Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Case report: A novel ACTA1 variant in a patient with nemaline rods and increased glycogen deposition.

Background: Congenital myopathies are a group of heterogeneous inherited disorders, mainly characterized by early-onset hypotonia and muscle weakness. The spectrum of clinical phenotype can be highly variable, going from very mild to severe presentations. The course also varies broadly resulting in a fatal outcome in the most severe cases but can either be benign or lead to an amelioration even in severe presentations. Muscle biopsy analysis is crucial for the identification of pathognomonic morphological features, such as core areas, nemaline bodies or rods, nuclear centralizations and congenital type 1 fibers disproportion. However, multiple abnormalities in the same muscle can be observed, making more complex the myopathological scenario. Case presentation: Here, we describe an Italian newborn presenting with severe hypotonia, respiratory insufficiency, inability to suck and swallow, requiring mechanical ventilation and gastrostomy feeding. Muscle biopsy analyzed by light microscopy showed the presence of vacuoles filled with glycogen, suggesting a metabolic myopathy, but also fuchsinophilic inclusions. Ultrastructural studies confirmed the presence of normally structured glycogen, and the presence of minirods, directing the diagnostic hypothesis toward a nemaline myopathy. An expanded Next Generation Sequencing analysis targeting congenital myopathies genes revealed the presence of a novel heterozygous c.965 T > A p. (Leu322Gln) variant in the ACTA1 gene, which encodes the skeletal muscle alpha-actin. Conclusion: Our case expands the repertoire of molecular and pathological features observed in actinopathies. We highlight the value of ultrastructural examination to investigate the abnormalities detected at the histological level. We also emphasized the use of expanded gene panels in the molecular analysis of neuromuscular patients, especially for those ones presenting multiple bioptic alterations.

Prominent muscle involvement in a familial form of mitochondrial disease due to a COA8 variant.

Isolated mitochondrial respiratory chain Complex IV (Cytochrome c Oxidase or COX) deficiency is the second most frequent isolated respiratory chain defect. Causative mutations are mainly identified in structural COX subunits or in proteins involved in the maturation and assembly of the COX holocomplex. We describe an Italian familial case of mitochondrial myopathy due to a variant in the COX assembly factor 8 gene (COA8). Patient 1 is a 52-year-old woman who presented generalized epilepsy and retinitis pigmentosa at 10 years of age. From her early adulthood she complained about cramps and myalgia after exercise, and bilateral hearing loss emerged. Last neurological examination (52 years of age) showed bilateral ptosis, muscle weakness, peripheral neuropathy, mild dysarthria and dysphonia, cognitive impairment. Muscle biopsy had shown the presence of ragged-red fibers. Patient 2 (Patient 1's sister) is a 53-year-old woman presenting fatigability, myalgia, and hearing loss. Neurological examination showed ptosis and muscle weakness. Muscle biopsy displayed a diffuse reduction of COX activity staining and ragged-red fibers. Both sisters presented secondary amenorrhea. After ruling out mtDNA mutations, Whole Exome Sequencing analysis identified the novel homozygous COA8 defect c.170_173dupGACC, p.(Pro59fs) in the probands. Loss-of-function COA8 mutations have been associated with cavitating leukoencephalopathy with COX deficiency in 9 reported individuals. Disease course shows an early-onset rapid clinical deterioration, affecting both cognitive and motor functions over months, followed by stabilization and slow improvement over several years. Our findings expand the clinical spectrum of COA8-related disease. We confirm the benign course of this rare disorder, highlighting its (intrafamilial) clinical variability.

The Profiling of 179 miRNA Expression in Serum from Limb Girdle Muscular Dystrophy Patients and Healthy Controls.

