Effect of epicatechin consumption on the inflammatory pathway and mitochondria morphology in PBMC from a R350P desminopathy patient: A case report.

Desminopathy R350P is a human myopathy that is characterized by the progressive loss of muscle fiber organization. This results in the loss of muscle size, mobility, and strength. In desminopathy, inflammation affects muscle homeostasis and repair, and contributes to progressive muscle deterioration. Mitochondria morphology was also suggested to affect desminopathy progression. Epicatechin (Epi)-a natural compound found in cacao-has been proposed to regulate inflammatory signaling and mitochondria morphology in human and animal models. Hence, we hypothesize chronic Epi consumption to improve inflammatory pathway and mitochondria morphology in the peripheral blood mononuclear cells (PBMCs) of a desminopathy R350P patient. We found that 12 weeks of Epi consumption partially restored TRL4 signaling, indicative of inflammatory signaling and mitochondria morphology in the desminopathy patient. Moreover, Epi consumption improved blood health parameters, including reduced HOMA-IR and IL-6 levels in the desminopathy patient. This indicates that Epi consumption could be a useful tool to slow disease progression in desminopathy patients.

Desmin gene expression is not ubiquitous in all upper airway myofibers and the pattern differs between healthy and sleep apnea subjects.

BACKGROUND: Desmin is a major cytoskeletal protein considered ubiquitous in mature muscle fibers. However, we earlier reported that a subgroup of muscle fibers in the soft palate of healthy subjects and obstructive sleep apnea patients (OSA) lacked immunoexpression for desmin. This raised the question of whether these fibers also lack messenger ribonucleic acid (mRNA) for desmin and can be considered a novel fiber phenotype. Moreover, some fibers in the OSA patients had an abnormal distribution and aggregates of desmin. Thus, the aim of the study was to investigate if these desmin protein abnormalities are also reflected in the expression of desmin mRNA in an upper airway muscle of healthy subjects and OSA patients. METHODS: Muscle biopsies from the musculus uvulae in the soft palate were obtained from ten healthy male subjects and six male patients with OSA. Overnight sleep apnea registrations were done for all participants. Immunohistochemistry, in-situ hybridization, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) techniques were used to evaluate the presence of desmin protein and its mRNA. RESULTS: Our findings demonstrated that a group of muscle fibers lacked expression for desmin mRNA and desmin protein in healthy individuals and OSA patients (12.0 ± 5.6% vs. 23.1 ± 10.8%, p = 0.03). A subpopulation of these fibers displayed a weak subsarcolemmal rim of desmin accompanied by a few scattered mRNA dots in the cytoplasm. The muscles of OSA patients also differed from healthy subjects by exhibiting muscle fibers with reorganized or accumulated aggregates of desmin protein (14.5 ± 6.5%). In these abnormal fibers, the density of mRNA was generally low or concentrated in specific regions. The overall quantification of desmin mRNA by RT-qPCR was significantly upregulated in OSA patients compared to healthy subjects (p = 0.01). CONCLUSIONS: Our study shows evidence that muscle fibers in the human soft palate lack both mRNA and protein for desmin. This indicates a novel cytoskeletal structure and challenges the ubiquity of desmin in muscle fibers. Moreover, the observation of reorganized or accumulated aggregates of desmin mRNA and desmin protein in OSA patients suggests a disturbance in the transcription and translation process in the fibers of the patients.

Role of the Alpha-B-Crystallin Protein in Cardiomyopathic Disease.

Alpha-B-crystallin, a member of the small heat shock family of proteins, has been implicated in a variety of cardiomyopathies and in normal cardiac homeostasis. It is known to function as a molecular chaperone, particularly for desmin, but also interacts with a wide variety of additional proteins. The molecular chaperone function is also enhanced by signal-dependent phosphorylation at specific residues under stress conditions. Naturally occurring mutations in CRYAB, the gene that encodes alpha-B-crystallin, have been suggested to alter ionic intermolecular interactions that affect dimerization and chaperone function. These mutations have been associated with myofibrillar myopathy, restrictive cardiomyopathy, and hypertrophic cardiomyopathy and promote pathological hypertrophy through different mechanisms such as desmin aggregation, increased reductive stress, or activation of calcineurin-NFAT signaling. This review will discuss the known mechanisms by which alpha-B-crystallin functions in cardiac homeostasis and the pathogenesis of cardiomyopathies and provide insight into potential future areas of exploration.

