Incorporation of desmocollin-2 into the plasma membrane requires N-glycosylation at multiple sites.

Brodehl A, Stanasiuk C, Anselmetti D, Gummert J, Milting H. Incorporation of desmocollin-2 into the plasma membrane requires N-glycosylation at multiple sites. FEBS open bio. 2019;9(4):996-1007.Desmocollin-2 (DSC2) is a desmosomal protein of the cadherin family. Desmosomes are multiprotein complexes, which are involved in cell adhesion of cardiomyocytes and of keratinocytes. The molecular structure of the complete extracellular domain (ECD) of DSC2 was recently described, revealing three disulfide bridges, four N-glycosylation sites, and four O-mannosylation sites. However, the functional relevance of these post-translational modifications for the protein trafficking of DSC2 to the plasma membrane is still unknown. Here, we generated a set of DSC2 mutants, in which we systematically exchanged all N-glycosylation sites, O-mannosylation sites, and disulfide bridges within the ECD and investigated the resulting subcellular localization by confocal laser scanning microscopy. Of note, all single and double N-glycosylation- deficient mutants were efficiently incorporated into the plasma membrane, indicating that the absence of these glycosylation sites has a minor effect on the protein trafficking of DSC2. However, the exchange of multiple N-glycosylation sites resulted in intracellular accumulation. Colocalization analysis using cell compartment trackers revealed that N-glycosylation- deficient DSC2 mutants were retained within the Golgi apparatus. In contrast, elimination of the four O-mannosylation sites or the disulfide bridges in the ECD has no obvious effect on the intracellular protein processing of DSC2. These experiments underscore the importance of N-glycosylation at multiple sites of DSC2 for efficient intracellular transport to the plasma membrane. © 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd

Incorporation of desmocollin-2 into the plasma membrane requires N\it N-glycosylation at multiple sites

Desmocollin-2 (DSC2) is a desmosomal protein of the cadherin family. Desmosomes are multiprotein complexes, which are involved in cell adhesion of cardiomyocytes and of keratinocytes. The molecular structure of the complete extracellular domain (ECD) of DSC2 was recently described, revealing three disulfide bridges, four $\it N$-glycosylation sites, and four $\it O$-mannosylation sites. However, the functional relevance of these post-translational modifications for the protein trafficking of DSC2 to the plasma membrane is still unknown. Here, we generated a set of DSC2 mutants, in which we systematically exchanged all $\it N$-glycosylation sites, $\it O$-mannosylation sites, and disulfide bridges within the ECD and investigated the resulting subcellular localization by confocal laser scanning microscopy. Of note, all single and double $\it N$-glycosylation- deficient mutants were efficiently incorporated into the plasma membrane, indicating that the absence of these glycosylation sites has a minor effect on the protein trafficking of DSC2. However, the exchange of multiple N-glycosylation sites resulted in intracellular accumulation. Colocalization analysis using cell compartment trackers revealed that N-glycosylation- deficient DSC2 mutants were retained within the Golgi apparatus. In contrast, elimination of the four $\it O$-mannosylation sites or the disulfide bridges in the ECD has no obvious effect on the intracellular protein processing of DSC2. These experiments underscore the importance of N-glycosylation at multiple sites of DSC2 for efficient intracellular transport to the plasma membrane

Restrictive cardiomyopathy is caused by a novel homozygous desmin (DES) mutation p.Y122H leading to a severe filament assembly defect

Here, we present a small Iranian family, where the index patient received a diagnosis of restrictive cardiomyopathy (RCM) in combination with atrioventricular (AV) block. Genetic analysis revealed a novel homozygous missense mutation in the DES gene (c.364T > C; p.Y122H), which is absent in human population databases. The mutation is localized in the highly conserved coil-1 desmin subdomain. In silico, prediction tools indicate a deleterious effect of the desmin (DES) mutation p.Y122H. Consequently, we generated an expression plasmid encoding the mutant and wildtype desmin formed, and analyzed the filament formation in vitro in cardiomyocytes derived from induced pluripotent stem cells and HT-1080 cells. Confocal microscopy revealed a severe filament assembly defect of mutant desmin supporting the pathogenicity of the DES mutation, p.Y122H, whereas the wildtype desmin formed regular intermediate filaments. According to the guidelines of the American College of Medical Genetics and Genomics, we classified this mutation, therefore, as a novel pathogenic mutation. Our report could point to a recessive inheritance of the DES mutation, p.Y122H, which is important for the genetic counseling of similar families with restrictive cardiomyopathy caused by DES mutations