Limb girdle muscular dystrophies (LGMDs) are a group of genetically inherited neuromuscular diseases with a very variable clinical presentation and overlapping traits. Over the last few years there has been an increasing interest in the use of non-invasive circulating biomarkers to monitor disease progression and to evaluate the efficacy of therapeutic approaches. Our aim was to identify the miRNA signature with potential value for LGMD patient screening and stratification. Using miRCURY LNA miRNA qPCR Serum/Plasma Panel, we analyzed 179 miRNAs from 16 patients, divided in four pools based on their genetic diagnosis, and from healthy controls. The miRNAs analysis showed a total of 107 dysregulated miRNAs in LGMD patients when compared to the healthy controls. After filtering via skeletal tissue expression and gene/pathways target analysis, the number of dysregulated miRNAs drastically reduced. Six selected miRNAs-let-7f-5p (in LGMDR1), miR-20a-5p (in LGMDR2), miR-130b-5p, miR-378a-5p (both in LGMDR3), miR-376c-3p and miR-382-5p (both in LGMDR4)-whose expression was significantly lower compared to controls in the different LGMD pools, were further investigated. The bioinformatic analysis of the target genes in each selected miRNA revealed ECM-receptor interaction and TGF-beta signaling as the most involved pathways. The correlation analysis showed a good correlation of let-7f-5p with fibrosis and with the cross sectional area of type I and type II fibers, while miR-130b-5p showed a good correlation with the age of onset of the disease. The receiver operating characteristic curves showed how single miRNAs were able to discriminate a specific group of LGMD patients and how the combination of six miRNAs was able to discriminate LGMD patients from controls.

Characterization of Skeletal Muscle Biopsy and Derived Myoblasts in a Patient Carrying Arg14del Mutation in Phospholamban Gene.

Phospholamban is involved in the regulation of the activity and storage of calcium in cardiac muscle. Several mutations have been identified in the PLN gene causing cardiac disease associated with arrhythmogenic and dilated cardiomyopathy. The patho-mechanism underlying PLN mutations is not fully understood and a specific therapy is not yet available. PLN mutated patients have been deeply investigated in cardiac muscle, but very little is known about the effect of PLN mutations in skeletal muscle. In this study, we investigated both histological and functional features in skeletal muscle tissue and muscle-derived myoblasts from an Italian patient carrying the Arg14del mutation in PLN. The patient has a cardiac phenotype, but he also reported lower limb fatigability, cramps and fasciculations. The evaluation of a skeletal muscle biopsy showed histological, immunohistochemical and ultrastructural alterations. In particular, we detected an increase in the number of centronucleated fibers and a reduction in the fiber cross sectional area, an alteration in p62, LC3 and VCP proteins and the formation of perinuclear aggresomes. Furthermore, the patient's myoblasts showed a greater propensity to form aggresomes, even more marked after proteasome inhibition compared with control cells. Further genetic and functional studies are necessary to understand whether a definition of PLN myopathy, or cardiomyopathy plus, can be introduced for selected cases with clinical evidence of skeletal muscle involvement. Including skeletal muscle examination in the diagnostic process of PLN-mutated patients can help clarify this issue.

A biallelic variant in COX18 cause isolated Complex IV deficiency associated with neonatal encephalo-cardio-myopathy and axonal sensory neuropathy.

Pathogenic variants impacting upon assembly of mitochondrial respiratory chain Complex IV (Cytochrome c Oxidase or COX) predominantly result in early onset mitochondrial disorders often leading to CNS, skeletal and cardiac muscle manifestations. The aim of this study is to describe a molecular defect in the COX assembly factor gene COX18 as the likely cause of a neonatal form of mitochondrial encephalo-cardio-myopathy and axonal sensory neuropathy. The proband is a 19-months old female displaying hypertrophic cardiomyopathy at birth and myopathy with axonal sensory neuropathy and failure to thrive developing in the first months of life. Serum lactate was consistently increased. Whole exome sequencing allowed the prioritization of the unreported homozygous substitution NM_001297732.2:c.667 G > C p.(Asp223His) in COX18. Patient's muscle biopsy revealed severe and diffuse COX deficiency and striking mitochondrial abnormalities. Biochemical and enzymatic studies in patient's myoblasts and in HEK293 cells after COX18 silencing showed a severe impairment of both COX activity and assembly. The biochemical defect was partially rescued by delivery of wild-type COX18 cDNA into patient's myoblasts. Our study identifies a novel defect of COX assembly and expands the number of nuclear genes involved in a mitochondrial disorder due to isolated COX deficiency.

Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes.