[Clinical and genetic analysis of a patient with Desminopathy manifesting initially with myalgia after lower limb activity].

OBJECTIVE: To explore the clinical characteristics and genetic variant of a patient with desminopathy manifesting with atypical symptoms. METHODS: A patient who was admitted to the Department of Neurology of Jing'an District Central Hospital on February 24, 2021 was selected as the study subject. Clinical data, laboratory tests, muscle pathology, muscle magnetic resonance imaging (MRI) and genetic testing of the patient were retrospectively analyzed. RESULTS: The patient had developed myalgia after lower limb activity, and gradually developed asymmetrical muscle weakness and atrophy of the lower limbs. Cardiac examination revealed atrioventricular block and decreased left ventricular diastolic function. Muscle MRI showed that semitendinosus, sartorius, gracilis, fibula, gastronemius and supinator muscles were selectively involved at the early stage. Muscle biopsy confirmed pathological changes of desmin positive myofibrils. Genetic testing revealed that the patient has harbored a c.1024A>G (p.n342d) missense variant in exon 6 of the DES gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was rated as likely pathogenic (PS4_moderate+PM2_supporting+PP3_moderate+PP1). CONCLUSION: Desmin disease has a great clinical heterogeneity. Postexercise myalgia of lower limbs is a rare clinical phenotype. For patients harboring the c.1024A>G (p.n342d) variant of the DES gene, in addition to semitendinosus and fibula, Cardiac involvement is relatively insidious and easy to be ignored in clinic. Timely muscle MRI, muscle biopsy and gene detection will help the early diagnosis of the disease.

Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation.

BACKGROUND: Beyond the observed alterations in cellular structure and mitochondria, the mechanisms linking rare genetic mutations to the development of heart failure in patients affected by desmin mutations remain unclear due in part, to the lack of relevant human cardiomyocyte models. METHODS: To shed light on the role of mitochondria in these mechanisms, we investigated cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DESE439K mutation that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cardiomyocytes were either cultured on an anisotropic micropatterned surface to obtain elongated and aligned cardiomyocytes, or as a cardiac spheroid to create a micro-tissue. Moreover, when applicable, results from cardiomyocytes were confirmed with heart biopsies of suddenly died patient of the same family harboring DESE439K mutation, and post-mortem heart samples from five control healthy donors. RESULTS: The heterozygous DESE439K mutation leads to dramatic changes in the overall cytoarchitecture of cardiomyocytes, including cell size and morphology. Most importantly, mutant cardiomyocytes display altered mitochondrial architecture, mitochondrial respiratory capacity and metabolic activity reminiscent of defects observed in patient's heart tissue. Finally, to challenge the pathological mechanism, we transferred normal mitochondria inside the mutant cardiomyocytes and demonstrated that this treatment was able to restore mitochondrial and contractile functions of cardiomyocytes. CONCLUSIONS: This work highlights the deleterious effects of DESE439K mutation, demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens up new potential therapeutic perspectives for this disease.

Desmin and its molecular chaperone, the αB-crystallin: How post-translational modifications modulate their functions in heart and skeletal muscles?

Maintenance of the highly organized striated muscle tissue requires a cell-wide dynamic network through protein-protein interactions providing an effective mechanochemical integrator of morphology and function. Through a continuous and complex trans-cytoplasmic network, desmin intermediate filaments ensure this essential role in heart and in skeletal muscle. Besides their role in the maintenance of cell shape and architecture (permitting contractile activity efficiency and conferring resistance towards mechanical stress), desmin intermediate filaments are also key actors of cell and tissue homeostasis. Desmin participates to several cellular processes such as differentiation, apoptosis, intracellular signalisation, mechanotransduction, vesicle trafficking, organelle biogenesis and/or positioning, calcium homeostasis, protein homeostasis, cell adhesion, metabolism and gene expression. Desmin intermediate filaments assembly requires αB-crystallin, a small heat shock protein. Over its chaperone activity, αB-crystallin is involved in several cellular functions such as cell integrity, cytoskeleton stabilization, apoptosis, autophagy, differentiation, mitochondria function or aggresome formation. Importantly, both proteins are known to be strongly associated to the aetiology of several cardiac and skeletal muscles pathologies related to desmin filaments disorganization and a strong disturbance of desmin interactome. Note that these key proteins of cytoskeleton architecture are extensively modified by post-translational modifications that could affect their functional properties. Therefore, we reviewed in the herein paper the impact of post-translational modifications on the modulation of cellular functions of desmin and its molecular chaperone, the αB-crystallin.

