiated cell expression. The fold-changes obtained for the unmutated control cells were considered as the basal levels of normal cells for each gene, whereas the fold-changes obtained for both samples were compared with basal levels. Genes showing an increase or decrease of at least 2-fold, in comparison with basal levels, were selected.

Plasmids, Cell Culture, and Confocal Microscopy

The expression plasmid pmRuby-N1-Desmin has been previously described.²⁵ This construct encodes a C-terminal fusion protein of human desmin and the fluorescence protein mRuby that was used for direct fluorescence microscopy. The mutations p.Glu401Asp and p.Glu401Lys were introduced using the QuickChange Lightning Site-Directed Mutagenesis Kit (210518, Agilent Technologies). The mutation insertions were verified by Sanger sequencing (Macrogen). SW13 cells were grown with ≈70% confluency on cover slips in DMEM (Gibco; 4.5 g/L glucose, 10% FBS, supplemented with penicillin/streptomycin). These cells were used because they do not express endogenous desmin or any other cytoplasmic intermediate filament protein. The cells were transiently transfected with Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Twenty-four hours after transfection, the cells were washed several times for 5 minutes with PBS and fixed for 5 minutes with Roti Histofix (Carl Roth). They were then extensively washed in PBS and permeabilized with Triton X-100 (0.1% in PBS). F-actin was stained with fluorescein isothiocyanate–conjugated phalloidin (Sigma-Aldrich) 50 µg/mL for 40 minutes at room temperature according to the manufacturer’s instructions. The cells were embedded in Mowiol 4 to 88 (Carl Roth). Confocal laser scanning microscopy was performed with the TCS SP8 system (Leica Microsystems) equipped with a HC PL API C52 (63×/1.30) glycerin objective, hybrid detectors, and Application Suite X software. The fluorescence molecules mRuby and fluorescein isothiocyanate were sequentially excited. Each cell culture experiment was performed in triplicate.

Statistical Analysis

Statistical calculations were performed using SPSS Statistics (version 20, IBM Corp). Clinical characteristics were compared using χ² or Fisher exact test for categorical variables and Student t test or Mann-Whitney U test for continuous variables. All categorical data were reported as frequencies and percentages, and continuous variables were expressed as mean value±SD for each measurement. A P value of <0.05 was considered to be statistically significant. Survival analysis was estimated by using the Kaplan-Meier method.

Results

Genetic Analysis

The genetic analysis of the index patient (IV:31, Figure 1) revealed the heterozygous missense mutation in DES (NM_001927.3:c.1203G>C; NP_01918.3:p.Glu401Asp or p.E401D). This variant had not been previously described in any publication or in public databases, such as the Human Gene Mutation Database,²⁶ the Single Nucleotide Polymorphism Database,²⁷ NHLBI GO Exome Sequencing Project,²⁸ and ClinVar,²⁹ or in the Exome Aggregation Consortium (ExAC) database³⁰ (as of 31/03/2017). Amino acid residue E401 belongs to the fourth α-helix (coil 2B, amino acids 296–412) of the α-helical central rod domain (109–412), a region that is essential for desmin filament assembly. The in silico prediction indicated that p.Glu401Asp substitution NC_000002.11:g220286241G>C affects a highly conserved amino acid residue among different species (Figure II in the online-only Data Supplement).

Clinical Analysis

The index patient, a 46-year-old man (IV:31, Figure 1), was referred for examination because of a family history of SCD of 2 brothers aged 31 (IV:29) and 18 (IV:26). Postmortem examination of one of these brothers (IV.29) revealed epicardial and midwall fibrosis from the inferior LV wall to the septum and lateral wall. The right ventricle looked macroscopically normal, and there was no evidence of coronary artery disease. Histological examination showed focal areas of subendocardial fibrosis in the free wall of the right ventricle. In the left ventricle, subepicardial and midwall fat and fibrous tissue were confirmed, presenting widespread spaces admixed with degenerative myocytes. Myocardial inflammation was absent. In addition, there were no pathological findings suggestive of skeletal myopathy such a myofiber necrosis or myofiber atrophy. In summary, the postmortem study strongly suggested dominant inherited arrhythmogenic left ventricular cardiomyopathy/dysplasia.³¹ The patient presented New York Heart Association functional class II symptoms with generalized T-wave inversion in all precordial leads and biventricular dilation and severely depressed biventricular ejection fraction, as well. These data were confirmed by CMR, which also showed late gadolinium enhancement at the subepicardial LV level. The 24-hour Holter study showed >500 of premature ventricular complexes with both left and right bundle-branch block morphologies. Finally, he was diagnosed to have inherited arrhythmogenic cardiomyopathy/dysplasia with biventricular involvement and received a cardiac defibrillator implantation for primary prevention of SCD.

