Hemi- and Homozygous Loss-of-Function Mutations in DSG2 (Desmoglein-2) Cause Recessive Arrhythmogenic Cardiomyopathy with an Early Onset.

About 50% of patients with arrhythmogenic cardiomyopathy (ACM) carry a pathogenic or likely pathogenic mutation in the desmosomal genes. However, there is a significant number of patients without positive familial anamnesis. Therefore, the molecular reasons for ACM in these patients are frequently unknown and a genetic contribution might be underestimated. Here, we used a next-generation sequencing (NGS) approach and in addition single nucleotide polymor-phism (SNP) arrays for the genetic analysis of two independent index patients without familial medical history. Of note, this genetic strategy revealed a homozygous splice site mutation (DSG2-c.378+1G>T) in the first patient and a nonsense mutation (DSG2-p.L772X) in combination with a large deletion in DSG2 in the second one. In conclusion, a recessive inheritance pattern is likely for both cases, which might contribute to the hidden medical history in both families. This is the first report about these novel loss-of-function mutations in DSG2 that have not been previously identi-fied. Therefore, we suggest performing deep genetic analyses using NGS in combination with SNP arrays also for ACM index patients without obvious familial medical history. In the future, this finding might has relevance for the genetic counseling of similar cases.

A homozygous DSC2 deletion associated with arrhythmogenic cardiomyopathy is caused by uniparental isodisomy.

AIMS: We aimed to unravel the genetic, molecular and cellular pathomechanisms of DSC2 truncation variants leading to arrhythmogenic cardiomyopathy (ACM). METHODS AND RESULTS: We report a homozygous 4-bp DSC2 deletion variant c.1913_1916delAGAA, p.Q638LfsX647hom causing a frameshift carried by an ACM patient. Whole exome sequencing and comparative genomic hybridization analysis support a loss of heterozygosity in a large segment of chromosome 18 indicating segmental interstitial uniparental isodisomy (UPD). Ultrastructural analysis of the explanted myocardium from a mutation carrier using transmission electron microscopy revealed a partially widening of the intercalated disc. Using qRT-PCR we demonstrated that DSC2 mRNA expression was substantially decreased in the explanted myocardial tissue of the homozygous carrier compared to controls. Western blot analysis revealed absence of both full-length desmocollin-2 isoforms. Only a weak expression of the truncated form of desmocollin-2 was detectable. Immunohistochemistry showed that the truncated form of desmocollin-2 did not localize at the intercalated discs. In vitro, transfection experiments using induced pluripotent stem cell derived cardiomyocytes and HT-1080 cells demonstrated an obvious absence of the mutant truncated desmocollin-2 at the plasma membrane. Immunoprecipitation in combination with fluorescence measurements and Western blot analyses revealed an abnormal secretion of the truncated desmocollin-2. CONCLUSION: In summary, we unraveled segmental UPD as the likely genetic reason for a small homozygous DSC2 deletion. We conclude that a combination of nonsense mediated mRNA decay and extracellular secretion is involved in DSC2 related ACM.

The novel αB-crystallin (CRYAB) mutation p.D109G causes restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare heart disease characterized by diastolic dysfunction and atrial enlargement. The genetic etiology of RCM is not completely known. We identified by a next-generation sequencing panel the novel CRYAB missense mutation c.326A>G, p.D109G in a small family with RCM in combination with skeletal myopathy with an early onset of the disease. CRYAB encodes αB-crystallin, a member of the small heat shock protein family, which is highly expressed in cardiac and skeletal muscle. In addition to in silico prediction analysis, our structural analysis of explanted myocardial tissue of a mutation carrier as well as in vitro cell transfection experiments revealed abnormal protein aggregation of mutant αB-crystallin and desmin, supporting the deleterious effect of this novel mutation. In conclusion, CRYAB appears to be a novel RCM gene, which might have relevance for the molecular diagnosis and the genetic counseling of further affected families in the future.

Molecular autopsy of sudden unexplained deaths reveals genetic predispositions for cardiac diseases among young forensic cases.