Intermediate filaments in the heart: The dynamic duo of desmin and lamins orchestrates mechanical force transmission.

The intermediate filament (IF) cytoskeleton supports cellular structural integrity, particularly in response to mechanical stress. The most abundant IF proteins in mature cardiomyocytes are desmin and lamins. The desmin network tethers the contractile apparatus and organelles to the nuclear envelope and the sarcolemma, while lamins, as components of the nuclear lamina, provide structural stability to the nucleus and the genome. Mutations in desmin or A-type lamins typically result in cardiomyopathies and recent studies emphasized the synergistic roles of desmin and lamins in the maintenance of nuclear integrity in cardiac myocytes. Here we explore the emerging roles of the interdependent relationship between desmin and lamins in providing resilience to nuclear structure while transducing extracellular mechanical cues into the nucleus.

Deep Characterization of a Greek Patient with Desmin-Related Myofibrillar Myopathy and Cardiomyopathy.

Desmin is a class III intermediate filament protein highly expressed in cardiac, smooth and striated muscle. Autosomal dominant or recessive mutations in the desmin gene (DES) result in a variety of diseases, including cardiomyopathies and myofibrillar myopathy, collectively called desminopathies. Here we describe the clinical, histological and radiological features of a Greek patient with a myofibrillar myopathy and cardiomyopathy linked to the c.734A>G,p.(Glu245Gly) heterozygous variant in the DES gene. Moreover, through ribonucleic acid sequencing analysis in skeletal muscle we show that this variant provokes a defect in exon 3 splicing and thus should be considered clearly pathogenic.

AAV9-mediated SMN gene therapy rescues cardiac desmin but not lamin A/C and elastin dysregulation in Smn2B/- spinal muscular atrophy mice.

Structural, functional and molecular cardiac defects have been reported in spinal muscular atrophy (SMA) patients and mouse models. Previous quantitative proteomics analyses demonstrated widespread molecular defects in the severe Taiwanese SMA mouse model. Whether such changes are conserved across different mouse models, including less severe forms of the disease, has yet to be established. Here, using the same high-resolution proteomics approach in the less-severe Smn2B/- SMA mouse model, 277 proteins were found to be differentially abundant at a symptomatic timepoint (post-natal day (P) 18), 50 of which were similarly dysregulated in severe Taiwanese SMA mice. Bioinformatics analysis linked many of the differentially abundant proteins to cardiovascular development and function, with intermediate filaments highlighted as an enriched cellular compartment in both datasets. Lamin A/C was increased in the cardiac tissue, whereas another intermediate filament protein, desmin, was reduced. The extracellular matrix (ECM) protein, elastin, was also robustly decreased in the heart of Smn2B/- mice. AAV9-SMN1-mediated gene therapy rectified low levels of survival motor neuron protein and restored desmin levels in heart tissues of Smn2B/- mice. In contrast, AAV9-SMN1 therapy failed to correct lamin A/C or elastin levels. Intermediate filament proteins and the ECM have key roles in cardiac function and their dysregulation may explain cardiac impairment in SMA, especially since mutations in genes encoding these proteins cause other diseases with cardiac aberration. Cardiac pathology may need to be considered in the long-term care of SMA patients, as it is unclear whether currently available treatments can fully rescue peripheral pathology in SMA.

Upregulated desmin/integrin ß1/MAPK axis promotes elastic cartilage regeneration with increased ECM mechanical strength.

Elastic cartilage tissue engineering is promising for providing available scaffolds for plastic reconstructive surgery. The insufficient mechanical strength of regenerative tissue and scarce resources of reparative cells are two obstacles for the preparation of tissue-engineered elastic cartilage scaffolds. Auricular chondrocytes are important reparative cells for elastic cartilage tissue engineering, but resources are scarce. Identifying auricular chondrocytes with enhanced capability of elastic cartilage formation is conducive to reducing the damage to donor sites by decreasing the demand on native tissue isolation. Based on the biochemical and biomechanical differences in native auricular cartilage, we found that auricular chondrocytes with upregulated desmin expressed more integrin ß1, forming a stronger interaction with the substrate. Meanwhile, activated MAPK pathway was found in auricular chondrocytes highly expressing desmin. When desmin was knocked down, the chondrogenesis and mechanical sensitivity of chondrocytes were both impaired, and the MAPK pathway was downregulated. Finally, auricular chondrocytes highly expressing desmin regenerated more elastic cartilage with increased ECM mechanical strength. Therefore, desmin/integrin ß1/MAPK signaling can not only serve as a selection standard but also a manipulation target of auricular chondrocytes to promote elastic cartilage regeneration.