In total, 66 family members were genetically sequenced for DES-p.Glu401Asp, of whom 23 presented the mutation; an additional 6 subjects were obligate carriers (deceased before sequencing), and another 2 nongenotyped cases, with, however, a previous SCD event, were considered probable carriers, corresponding to a total of 31 affected family members. Cosegregation analysis was performed to correlate genotype and phenotype, clearly confirming the pathogenic impact of this specific DES mutation. Evidence of primary myocardial disease was present in 100% of carriers, with variable clinical expression, whereas noncarriers showed no traces of nonischemic cardiomyopathy (Table II in the online-only Data Supplement). One noncarrier patient presented LV systolic dysfunction and atrioventricular block of ischemic origin.

Of the 23 carriers studied, 9 fulfilled the modified diagnostic criteria. Main clinical and genetic data of DES mutation carriers are summarized in the Table. The most common ECG finding was T-wave inversion in at least 3 consecutive ECG leads (14 cases) and generalized low voltage (12 cases). The ECG was abnormal in 97% of the carriers for whom it was available, mostly because of the presence of negative T waves in inferior (II, III, aVF) and anterolateral leads (V₂ through V₆) (Figure 2A). No change was detected in the PR interval, which was similar in both groups, although a wider QRS was identified in the carrier group (Table II in the online-only Data Supplement). Two of the 3 affected children (7, 11, and 13 years old) presented negative T wave beyond V₃. One of them (VI.1) showed low voltages in limb leads in the ECG and subepicardial late gadolinium enhacement in inferior wall in the CMR. The 24-hour Holter study revealed nonsustained ventricular tachycardias in 32% of carriers and high-density ventricular ectopy in 23% of them. There was only 1 case of atrioventricular block in an 84-year-old man. We found no evidence of skeletal myopathy, and creatine phosphokinase determination was normal in all mutation carriers.

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The most characteristic finding of the cardiac structure and function studied by echocardiography and CMR was near-exclusive LV affection, with hypokinesia localized on the midapical inferolateral wall of the LV, mildly depressed LV ejection fraction (46±8.2%), and no ventricular dilation (51.52±5.87 mm, normal in terms of body surface in most cases). Right ventricular involvement was demonstrated only in 5 cases that presented concomitant moderate/severe LV ejection fraction impairment, suggesting a primary involvement of the LV and subsequent right ventricular affection. Furthermore, CMR revealed an extensive late gadolinium enhancement with a typical circumferential subepicardial pattern in most of the cases, and midapical lateral and inferolateral subepicardial distribution in the remaining patients (Figure 2B).

During familial screening, 15 of affected individuals were asymptomatic at diagnosis, 4 presented with palpitations, 3 with heart failure symptoms, and 1 with syncope. None showed myocarditis-like symptoms. It is noteworthy that patients with heart failure showed severe LV ejection fraction impairment at presentation (2 of them had a previous diagnosis of idiopathic dilated cardiomyopathy). A high incidence of cardiac events, mainly arrhythmic-related, was observed among carriers retrospectively and during follow-up (Table II in the online-only Data Supplementand Figure 3): 4 SCD (18, 31, 33, and 64 years old), 2 heart failure deaths (both at 41 years old), 1 atrioventricular block (84 years old), 2 heart transplantations (39 and 60 years old), 1 sustained monomorphic ventricular tachycardia (37 years old), and 5 implantable cardioverter defibrillators for primary prevention of SCD with 2 episodes of appropriate therapies on follow-up (21 and 47 years old). These findings support a high risk for malignant arrhythmias and SCD as first clinical manifestation.