Collagen VI is a heterotrimeric protein expressed in several tissues and involved in the maintenance of cell integrity. It localizes at the cell surface, creating a microfilamentous network that links the cytoskeleton to the extracellular matrix. The heterotrimer consists of three chains encoded by COL6A1, COL6A2 and COL6A3 genes. Recessive and dominant molecular defects cause two main disorders, the severe Ullrich congenital muscular dystrophy and the relatively mild and slowly progressive Bethlem myopathy. We analyzed the clinical aspects, pathological features and mutational spectrum of 15 COL6-mutated patients belonging to our cohort of muscular dystrophy probands. Patients presented a heterogeneous phenotype ranging from severe forms to mild adult-onset presentations. Molecular analysis by NGS detected 14 different pathogenic variants, three of them so far unreported. Two changes, localized in the triple-helical domain of COL6A1, were associated with a more severe phenotype. Histological, immunological and ultrastructural techniques were employed for the validation of the genetic variants; they documented the high variability in COL6 distribution and the extracellular matrix disorganization, highlighting the clinical heterogeneity of our cohort. The combined use of these different technologies is pivotal in the diagnosis of COL6 patients.

MERRF Mutation A8344G in a Four-Generation Family without Central Nervous System Involvement: Clinical and Molecular Characterization.

A 53-year-old man approached our Neuromuscular Unit following an incidental finding of hyperckemia. Similar to his mother who had died at the age of 77 years, he was diabetic and had a few lipomas. The patient's two sisters, aged 60 and 50 years, did not have any neurological symptoms. Proband's skeletal muscle biopsy showed several COX-negative fibers, many of which were "ragged red". Genetic analysis revealed the presence of the A8344G mtDNA mutation, which is most commonly associated with a maternally inherited multisystem mitochondrial disorder known as MERRF (myoclonus epilepsy with ragged-red fibers). The two sisters also carry the mutation. Family members on the maternal side were reported healthy. Although atypical phenotypes have been reported in association with the A8344G mutation, central nervous system (CSN) manifestations other than myoclonic epilepsy are always reported in the family tree. If present, our four-generation family manifestations are late-onset and do not affect CNS. This could be explained by the fact that the mutational load remains low and therefore prevents tissues/organs from reaching the pathologic threshold. The fact that this occurs throughout generations and that CNS, which has the highest energetic demand, is clinically spared, suggests that regulatory genes and/or pathways affect mitochondrial segregation and replication, and protect organs from progressive dysfunction.

Case report: A novel patient presenting TRIM32-related limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy autosomal recessive 8 (LGMDR8) is a rare clinical manifestation caused by the presence of biallelic variants in the TRIM32 gene. We present the clinical, molecular, histopathological, and muscle magnetic resonance findings of a novel 63-years-old LGMDR8 patient of Italian origins, who went undiagnosed for 24 years. Clinical exome sequencing identified two TRIM32 missense variants, c.1181G > A p.(Arg394His) and c.1781G > A p.(Ser594Asp), located in the NHL1 and NHL4 structural domains, respectively, of the TRIM32 protein. We conducted a literature review of the clinical and instrumental data associated to the so far known 26 TRIM32 variants, carried biallelically by 53 LGMDR8 patients reported to date in 20 papers. Our proband's variants were previously identified only in three independent LGMDR8 patients in homozygosis, therefore our case is the first in literature to be described as compound heterozygous for such variants. Our report also provides additional data in support of their pathogenicity, since p.(Arg394His) is currently classified as a variant of uncertain significance, while p.(Ser594Asp) as likely pathogenic. Taken together, these findings might be useful to improve both the genetic counseling and the diagnostic accuracy of this rare neuromuscular condition.

Megaconial congenital muscular dystrophy due to novel CHKB variants: a case report and literature review.