Desmin mutations impact the autophagy flux in C2C12 cell in mutation-specific manner.

Desmin is the main intermediate filament of striated and smooth muscle cells and plays a crucial role in maintaining the stability of muscle fiber during contraction and relaxation cycles. Being a component of Z-disk area, desmin integrates autophagic pathways, and the disturbance of Z-disk proteins' structure negatively affects chaperone-assisted selective autophagy (CASA). In the present study, we focused on alteration of autophagy flux in myoblasts expressing various Des mutations. We applied Western blotting, immunocytochemistry, RNA sequencing, and shRNA approach to demonstrate that DesS12F, DesA357P, DesL345P, DesL370P, and DesD399Y mutations. Mutation-specific effect on autophagy flux being most severe in aggregate-prone Des mutations such as DesL345P, DesL370P, and DesD399Y. RNA sequencing data confirmed the most prominent effect of these mutations on expression profile and, in particular, on autophagy-related genes. To verify CASA contribution to desmin aggregate formation, we suppressed CASA by knocking down Bag3 and demonstrated that it promoted aggregate formation and lead to downregulation of Vdac2 and Vps4a and upregulation of Lamp, Pink1, and Prkn. In conclusion, Des mutations showed a mutation-specific effect on autophagy flux in C2C12 cells with either a predominant impact on autophagosome maturation or on degradation and recycling processes. Aggregate-prone desmin mutations lead to the activation of basal autophagy level while suppressing the CASA pathway by knocking down Bag3 can promote desmin aggregate formation.

Muscle Specific Promotors for Gene Therapy - A Comparative Study in Proliferating and Differentiated Cells.

BACKGROUND: Depending on the therapy approach and disease background, the heterogeneity of muscular tissues complicates the development of targeted gene therapy, where either expression in all muscle types or restriction to only one muscle type is warranted. Muscle specificity can be achieved using promotors mediating tissue specific and sustained physiological expression in the desired muscle types but limited activity in non-targeted tissue. Several muscle specific promotors have been described, but direct comparisons between them are lacking. OBJECTIVE: Here we present a direct comparison of muscle specific Desmin-, MHCK7, microRNA206- and Calpain3 promotor. METHODS: To directly compare these muscle specific promotors we utilized transfection of reporter plasmids using an in vitro model based on electrical pulse stimulation (EPS) to provoke sarcomere formation in 2D cell culture for quantification of promotor activities in far differentiated mouse and human myotubes. RESULTS: We found that Desmin- and MHCK7 promotors showed stronger reporter gene expression levels in proliferating and differentiated myogenic cell lines than miR206 and CAPN3 promotor. However, Desmin and MHCK7 promotor promoted gene expression also cardiac cells whereas miR206 and CAPN3 promotor expression was restricted to skeletal muscle. CONCLUSIONS: Our results provides direct comparison of muscle specific promotors with regard to expression strengths and specificity as this is important feature to avoid undesired transgene expression in non-target muscle cells for a desired therapy approach.

Z-Disk-Associated Plectin (Isoform 1d): Spatial Arrangement, Interaction Partners, and Role in Filamin C Homeostasis.

Plectin, a highly versatile cytolinker protein, is crucial for myofiber integrity and function. Accordingly, mutations in the human gene (PLEC) cause several rare diseases, denoted as plectinopathies, with most of them associated with progressive muscle weakness. Of several plectin isoforms expressed in skeletal muscle and the heart, P1d is the only isoform expressed exclusively in these tissues. Using high-resolution stimulated emission depletion (STED) microscopy, here we show that plectin is located within the gaps between individual α-actinin-positive Z-disks, recruiting and bridging them to desmin intermediate filaments (Ifs). Loss of plectin in myofibril bundles led to a complete loss of desmin Ifs. Loss of Z-disk-associated plectin isoform P1d led to disorganization of muscle fibers and slower relaxation of myofibrils upon mechanical strain, in line with an observed inhomogeneity of muscle ultrastructure. In addition to binding to α-actinin and thereby providing structural support, P1d forms a scaffolding platform for the chaperone-assisted selective autophagy machinery (CASA) by directly interacting with HSC70 and synpo2. In isoform-specific knockout (P1d-KO) mouse muscle and mechanically stretched plectin-deficient myoblasts, we found high levels of undigested filamin C, a bona fide substrate of CASA. Similarly, subjecting P1d-KO mice to forced swim tests led to accumulation of filamin C aggregates in myofibers, highlighting a specific role of P1d in tension-induced proteolysis activated upon high loads of physical exercise and muscle contraction.