AIMS: Coronary artery disease accounts for the majority of sudden cardiac deaths (SCD) in the older population whereas cardiomyopathies and arrhythmogenic abnormalities predominate in younger SCD victims (<35 years) with a significant genetic component. The elucidation of the pathogenetic cause of death might be relevant for the prevention of further deaths within affected families. Aim of this study was to determine the portion of underlying genetic heart diseases among unexplained putative SCD cases from a large German forensic department. METHODS AND RESULTS: We included 10 forensic cases of sudden unexplained death (SUD) victims aged 19-40 years, who died by SCD due to forensic autopsy. DNA was analysed by next generation panel sequencing of 174 candidate genes for channelopathies and cardiomyopathies. Cardiological examinations, genetic counselling, and subsequent genetic testing were offered to all affected families. We identified within 1 year 10 cases of SUD among 172 forensic cases. Evidence for a genetic disposition was found in 8 of 10 (80%) cases, with pathogenic mutations in 3 and variants of uncertain significance in 5 of SCD cases. Subsequent selective screening of family members revealed two additional mutation carriers. CONCLUSION: The study provides strong evidence that molecular genetics improves the post mortem diagnosis of fatal genetic heart diseases among SUD victims. Molecular genetics should be integrated in forensic and pathological routine practice.

Functional characterization of the novel DES mutation p.L136P associated with dilated cardiomyopathy reveals a dominant filament assembly defect.

BACKGROUND: Dilated cardiomyopathy (DCM) could be caused by mutations in more than 40 different genes. However, the pathogenic impact of specific mutations is in most cases unknown complicating the genetic counseling of affected families. Therefore, functional studies could contribute to distinguish pathogenic mutations and benign variants. Here, we present a novel heterozygous DES missense variant (c.407C>T; p.L136P) identified by next generation sequencing in a DCM patient. DES encodes the cardiac intermediate filament protein desmin, which has important functions in mechanical stabilization and linkage of the cell structures in cardiomyocytes. METHODS AND RESULTS: Cell transfection experiments and assembly assays of recombinant desmin in combination with atomic force microscopy were used to investigate the impact of this novel DES variant on filament formation. Desmin-p.L136P forms cytoplasmic aggregates indicating a severe intrinsic filament assembly defect of this mutant. Co-transfection experiments of wild-type and mutant desmin conjugated to different fluorescence proteins revealed a dominant affect of this mutant on filament assembly. These experiments were complemented by apertureless scanning near-field optical microscopy. CONCLUSION: In vitro analysis demonstrated that desmin-p.L136P is unable to form regular filaments and accumulate instead within the cytoplasm. Therefore, we classified DES-p.L136P as a likely pathogenic mutation. In conclusion, the functional characterization of DES-p.L136P might have relevance for the genetic counseling of affected families with similar DES mutations and could contribute to distinguish pathogenic mutations from benign rare variants.

The TMEM43 Newfoundland mutation p.S358L causing ARVC-5 was imported from Europe and increases the stiffness of the cell nucleus.

AIMS: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetic condition caused predominantly by mutations within desmosomal genes. The mutation leading to ARVC-5 was recently identified on the island of Newfoundland and caused by the fully penetrant missense mutation p.S358L in TMEM43. Although TMEM43-p.S358L mutation carriers were also found in the USA, Germany, and Denmark, the genetic relationship between North American and European patients and the disease mechanism of this mutation remained to be clarified. METHODS AND RESULTS: We screened 22 unrelated ARVC patients without mutations in desmosomal genes and identified the TMEM43-p.S358L mutation in a German ARVC family. We excluded TMEM43-p.S358L in 22 unrelated patients with dilated cardiomyopathy. The German family shares a common haplotype with those from Newfoundland, USA, and Denmark, suggesting that the mutation originated from a common founder. Examination of 40 control chromosomes revealed an estimated age of 1300-1500 years for the mutation, which proves the European origin of the Newfoundland mutation. Skin fibroblasts from a female and two male mutation carriers were analysed in cell culture using atomic force microscopy and revealed that the cell nuclei exhibit an increased stiffness compared with TMEM43 wild-type controls. CONCLUSION: The German family is not affected by a de novo TMEM43 mutation. It is therefore expected that an unknown number of European families may be affected by the TMEM43-p.S358L founder mutation. Due to its deleterious clinical phenotype, this mutation should be checked in any case of ARVC-related genotyping. It appears that the increased stiffness of the cell nucleus might be related to the massive loss of cardiomyocytes, which is typically found in ventricles of ARVC hearts.

Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants.

Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.

The Desmin Mutation DES-c.735G>C Causes Severe Restrictive Cardiomyopathy by Inducing In-Frame Skipping of Exon-3.

Currently, little is known about the genetic background of restrictive cardiomyopathy (RCM). Herein, we screened an index patient with RCM in combination with atrial fibrillation using a next generation sequencing (NGS) approach and identified the heterozygous mutation DES-c.735G>C. As DES-c.735G>C affects the last base pair of exon-3, it is unknown whether putative missense or splice site mutations are caused. Therefore, we applied nanopore amplicon sequencing revealing the expression of a transcript without exon-3 in the explanted myocardial tissue of the index patient. Western blot analysis verified this finding at the protein level. In addition, we performed cell culture experiments revealing an abnormal cytoplasmic aggregation of the truncated desmin form (p.D214-E245del) but not of the missense variant (p.E245D). In conclusion, we show that DES-c.735G>C causes a splicing defect leading to exon-3 skipping of the DES gene. DES-c.735G>C can be classified as a pathogenic mutation associated with RCM and atrial fibrillation. In the future, this finding might have relevance for the genetic understanding of similar cases.

A novel desmin (DES) indel mutation causes severe atypical cardiomyopathy in combination with atrioventricular block and skeletal myopathy.

BACKGROUND: DES mutations cause different cardiac and skeletal myopathies. Most of them are missense mutations. METHODS: Using a next-generation sequencing cardiac 174 gene panel, we identified a novel heterozygous in-frame indel mutation (DES-c.493_520del28insGCGT, p.Q165_A174delinsAS) in a Caucasian patient with cardiomyopathy in combination with atrioventricular block and skeletal myopathy. This indel mutation is located in the coding region of the first exon. Family anamnesis revealed a history of sudden cardiac death. We performed cell transfection experiments and in vitro assembly experiments to prove the pathogenicity of this novel DES indel mutation. RESULTS: These experiments revealed a severe filament formation defect of mutant desmin supporting the pathogenicity. In addition, we labeled a skeletal muscle biopsy from the mutation carrier revealing cytoplasmic desmin positive protein aggregates. In summary, we identified and functionally characterized a pathogenic DES indel mutation causing cardiac and skeletal myopathy. CONCLUSION: Our study has relevance for the clinical and genetic interpretation of further DES indel mutations causing cardiac or skeletal myopathies and might be helpful for risk stratification.

Composite polymorphisms in the ryanodine receptor 2 gene associated with arrhythmogenic right ventricular cardiomyopathy.

OBJECTIVE: Mutations in the cardiac ryanodine receptor (RYR2) gene have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC). The molecular mechanisms by which genetic modifications lead to ARVC are still not well understood. METHODS: ARVC patients were screened for mutations in the RYR2 gene by denaturing HPLC and DNA sequencing. Single channel measurements were carried out with RyR2 channels purified from explanted hearts of ARVC patients. RESULTS: None of the published RYR2 mutations were found in our ARVC-cohort. However, we identified two single nucleotide polymorphisms (SNPs) in exon 37 of the human RYR2 gene which lead to the amino acid exchanges G1885E and G1886S, respectively. Both SNPs together were found exclusively in 3 out of 85 ARVC patients in a composite heterozygous fashion (genotype T4). This genotype was associated with ARVC (p<0.05) but not with dilated cardiomyopathy (DCM, 79 patients) or none-failing controls (463 blood donors). However, either one of the two SNPs were identified in further 7 ARVC patients, in 11 DCM patients, and in 64 blood donors. The SNP leading to G1886S may create a protein kinase C phosphorylation site in the human RyR2. Single channel recordings at pCa4.3 revealed four conductance states for the RyR2 of genotype T4 and a single open state for the wild type RyR2. At pCa7.7, the lowest subconductance state of the RyR2 channel of genotype T4 persisted with a greatly enhanced open probability indicating a leaky channel. CONCLUSION: The RyR2 channel leak under diastolic conditions could cause SR-Ca2+ depletion, concomitantly arrhythmogenesis and heart failure in a subgroup of ARVC patients of genotype T4. A change in the RyR2 subunit composition due to the combined expression of both SNPs alters the behaviour of the tetrameric channel complex.