BACKGROUND: Choline kinase beta (CHKB) catalyzes the first step in the de novo biosynthesis of phosphatidyl choline and phosphatidylethanolamine via the Kennedy pathway. Derangement of this pathway might also influence the homeostasis of mitochondrial membranes. Autosomal recessive CHKB mutations cause a rare form of congenital muscular dystrophy known as megaconial congenital muscular dystrophy (MCMD). CASE PRESENTATION: We describe a novel proband presenting MCMD due to unpublished CHKB mutations. The patient is a 6-year-old boy who came to our attention for cognitive impairment and slowly progressive muscular weakness. He was the first son of non-consanguineous healthy parents from Sri Lanka. Neurological examination showed proximal weakness at four limbs, weak osteotendinous reflexes, Gowers' maneuver, and waddling gate. Creatine kinase levels were mildly increased. EMG and brain MRI were normal. Left quadriceps skeletal muscle biopsy showed a myopathic pattern with nuclear centralizations and connective tissue increase. Histological and histochemical staining suggested subsarcolemmal localization and dimensional increase of mitochondria. Ultrastructural analysis confirmed the presence of enlarged ("megaconial") mitochondria. Direct sequencing of CHKB identified two novel defects: the c.1060G > C (p.Gly354Arg) substitution and the c.448-56_29del intronic deletion, segregating from father and mother, respectively. Subcloning of RT-PCR amplicons from patient's muscle RNA showed that c.448-56_29del results in the partial retention (14 nucleotides) of intron 3, altering physiological splicing and transcript stability. Biochemical studies showed reduced levels of the mitochondrial fission factor DRP1 and the severe impairment of mitochondrial respiratory chain activity in patient's muscle compared to controls. CONCLUSIONS: This report expands the molecular findings associated with MCMD and confirms the importance of considering CHKB variants in the differential diagnosis of patients presenting with muscular dystrophy and mental retardation. The clinical outcome of MCMD patients seems to be influenced by CHKB molecular defects. Histological and ultrastructural examination of muscle biopsy directed molecular studies and allowed the identification and characterization of an intronic mutation, usually escaping standard molecular testing.

Antisense Morpholino-Based In Vitro Correction of a Pseudoexon-Generating Variant in the SGCB Gene.

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities.

Immunofluorescence signal intensity measurements as a semi-quantitative tool to assess sarcoglycan complex expression in muscle biopsy.

Sarcoglycanopathies are highly heterogeneous in terms of disease progression, muscular weakness, loss of ambulation and cardiac/respiratory involvement. Their clinical severity usually correlates with the residual protein amount, which makes protein quantification extremely relevant. Sarcoglycanopathy diagnosis is genetic, but skeletal muscle analysis - by both immunohistochemistry and Western blot (WB) - is still mandatory to establish the correct diagnostic process. Unfortunately, however, WB analysis cannot be performed if the bioptic specimen is scarce. This study provides a sensitive tool for semi-quantification of residual amount of sarcoglycans in patients affected by sarcoglycanopathies, based on immunofluorescence staining on skeletal muscle sections, image acquisition and software elaboration. We applied this method to eleven sarcoglycanopathies, seven Becker muscular dystrophies and four age-matched controls. Fluorescence data analysed in patients and compared to age-matched controls showed a significant reduction of the mutated sarcoglycan expression and a variable reduction of the other sarcoglycans. Fluorescence normalized data analysed in relation to the age of onset of the disease, showed a negative correlation of α-sarcoglycan fluorescent signal versus fibrosis in patients with an early age of onset and a negative correlation between δ-sarcoglycan signal and fibrosis in both intermediate and late age of onset groups. The availability of a method that allows objective quantification of the sarcolemmal proteins, faster and less consuming than WB analysis and able to detect low residual sarcoglycan expression with great sensitivity, proves useful to better define both patient prognosis and expected disease evolution. The proposed method could be employed also to monitor the efficacy of therapeutic interventions and during clinical trials.