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Histological Analysis of Cardiac Tissue Affected by Desmin Mutation

Histological analysis of the heart from a desmin mutation carrier (III:21) showed major differences in comparison with the control heart. Picrosirius staining in the control tissue revealed that all cardiomyocytes showed their typical branched cylindrical shape, a single and centrally located nucleus, a highly dense and strongly stained sarcoplasm, and several intercalated discs between adjacent cells (Figure 4A). With regard to cardiac tissue affected by desmin mutation p.Glu401Asp, it was possible to recognize zones affected by typical degenerative changes and others with a more conserved myocardium. Degenerative findings were found associated with the subepicardial myocardium, where picrosirius staining revealed an increase of disorganized collagen fibers, the presence of infiltrating adipose tissue, and groups of cardiomyocytes with degenerative changes. However, these findings were not accompanied by an inflammatory reaction (Figure 4B). In the myocardial zones without degenerative changes, the tissue organization was preserved and comparable to the control. However, cell thickness was irregular, and an increased perinuclear space, accompanied by a relatively normal nucleus, was observed. Curiously, the sarcoplasm of these cells was weakly stained, less dense with less prominent intercalar discs than the control tissue (Figure 4). Heidenhain hematoxylin staining, which specifically stains myofibrils, confirmed the presence of fewer contractile myofibrils in the desmin mutation carrier’s myocardium in comparison with the control. Myofibrils were less abundant, poorly organized, and less compacted, with spaces observed among them. Immunohistochemical analysis of desmin showed an intense and homogeneously distributed staining pattern in the sarcoplasm of both cardiac tissues, although it was more intense in the control intercalated discs. In addition, we did not observe any prominent desmin aggregates in the sarcoplasm of the cardiac tissue from this mutation carrier. After analyzing some important structural and functional proteins of cell–cell junctional complexes at the intercalated discs, desmoplakin, plakoglobin, and connexin-43 were observed to be positive in both cardiac tissues, thus confirming the presence of these junctional complexes. However, desmoplakin and plakoglobin were weakly stained in the myocardial tissue from the mutation carrier in comparison with the control. The connexin-43 intensity and pattern were comparable in control and affected tissue, as previously described with regard to another DES mutation, p.A120D³² (Figure 4C).

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ADMSC Differentiation and Gene Expression Analysis

Cell cultures established from different family members showed that all cell types were able to grow and proliferate ex vivo under standard culture conditions. Control cells isolated from the control family members without the DES mutation (healthy control) showed a typical mesenchymal morphology (elongated, spindle-shaped, flattened cells). As expected, confluent cells managed to fully cover the culture flask surface, which is typical of mesenchymal stem cells (Figure 5). In a similar manner, cells isolated from patients with the DES mutation showing mild symptomatology showed similar cell morphology and growth pattern to controls, although they tended to grow and proliferate more slowly. It is curious that cells from the affected DES mutation carrier patient (IV.31) grew much more slowly than the mild phenotype cell line and especially than the healthy control cell line. In addition, these cells presented a typical mesenchymal morphology, although their growth pattern was very different. In fact, they had difficulty adhering to the culture surface, resulting in a reticular pattern, with cells tending to adhere to one another rather than to the culture surface (Figure 5).

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When ADMSCs were differentiated into cardiomyocytes, some cells could be observed to acquire a typical cardiomyocyte morphology, although the percentage of cells showing this phenotype was low. Differentiated cells were characterized by a large and polygonal cytoplasm with a thick, well-organized cytoskeleton (see representative images in Figure IIIA in the online-only Data Supplement). Differentiation was confirmed by immunofluorescence for the TNNT2 cardiac marker, which demonstrated that most cells were positive. Quantitative analysis using real-time quantitative PCR (Figure 6 and Table III in the online-only Data Supplement) showed that desmin gene expression increased more than 23-fold in differentiated cells. However, cells carrying the DES-p.Glu401Asp did not show increased DES expression after cardiomyogenic differentiation. Analysis of the genes selected in this study showed that 9 genes were overexpressed at least 2-fold in cells from probands with mild symptomatology in comparison with healthy control samples, and 40 genes were downregulated at least 2-fold in the same samples. In turn, samples from the proband with advanced cardiomyopathy showed 11 upregulated and 37 downregulated genes in comparison with healthy controls. It is interesting to note that 4 of these genes were overexpressed and 24 downregulated in both the mild-phenotype and severe-phenotype carrier samples in comparison with healthy control samples.