The Myocardial Accumulation of Aggregated Desmin Protein in a Case of Desminopathy with a de novo DES p.R406W Mutation.

Desminopathy is a cardiac and skeletal myopathy caused by disease-causing variants in the desmin (DES) gene and represents a subgroup of myofibrillar myopathies, where cytoplasmic desmin-postive immunoreactivity is the pathological hallmark. We herein report a 28-year-old Japanese man who was initially diagnosed with sporadic hypertrophic cardiomyopathy with atrioventricular block at 9 years old and developed weakness in the soft palate and extremities. The myocardial tissue dissected during implantation of the ventricular-assisted device showed a dilated phase of hypertrophic cardiomyopathy and intracellular accumulation of proteinase K-resistant desmin aggregates. Genetic testing confirmed a de novo mutation of DES, which has already been linked to desminopathy. As the molecular diagnosis of desminopathy is challenging, particularly if patients show predominantly cardiac signs and a routine skeletal muscle biopsy is unavailable, these characteristic pathological findings of endomyocardial proteinase K-resistant desmin aggregates might aid in clinical practice.

The N-Terminal Part of the 1A Domain of Desmin Is a Hot Spot Region for Putative Pathogenic DES Mutations Affecting Filament Assembly.

Desmin is the major intermediate filament protein of all three muscle cell types, and connects different cell organelles and multi-protein complexes such as the cardiac desmosomes. Several pathogenic mutations in the DES gene cause different skeletal and cardiac myopathies. However, the significance of the majority of DES missense variants is currently unknown, since functional data are lacking. To determine whether desmin missense mutations within the highly conserved 1A coil domain cause a filament assembly defect, we generated a set of variants with unknown significance and systematically analyzed the filament assembly using confocal microscopy in transfected SW-13, H9c2 cells and cardiomyocytes derived from induced pluripotent stem cells. We found that mutations in the N-terminal part of the 1A coil domain affect filament assembly, leading to cytoplasmic desmin aggregation. In contrast, mutant desmin in the C-terminal part of the 1A coil domain forms filamentous structures comparable to wild-type desmin. Our findings suggest that the N-terminal part of the 1A coil domain is a hot spot for pathogenic desmin mutations, which affect desmin filament assembly. This study may have relevance for the genetic counselling of patients carrying variants in the 1A coil domain of the DES gene.

Bi-Allelic DES Gene Variants Causing Autosomal Recessive Myofibrillar Myopathies Affecting Both Skeletal Muscles and Cardiac Function.

Mutations in the human desmin gene (DES) may cause both autosomal dominant and recessive cardiomyopathies leading to heart failure, arrhythmias and atrio-ventricular blocks, or progressive myopathies. Cardiac conduction disorders, arrhythmias and cardiomyopathies usually associated with progressive myopathy are the main manifestations of autosomal dominant desminopathies, due to mono-allelic pathogenic variants. The recessive forms, due to bi-allelic variants, are very rare and exhibit variable phenotypes in which premature sudden cardiac death could also occur in the first or second decade of life. We describe a further case of autosomal recessive desminopathy in an Italian boy born of consanguineous parents, who developed progressive myopathy at age 12, and dilated cardiomyopathy four years later and died of intractable heart failure at age 17. Next Generation Sequencing (NGS) analysis identified the homozygous loss-of-function variant c.634C>T; p.Arg212*, which was likely inherited from both parents. Furthermore, we performed a comparison of clinical and genetic results observed in our patient with those of cases so far reported in the literature.

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

The gene variant for desmin rs1058261 may protect against combined cancer and cardiovascular death, the Tampere adult population cardiovascular risk study.