MicroRNAs as serum biomarkers in Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is an X-linked neuromuscular disorder due to mutation in the DMD gene, encoding dystrophin. Despite a wide clinical variability, BMD is characterized by progressive muscle degeneration and proximal muscle weakness. Interestingly, a dysregulated expression of muscle-specific microRNAs (miRNAs), called myomirs, has been found in patients affected with muscular dystrophies, although few studies have been conducted in BMD. We analysed the serum expression levels of a subset of myomirs in a cohort of 29 ambulant individuals affected by BMD and further classified according to the degree of alterations at muscle biopsy and in 11 age-matched healthy controls. We found a significant upregulation of serum miR-1, miR-133a, miR-133b and miR-206 in our cohort of BMD patients, supporting the role of these miRNAs in the pathophysiology of the disease, and we identified serum cut-off levels discriminating patients from healthy controls, confiming the potential of circulating miRNAs as promising noninvasive biomarkers. Moreover, serum levels of miR-133b were found to be associated with fibrosis at muscle biopsy and with patients' motor performances, suggesting that miR-133b might be a useful prognostic marker for BMD patients. Taken together, our data showed that these serum myomirs may represent an effective tool that may support stratification of BMD patients, providing the opportunity of both monitoring disease progression and assessing the treatment efficacy in the context of clinical trials.

A novel RRM2B mutation associated with mitochondrial DNA depletion syndrome.

Mitochondrial DNA (mtDNA) depletion syndromes are disorders characterized by infantile-onset, severe progression, and the drastic loss of mtDNA content in affected tissues. In a patient who showed severe hypotonia, proximal tubulopathy and sensorineural hearing loss after birth, we observed severe mtDNA depletion and impaired respiratory chain activity in muscle due to heterozygous variants c.686G > T and c.551-2A > G in RRM2B, encoding the p53R2 subunit of the ribonucleotide reductase.

Case Report: Rare Homozygous RNASEH1 Mutations Associated With Adult-Onset Mitochondrial Encephalomyopathy and Multiple Mitochondrial DNA Deletions.

Mitochondrial DNA (mtDNA) maintenance disorders embrace a broad range of clinical syndromes distinguished by the evidence of mtDNA depletion and/or deletions in affected tissues. Among the nuclear genes associated with mtDNA maintenance disorders, RNASEH1 mutations produce a homogeneous phenotype, with progressive external ophthalmoplegia (PEO), ptosis, limb weakness, cerebellar ataxia, and dysphagia. The encoded enzyme, ribonuclease H1, is involved in mtDNA replication, whose impairment leads to an increase in replication intermediates resulting from mtDNA replication slowdown. Here, we describe two unrelated Italian probands (Patient 1 and Patient 2) affected by chronic PEO, ptosis, and muscle weakness. Cerebellar features and severe dysphagia requiring enteral feeding were observed in one patient. In both cases, muscle biopsy revealed diffuse mitochondrial abnormalities and multiple mtDNA deletions. A targeted next-generation sequencing analysis revealed the homozygous RNASEH1 mutations c.129-3C>G and c.424G>A in patients 1 and 2, respectively. The c.129-3C>G substitution has never been described as disease-related and resulted in the loss of exon 2 in Patient 1 muscle RNASEH1 transcript. Overall, we recommend implementing the use of high-throughput sequencing approaches in the clinical setting to reach genetic diagnosis in case of suspected presentations with impaired mtDNA homeostasis.

Peculiar histological and ultrastructural skeletal muscle alterations in a patient with CMV infection and autoimmune myositis: case evaluation and brief literature review.

We report the case of a young woman with CMV infection, high level of creatine kinase and myopathy. Electromyography showed a myopathic pattern. Muscle biopsy showed a marked increase of NADH enzymatic activity in the central area of almost all type I fibres, few degenerative and necrotic fibres and scattered mononuclear cell infiltrates. Ultrastructural analysis showed a marked disarrangement of sarcomeric structure and large inclusions of thin filaments in some fibres, while immunohistochemistry evidenced alteration in desmin, actin and αB-crystallin protein signals. PCR for CMV detection on muscle sections was negative. Histological, immunological and ultrastructural evaluations were compatible with a necrotic inflammatory myopathy. The correlations between CMV liver infection and the myopathic pattern are discussed. This case underscores the need to consider CMV infection in the differential diagnosis of myopathy with undetermined aetiology, quickly providing directions for a targeted muscle pharmacological intervention.