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Plasmids and Cell Culture

Given the effect of DES mutations on filament formation in vitro,³³ we attempted to determine whether this is also the case for the novel mutation DES-p.Glu401Asp. We used SW13 cells without any endogenous cytoplasmic intermediate filament protein expression.³⁴ These experiments showed that both mutations p.Glu401Asp and p.Glu401Lys, which have been previously described,³⁵ disrupted the intermediate filament structure in the transiently transfected cells. Both mutant desmins formed cytoplasmic aggregates, whereas wild-type desmins formed regular filaments (Figure 7).

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Discussion

The genes most commonly responsible for classic iARVC/D are those that encode for cardiac desmosomal proteins, being LV involvement and SCD commonly reported because of DSP variants.³⁶ Nevertheless, with the development of the advanced imaging techniques available today, we know that biventricular impairment is common, with an even worse prognosis. Also, with the advent of massive parallel sequencing techniques, nondesmosomal genes, such as TMEM43,¹³TGFB3,³⁷PLN,³⁸ and DES,³⁹ whose mutations generate an iARVC/D phenotype, have been discovered. However, DES mutations more frequently produce a dilated or restrictive cardiomyopathy phenotype, atrioventricular conduction disorder, and skeletal myopathy.³⁹ This study provides strong evidence on the pathogenicity of this novel DES mutation as a cause of biventricular inherited arrhythmogenic cardiomyopathy/dysplasia with dominant primary LV affection, without conduction system abnormality or traces of skeletal muscular involvement. To our knowledge, this study involves the largest family of a single DES mutation and provides accurate genotype-phenotype cosegregation data. It is based on an interesting functional analysis that suggests the presence of a pathogenic mechanism by which desminopathy is capable of producing an iARVC/D phenotype. In addition, this mutation was not present in healthy controls and does not appear in public databases such as ClinVar or the Exome Aggregated Consortium database. Our results allow us to classify DES p.Glu401Asp as pathogenic according to American College of Medical Genetics and Genomics guidelines on the interpretation of genetic variants.²⁰

DES mutations as a cause of iARVC/D were first described in 2009 as typically affecting the right ventricle or causing severe biventricular dysplasia.^40,41 Later, isolated cases of single mutations causing iARVC/D, which involved limited or even with no familial study, were presented.^42–44 Information on the pathogenic mechanisms of DES mutations to produce the inherited arrhythmogenic cardiomyopathy/dysplasia phenotype is rare. Likewise, the reasons for their cardiac and skeletal expression variability are poorly understood. Given the ability of desmin to form a network between the cytoskeleton and desmosomes, the abnormal interaction between both cellular multiprotein complexes may lead to cellular adhesion defects. The desmin molecule contains 3 domains: a central α-helical rod domain flanked by 2 globular N- and C-terminal domains. The p.Glu401Asp mutation is located in segment 2B of the central rod domain, a highly conserved region in vertebrates, where most DES mutations were found previously.⁴⁵ Mutations at this position could eventually produce a critical break in the intra- and interhelical ionic bridges between desmin dimers, as already reported with regard to the mutation p.Glu401Lys.³⁵ The presence of this mutation could potentially lead to a loss of structural integrity and, consequently, of cellular adhesion, which is considered a crucial factor in the pathogenesis of iARVC/D. Our results corroborate the hypothesis of a disruption in the cellular membrane structural integrity of cells affected by mutation p.Glu401Asp. It is interesting to note that the phenotype exhibited by mutation p.Glu401Asp differs considerably from that associated with mutation p.Glu401Lys, which was described in a 20-year-old man with advanced atrioventricular block and skeletal myopathy.³⁵ This suggests that aspartic acid in position 401 is likely involved in the specific development of the ARVC/D phenotype with no conduction or skeletal muscle system involvement.