Distinct Myocardial Transcriptomic Profiles of Cardiomyopathies Stratified by the Mutant Genes

Sielemann K, Elbeck Z, Gärtner A, et al. Distinct Myocardial Transcriptomic Profiles of Cardiomyopathies Stratified by the Mutant Genes. Genes. 2020;11(12): 1430.Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely LMNA (lamin A/C), RBM20 (RNA binding motif protein 20), TTN (titin) and PKP2 (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in RBM20 and TTN as the splice factor RBM20 targets amongst other genes TTN, leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in LMNA is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in PKP2 lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies

The Desmin (<i>DES</i>) Mutation p.A337P Is Associated with Left-Ventricular Non-Compaction Cardiomyopathy

Here, we present a small Russian family, where the index patient received a diagnosis of left-ventricular non-compaction cardiomyopathy (LVNC) in combination with a skeletal myopathy. Clinical follow-up analysis revealed a LVNC phenotype also in her son. Therefore, we applied a broad next-generation sequencing gene panel approach for the identification of the underlying mutation. Interestingly, DES-p.A337P was identified in the genomes of both patients, whereas only the index patient carried DSP-p.L1348X. DES encodes the muscle-specific intermediate filament protein desmin and DSP encodes desmoplakin, which is a cytolinker protein connecting desmosomes with the intermediate filaments. Because the majority of DES mutations cause severe filament assembly defects and because this mutation was found in both affected patients, we analyzed this DES mutation in vitro by cell transfection experiments in combination with confocal microscopy. Of note, desmin-p.A337P forms cytoplasmic aggregates in transfected SW-13 cells and in cardiomyocytes derived from induced pluripotent stem cells underlining its pathogenicity. In conclusion, we suggest including the DES gene in the genetic analysis for LVNC patients in the future, especially if clinical involvement of the skeletal muscle is present

The desmin (DES) mutation p.A337P is associated with left-ventricular non-compaction cardiomyopathy

Here, we present a small Russian family, where the index patient received a diagnosis of left-ventricular non-compaction cardiomyopathy (LVNC) in combination with a skeletal myopathy. Clinical follow-up analysis revealed a LVNC phenotype also in her son. Therefore, we applied a broad next-generation sequencing gene panel approach for the identification of the underlying mutation. Interestingly, $\it DES$-p.A337P was identified in the genomes of both patients, whereas only the index patient carried $\it DSP$-p.L1348X. $\it DES$ encodes the muscle-specific intermediate filament protein desmin and $\it DSP$ encodes desmoplakin, which is a cytolinker protein connecting desmosomes with the intermediate filaments. Because the majority of $\it DES$ mutations cause severe filament assembly defects and because this mutation was found in both affected patients, we analyzed this $\it DES$ mutation in vitro by cell transfection experiments in combination with confocal microscopy. Of note, desmin-p.A337P forms cytoplasmic aggregates in transfected SW-13 cells and in cardiomyocytes derived from induced pluripotent stem cells underlining its pathogenicity. In conclusion, we suggest including the $\it DES$ gene in the genetic analysis for LVNC patients in the future, especially if clinical involvement of the skeletal muscle is present

Distinct myocardial transcriptomic profiles of cardiomyopathies stratified by the mutant genes

Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely $\it LMNA$ (lamin A/C), $\it RBM20$ (RNA binding motif protein 20), $\it TTN$ (titin) and $\it PKP2$ (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in $\it RBM20$ and $\it TTN$ as the splice factor RBM20 targets amongst other genes $\it TTN$, leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in $\it LMNA$ is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in $\it PKP2$ lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies

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