Muscle histological changes in a large cohort of patients affected with Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is a severe X-linked muscle disease. Age of onset, clinical variability, speed of progression and affected tissues display wide variability, making a clinical trial design for drug development very complex. The histopathological changes in skeletal muscle tissue are central to the pathogenesis, but they have not been thoroughly elucidated yet. Here we analysed muscle biopsies from a large cohort of BMD patients, focusing our attention on the histopathological muscle parameters, as fibrosis, fatty replacement, fibre cross sectional area, necrosis, regenerating fibres, splitting fibres, internalized nuclei and dystrophy evaluation. We correlated histological parameters with both demographic features and clinical functional evaluations. The most interesting results of our study are the accurate quantification of fibroadipose tissue replacement and the identification of some histopathological aspects that well correlate with clinical performances. Through correlation analysis, we divided our patients into three clusters with well-defined histological and clinical features. In conclusion, this is the first study that analyses in detail the histological characteristics of muscle biopsies in a large cohort of BMD patients, correlating them to a functional impairment. The collection of these data help to better understand the histopathological progression of the disease and can be useful to validate any pharmacological trial in which the modification of muscle biopsy is utilized as outcome measure.

Traumatic Brain Injury Triggers Neurodegeneration in a Mildly Symptomatic MELAS Patient: Implications on the Detrimental Role of Damaged Mitochondria in Determining Head Trauma Sequalae in the General Population.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a maternally inherited genetic mitochondrial disease with a typical onset in the first two decades of life and a major involvement of central nervous system (CNS). We present the case of a man affected with an oligosymptomatic, genetically determined MELAS syndrome, whose clinical picture dramatically and irreversibly worsened following a mild head injury. We hypothesize that the CNS metabolic stress induced by the brain injury activated an irreversible cascade of events leading to progressive neurodegeneration because damaged mitochondria were unable to restore the balance between energy requirements and availability.

Genetic defects are common in myopathies with tubular aggregates.

OBJECTIVE: A group of genes have been reported to be associated with myopathies with tubular aggregates (TAs). Many cases with TAs still lack of genetic clarification. This study aims to explore the genetic background of cases with TAs in order to improve our knowledge of the pathogenesis of these rare pathological structures. METHODS: Thirty-three patients including two family members with biopsy confirmed TAs were collected. Whole-exome sequencing was performed on 31 unrelated index patients and a candidate gene search strategy was conducted. The identified variants were confirmed by Sanger sequencing. The wild-type and the mutant p.Ala11Thr of ALG14 were transfected into human embryonic kidney 293 cells (HEK293), and western blot analysis was performed to quantify protein expression levels. RESULTS: Eleven index cases (33%) were found to have pathogenic variant or likely pathogenic variants in STIM1, ORAI1, PGAM2, SCN4A, CASQ1 and ALG14. Among them, the c.764A>T (p.Glu255Val) in STIM1 and the c.1333G>C (p.Val445Leu) in SCN4A were novel. Western blot analysis showed that the expression of ALG14 protein was severely reduced in the mutant ALG14 HEK293 cells (p.Ala11Thr) compared with wild type. The ALG14 variants might be associated with TAs in patients with complex multisystem disorders. INTERPRETATION: This study expands the phenotypic and genotypic spectrums of myopathies with TAs. Our findings further confirm previous hypothesis that genes related with calcium signalling pathway and N-linked glycosylation pathway are the main genetic causes of myopathies with TAs.

Engineering an Environment for the Study of Fibrosis: A 3D Human Muscle Model with Endothelium Specificity and Endomysium.

The integration of vascular structures into in vitro cultured tissues provides realistic models of complex tissue-vascular interactions. Despite the incidence and impact of muscle-wasting disorders, advanced in vitro systems are still far from recapitulating the environmental complexity of skeletal muscle. Our model comprises differentiated human muscle fibers enveloped by a sheath of human muscle-derived fibroblasts and supported by a vascular network with mural-like cells. Here, we demonstrate the induction of muscle-specific endothelium and the self-organization of endomysial muscle fibroblasts mediated by endothelial cells. We use this model to mimic the fibrotic environment characterizing muscular dystrophies and to highlight key signatures of fibrosis that are neglected or underestimated in traditional 2D monocultures. Overall, this vascularized meso-scale cellular construct finely recapitulates the human skeletal muscle environment and provides an advanced solution for in vitro studies of muscle physiology and pathology.