The histological study of the explanted heart of subject III.21 reveals affected (fibrofatty tissue replacement) and more preserved myocardial zones. It is interesting to note that, in the most preserved areas, cardiomyocytes showed an abnormal organization of their contractile apparatus, which was less abundant and poorly organized than in the control myocardium. Furthermore, the immunohistochemical study showed lower expression of desmoplakin and plakoglobin in cell junctions and did not detect any differences in connexin 43, as described elsewhere.⁴⁶ The interaction between the rod region and tail of desmin, which contain recognition binding sites implicated in the interaction with desmoplakin at the desmosome, has been previously described.⁴⁷ This means that the position of the mutation and the reduced presence of desmoplakin in the heart tissue are in line with previously published data and could be related to the increased risk of arrhythmic complications observed and to the iARVC/D phenotype. Regarding the slight decrease of plakoglobin in the intercalated disc, it could be supported by previous studies, where a decrease of plakoglobin immunoreaction is considered a consistent feature in patients with iARVC/D.⁴⁸ Surprisingly, in our study, we did not observe desmin cytoplasmic aggregates in the cardiac tissue of heterozygous carrier III.21, which appeared in transfected cells in vitro. It is well known that most of the desmin mutations are characterized by desmin-positive sarcoplasmic aggregates.^17,49 However, it was demonstrated that desmin mutations (for example, the c.1289-2A>G mutation) can manifest without the formation of these aggregates.^17,50 In our case, this absence could be related to the patient’s heterozygous condition. Furthermore, we used a strong cytomegalovirus promoter in vitro, which may provoke misfolding of the mutant desmin in cell cultures. In addition, the patient’s disease stage could be characterized by Z-disc disorganization and disruption of the intermediate filament network, but, with no desmin aggregates, may also explain the discrepancy of in vivo and cell culture findings of this study.⁴⁷ Finally, it is probable that this novel DES p.Glu401Asp mutation does not result in the formation of desmin aggregates, but its pathogenic mechanism could be related to an impairment of the adequate myofibril formation and 3-dimensional organization in the sarcoplasm. However, future studies are needed to elucidate the pathogenic mechanism related to this novel desmin mutation.

Analysis of ADMSC cultures revealed early differences between control, mild, and severe phenotypes, with striking cellular growth and adhesion defects in the patient with the severe phenotype. Following its differentiation into cardiomyocytes, RNA analysis showed that desmin expression was lower in the severe phenotype carrier. In addition, significant differences were detected in the RNA expression of different genes associated with cardiomyopathies or channelopathies. It is noteworthy that the lower RNA expression levels of a wide variety of potassium channels in both carrier groups, and the higher expression of SCNIB, a sodium channel with great arrhythmic potential, as well, were found. We also detected differences in the RNA expression of certain proteins between the mild and severe phenotype carriers (Figure 6 and Table III in the online-only Data Supplement). This could explain the variable clinical penetrance of iARVC/D observed in this family. These findings must be interpreted with caution, because their influence in vivo on the development of the disease remains unknown. In addition, RNA levels do not necessarily correspond to those of functional proteins in the myocardium. Incomplete clinical penetrance observed in this family is a common feature of iARVC/D. Although genetic, epigenetic, and environmental factors have been proposed, the origin of the incomplete penetrance observed in this family mentioned above is still unclear. Recent data suggest that a specific genetic background can lead to an increased susceptibility to myocarditis as part of an active phase of the disease. In our study, we did not find any myocarditis-like presentation, or suggestive data from histological analysis. However, we cannot definitely discard this possibility in this case. Future investigations are needed to address this issue.⁵¹ It has even been suggested that sex could play a role, because the disease is usually more severe in males.⁵² This was also found in the case of the family described in our study; the 4 SCD events and the 2 cardiac transplants occurred in men and, as Figure 3 shows, at an earlier age in males than in females, something described not only for p.Glu401Asp but also for other DES mutations.^53,54 According to our data, cardiac events in p.Glu401Asp carriers are particularly prevalent in men from 20 to 40 years, especially SCD, and life-saving therapies such as cardiac defibrillators should be promptly indicated in these cases.

In conclusion, DES mutation p.Glu401Asp causes iARVC/D with predominant LV involvement in the absence of skeletal myopathy and conduction system disturbance, with a high incidence of adverse clinical events. The pathogenic mechanism likely corresponds to alterations in desmin dimer assembly and its connection with membrane proteins that disrupt the membrane and cell–cell adhesion integrity.

Acknowledgments

The authors wish to thank Drs Rosa Ortega and Salvador Arias-Santiago for collecting tissue from the explanted heart and the adipose biopsies, respectively, for research purposes, and Laura Cortiñas for her assistance in image collecting. These results are from Dr Bermudez-Jiménez’s PhD program.

Footnotes