Acquired sporadic late onset nemaline myopathy (SLONM) and inherited nemaline myopathy (iNM) both feature accumulation of nemaline rods in muscle fibers. Unlike iNM, SLONM is amenable to therapy. The distinction between these disorders is therefore crucial when the diagnosis remains ambiguous after initial investigations. We sought to identify biomarkers facilitating this distinction and to investigate the pathophysiology of nemaline rod formation in these different disorders. Twenty-two muscle samples from patients affected by SLONM or iNM underwent quantitative histological analysis, laser capture microdissection for proteomic analysis of nemaline rod areas and rod-free areas, and transcriptomic analysis. In all iNM samples, nemaline rods were found in subsarcolemmal or central aggregates, whereas they were diffusely distributed within muscle fibers in most SLONM samples. In SLONM, muscle fibers harboring nemaline rods were smaller than those without rods. Necrotic fibers, increased endomysial connective tissue, and atrophic fibers filled with nemaline rods were more common in SLONM. Proteomic analysis detected differentially expressed proteins between nemaline rod areas and rod-free areas, as well as between SLONM and iNM. These differentially expressed proteins implicated immune, structural, metabolic, and cellular processes in disease pathophysiology. Notably, immunoglobulin overexpression with accumulation in nemaline rod areas was detected in SLONM. Transcriptomic analysis corroborated proteomic findings and further revealed substantial gene expression differences between SLONM and iNM. Overall, we identified unique pathological and molecular signatures associated with SLONM and iNM, suggesting distinct underlying pathophysiological mechanisms. These findings represent a step towards enhanced diagnostic tools and towards development of treatments for SLONM.

Nemaline myopathy (NM) is a slowly progressive or nonprogressive neuromuscular disorder caused by mutations in genes encoding skeletal muscle sarcomeric thin filament proteins. It is characterized by great heterogeneity at the clinical, histopathological, and genetic level. Although multiple molecular pathways are commonly affected in all NM patients, little is known about the molecular characteristics of muscles from patients in different NM subgroups. We have analyzed a group of global gene expression data sets for transcriptional patterns characteristic of particular nemaline myopathy classes. Differential expression between disease subgroups was primarily seen in mitochondrial-, structural-, and transcription-related genes. Multiple lines of evidence support the hypothesis that muscles from cases with "nontyping" NM, although clinically classified as typical NM, share a unique pathophysiological state and are characterized by distinct patterns of gene expression. Determination of the specific molecular differences in NM subgroups may eventually lead to improved prognostic determinations and treatment of these patients.

The congenital myopathies are a heterogeneous group of inherited neuromuscular disorders characterized by early-onset muscular weakness, hypotonia, and developmental delay. The congenital myopathies are further classified into centranuclear myopathies, nemaline myopathies, core myopathies, and congenital fiber-type disproportion based on major pathological features found in muscle biopsies. There is no evidence at present to show that cleft palate is associated with severe nemaline myopathies and fetal akinesia. The results of our study suggest that KLHL40 mutation associated with Cleft palate, nemaline myopathy and fetal akinesia.

Nemaline myopathies (NM) are rare and severe muscle diseases characterized by the presence of nemaline bodies (rods) in muscle fibers. Although ten genes have been implicated in the etiology of NM, an important number of patients remain without a molecular diagnosis.

The nemaline myopathies are characterized by weakness and eosinophilic, rodlike (nemaline) inclusions in muscle fibers. Amish nemaline myopathy is a form of nemaline myopathy common among the Old Order Amish. In the first months of life, affected infants have tremors with hypotonia and mild contractures of the shoulders and hips. Progressive worsening of the proximal contractures, weakness, and a pectus carinatum deformity develop before the children die of respiratory insufficiency, usually in the second year. The disorder has an incidence of approximately 1 in 500 among the Amish, and it is inherited in an autosomal recessive pattern. Using a genealogy database, automated pedigree software, and linkage analysis of DNA samples from four sibships, we identified an approximately 2-cM interval on chromosome 19q13.4 that was homozygous in all affected individuals. The gene for the sarcomeric thin-filament protein, slow skeletal muscle troponin T (TNNT1), maps to this interval and was sequenced. We identified a stop codon in exon 11, predicted to truncate the protein at amino acid 179, which segregates with the disease. We conclude that Amish nemaline myopathy is a distinct, heritable, myopathic disorder caused by a mutation in TNNT1.

Nemaline myopathy (NM), the most common of the congenital myopathies, is caused by various genetic mutations. In this study, we attempted to identify the causative mutations of NM and to reveal any specific genotype-phenotype relationship in Korean patients with this disease. We investigated the clinical features and genotypes in 15 pathologically diagnosed NM patients, using whole exome sequencing (WES) combined with targeted sequencing and array-based comparative genomic hybridization. This strategy revealed pathogenic causative mutations in seven patients (46.7%), among whom mutations in the nebulin gene (NEB) were the most frequent (5 patients, 33.3%). Copy number variation (CNV) abnormality in NEB was not observed in any of our patients. In those with NEB-associated NM, the clinical spectrum was highly variable regardless of the mutation type. However, the majority of patients showing anterior lower leg weakness were associated with mutations located between NEB exons 166 and 177. We concluded that the combination of WES and targeted Sanger sequencing is an effective strategy for analyzing genotypes in patients with NM, and that CNV in NEB may not be a frequent cause of this disease among Koreans.