Desmin-containing intermediate filaments are a part of muscle cytoskeleton. We have previously reported that the wild-type cytosine/cytosine genotype of a common Desmin synonymous single nucleotide polymorphism (C > T) (rs1058261) associated with cardiovascular diseases in a cohort of subjects from the Tampere adult population cardiovascular risk study. We now examined whether rs1058261 also associates with early death by following the cohort of 801 subjects from the age of 50 up to the age of 65. Outcomes for death were collected from the National Statistics Centre. Linkage disequilibrium analysis and gene expression correlations for rs1058261 were done in silico. With follow-up, subjects with wild-type cytosine/cytosine genotype had higher incidence of cancer deaths (odds ratio [OR] 5.27, confidence interval [CI] 1.160-23.946, P = .031), combined deaths from cardiovascular diseases or cancers (OR 3.92, CI 1.453-10.596, P = .007), and "hard" acute cardiovascular disease events (early myocardial infarction and/or death) (OR 3.90, CI 1.287-11.855, P = .016) compared to subjects with the T-allele. The in silico results of linkage disequilibrium and gene expression analyses showed negative gene expression sizes associated with rs1058261, which theoretically decreases desmin expression. Our findings suggest that variation rs1058261 in Desmin may serve as a surrogate marker for other variations involved in decrease of deaths from combined cancer and cardiovascular disease.

DICER1-associated central nervous system sarcoma with neural lineage differentiation: a case report.

BACKGROUND: DICER1-associated central nervous system sarcoma (DCS) without evidence of other cancer-related syndromes is rare. Though the morphology of DCS was highly variable, the immunophenotype was predominant myogenic phenotype. Other lineage markers were consistently negative. CASE PRESENTATION: We report a case of DCS with neurogenic differentiation proved by immunohistochemical staining and whole-exome sequencing (WES). An 8-year-old female patient presented with 8-day history of headache, nausea and vomiting. Magnetic resonance imaging (MRI) revealed a heterogeneous mass in the left parietal lobe. The patient underwent the craniotomy via left parietal approach to resect the tumor completely. Histologically, the tumor predominately showed fibrosarcoma-like spindle cells with obvious cytoplasmic eosinophilic globules. Immunohistochemically, the tumor stained positively for DICER1, Desmin, and several neurogenic markers. DICER1 somatic hotspot mutation was confirmed by WES, as well as TP53 and RAF1 mutations which were commonly found in DCS, and other sarcoma-associated genes including AR, AXL and ETV5 mutations. Subsequently, the result of Gene Ontology (GO) analysis showed that the mutated genes in this case were involved in neuron development. All of these findings indicated the diagnosis of DCS with neurogenic differentiation. Postoperatively, the patient received high-dose radiotherapy (60 Gy) and chemotherapy. There was no MRI evidence of tumor recurrence at the 21-month postoperative follow-up. CONCLUSIONS: This unusual DCS case with neuronal differentiation is an important addition to the immuno-phenotypic spectrum of DCS. Although the prognosis for DCS is poor, gross tumor resection with high dose radiotherapy and chemotherapy may assist in prolonging survival.

Expression patterns and correlation analyses of muscle-specific genes in the process of sheep myoblast differentiation.

The purpose of this study was to establish a system for the isolation, culture, and differentiation of sheep myoblasts, and to explore the expression patterns as well as mutual relationships of muscle-specific genes. Sheep fetal myoblasts (SFMs) were isolated by two-step enzymatic digestion, purified by differential adhesion and identified using immunofluorescence techniques. Two percent horse serum was used to induce differentiation in SFMs. Real-time quantitative and Western blot analyses were respectively used to detect the mRNA and protein expressions of muscle-specific genes including MyoD, MyoG, Myf5, Myf6, PAX3, PAX7, myomaker, desmin, MYH1, MYH2, MYH4, MYH7, and MSTN during the differentiation of SFMs. Finally, the correlation between muscle-specific genes was analyzed by the Pearson correlation coefficient method. The results showed that the isolated and purified SFMs could form myotubes after the induction for differentiation. The marker factors including MyoD, MyoG, myomaker, desmin, and MyHC were positively stained in SFMs. The mRNA expressions of MyoD, MyoG, and myomaker increased and then decreased, while Myf5, PAX3, and PAX7 decreased; Myf6, desmin, MYH1, MYH2, MYH4, and MYH7 increased; and MSTN fluctuated up and down during the differentiation of SFMs. The expression patterns of protein were basically consistent with those of mRNA except MSTN. There existed significant or highly significant correlations at mRNA or protein level among some genes. Some transcription factor proteins (MyoD, Myf5, Myf6, PAX3, PAX7) showed significant or highly significant correlations with the mRNA level of some other genes and/or themselves. In conclusion, SFMs with good myogenic differentiation ability were successfully isolated, and the expression patterns and correlations of muscle-specific genes during SFM differentiation were revealed, which laid an important foundation for elucidating the gene regulation mechanism of sheep myogenesis.