Influence of ACE-inhibition and mechanical unloading on the regulation of extracellular matrix proteins in the myocardium of heart transplantation candidates bridged by ventricular assist devices.

BACKGROUND: Whether adverse structural changes in the myocardium due to remodelling can be reversed by ventricular assist device (VAD) support in patients with end-stage heart failure is controversial. AIMS: To investigate the effect of VAD support on the extra-cellular matrix. METHODS: We analysed the collagen content in terminal failing ventricles of VAD-patients and donor hearts using 4-hydroxyproline for total collagen and real time RT-PCR for fibronectin (FN), collagen I alpha 1 (Col1A1), III alpha 1 (Col3A1) and TGF beta 1 analysis. RESULTS: Compared to donor hearts we found similar increases in Col1A1 and TGF beta1 but not Col3A1 and FN mRNAs, which were similar in the myocardium from patients receiving a VAD or heart transplant. However, patients receiving ACE-I during VAD-support had lower Col1A1 mRNA content at transplantation. The total collagen content was not influenced by mechanical unloading or by ACE-I medication. CONCLUSION: Mechanical unloading by VAD does not reduce the collagen content of the terminal failing ventricle possibly due to increased TGF beta1 levels. However, Col1A1 production may be reduced by ACE-I medication during VAD support.

The novel desmin mutant p.A120D impairs filament formation, prevents intercalated disk localization, and causes sudden cardiac death.

BACKGROUND: The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. METHODS AND RESULTS: We identified the 2 novel variants DES-p.A120D (c.359C>A) and DES-p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in cell lines and of the isolated recombinant protein. Model variants of codon 120 indicated that ionic interactions contribute to this filament formation defect. Ex vivo analysis of ventricular tissue slices revealed a loss of desmin staining within the intercalated disk and severe cytoplasmic aggregate formation, whereas z-band localization was not affected. The functional experiments of desmin-p.H326R did not demonstrate any differences from wild type. CONCLUSIONS: Because of the functional in vivo and in vitro characterization, DES-p.A120D has to be regarded as a pathogenic mutation and DES-p.H326R as a rare variant with unknown significance. Presumably, the loss of the desmin-p. A120D filament localization at the intercalated disk explains its clinical arrhythmogenic potential.

No association between single nucleotide polymorphisms and the development of nephrotoxicity after orthotopic heart transplantation.

BACKGROUND: Survival for heart transplantation (HTx) patients is limited by nephrotoxicity of the calcineurin inhibitors cyclosporine and tacrolimus. To determine whether genetic factors are involved in the development of renal dysfunction under immunosuppressive therapy, we screened various genes for sequence variations. METHODS: In a case-control study we analyzed in parallel polymorphisms within the transforming growth factor-beta1 gene (TGF-beta1; L10P, R25P), the multidrug resistance gene MDR 1 (A893T/S) and the CYP3A5 gene (CYP3A5*1/*3 allele). In total, we included 53 cardiac allograft recipients with renal insufficiency (serum creatinine >or=1.8 mg/dl and glomerular filtration rate <50 ml/min/1.73 m(2)) and 53 patients with normal renal function as controls. The controls were matched with patients for age, gender and post-HTx time. The polymorphisms were assessed by denaturing high-performance liquid chromatography (dHPLC) and direct sequencing. We performed univariate and multivariate logistic regression analysis to assess the association between different gene variants and renal dysfunction. RESULTS: No significant (p > 0.05) relationship was found between the polymorphisms investigated and the susceptibility of renal insufficiency under immunosuppressive therapy. CONCLUSIONS: Our data do not justify genotyping of the investigated single nucleotide polymorphisms (SNPs) to assess the development of renal dysfunction post-HTx.