Nemaline myopathies are heterogeneous congenital muscle disorders causing skeletal muscle weakness and, in some cases, death soon after birth. Mutations in nebulin, encoding a large sarcomeric protein required for thin filament function, are responsible for approximately 50% of nemaline myopathy cases. Despite the severity of the disease there is no effective treatment for nemaline myopathy with limited research to develop potential therapies. Several supplements, including L-tyrosine, have been suggested to be beneficial and consequently self-administered by nemaline myopathy patients without any knowledge of their efficacy. We have characterized a zebrafish model for nemaline myopathy caused by a mutation in nebulin. These fish form electron-dense nemaline bodies and display reduced muscle function akin to the phenotypes observed in nemaline myopathy patients. We have utilized our zebrafish model to test and evaluate four treatments currently self-administered by nemaline myopathy patients to determine their ability to increase skeletal muscle function. Analysis of muscle pathology and locomotion following treatment with L-tyrosine, L-carnitine, taurine, or creatine revealed no significant improvement in skeletal muscle function emphasizing the urgency to develop effective therapies for nemaline myopathy.

Nemaline myopathy, one of the most common congenital myopathies, is associated with mutations in various genes including ACTA1. This disease is also characterized by various forms/degrees of muscle weakness, with most cases being severe and resulting in death in infancy. Recent findings have provided valuable insight into the underlying pathophysiological mechanisms. Mutations in ACTA1 directly disrupt binding interactions between actin and myosin, and consequently the intrinsic force-generating capacity of muscle fibers. ACTA1 mutations are also associated with variations in myofiber size, the mechanisms of which have been unclear. In the present study, we sought to test the hypotheses that the compromised functional and morphological attributes of skeletal muscles bearing ACTA1 mutations (1) would be directly due to the inefficient actomyosin complex and (2) could be restored by manipulating myosin expression.

Nemaline Myopathy (NM) is a rare genetic disorder that encompasses a large spectrum of myopathies characterized by hypotonia and generalized muscle weakness. To date, mutations in thirteen different genes have been associated with NM. The most frequently responsible genes are NEB (50% of cases) and ACTA1 (15-25% of cases). In this report all known NM related genes were screened by Next Generation Sequencing in five Spanish patients in order to genetically confirm the clinical and histological diagnosis of NM. Four mutations in NEB (c.17779_17780delTA, c.11086A>C, c.21076C>T and c.2310+5G>A) and one mutation in ACTA1 (c.871A>T) were found in four patients. Three of the four mutations in NEB were novel. A cDNA sequencing assay of the novel variants c.17779_17780delTA, c.11086A>C and c.2310+5G>A revealed that the intronic variant c.2310+5G>A affected the splicing process. Mutations reported here could help clinicians and geneticists in NM diagnosis.

Nemaline myopathies are a clinically and genetically heterogeneous group of congenital myopathies, mainly characterized by muscle weakness, hypotonia and respiratory insufficiency. Here, we report a male foetus of consanguineous parents with a severe congenital syndrome characterized by arthrogryposis detected at 13 weeks of gestation. We describe severe complex dysmorphic facial and musculoskeletal features by post mortem fetal examination confirming the prenatal diagnosis. Histomorphological and ultrastructural studies of skeletal muscle reveal mini-rods in myotubes caused by a novel homozygous splice-site mutation in NEB (NM_001164508, chr2:g.152,417,623C>A GRCh37.p11 | c.19,102-1G>T ENST00000397345.3). No rods were seen in the myocardium. We discuss the relevance of this mutation in the context of nemaline myopathies associated with early developmental musculoskeletal disorders.

We have studied the expression and distribution patterns of the intermediate filament proteins desmin and vimentin, the sarcomere components titin, nebulin and myosin, the basement membrane constituents collagen type IV and laminin, and the reticular layer component collagen type VI in skeletal muscle of patients with "classic" congenital myopathies (CM), using indirect immunofluorescence assays. In all biopsy specimens obtained from patients with central core disease (CCD), nemaline myopathy (NM), X-linked myotubular myopathy (XLMTM) and centronuclear myopathy (CNM), disease-specific desmin disturbances were observed. Vimentin was present in immature fibres in severe neonatal NM, and as sarcoplasmic aggregates in one case of CNM, while the amounts of vimentin and embryonic myosin, observed in XLMTM, decreased with age of the patients. Abnormal expression of myosin isoforms was found in several CM biopsies, although the organization of myosin and other sarcomere components was rarely disturbed. Basement membrane and reticular layer proteins were often prominently increased in severe cases of CM. We conclude that (i) desmin is a marker for individual types of CM and might be used for diagnostic purposes; (ii) the expression patterns of the differentiation markers desmin, vimentin and embryonic myosin in XLMTM, point either to a postnatal muscle fibre maturation or to a variable time-point of maturational arrest in individual patients; (iii) the correlation between the distribution patterns of extracellular matrix proteins and clinical presentation points to a role of these proteins in pathophysiology of CM.

Nemaline myopathy is among the most common non-dystrophic congenital myopathies, and is characterized by the presence of nemaline rods in skeletal muscles fibers, general muscle weakness, and hypotonia. Although respiratory failure is the main cause of death in nemaline myopathy, only little is known regarding the contractile strength of the diaphragm, the main muscle of inspiration. To investigate diaphragm contractility, in the present study we took advantage of a mouse model for nebulin-based nemaline myopathy that we recently developed. In this mouse model, exon 55 of Neb is deleted (NebΔExon55), a mutation frequently found in patients. Diaphragm contractility was determined in permeabilized muscle fibers and was compared to the contractility of permeabilized fibers from three peripheral skeletal muscles: soleus, extensor digitorum longus, and gastrocnemius. The force generating capacity of diaphragm muscle fibers of NebΔExon55 mice was reduced to 25% of wildtype levels, indicating severe contractile weakness. The contractile weakness of diaphragm fibers was more pronounced than that observed in soleus muscle, but not more pronounced than that observed in extensor digitorum longus and gastrocnemius muscles. The reduced muscle contractility was at least partly caused by changes in cross-bridge cycling kinetics which reduced the number of bound cross-bridges. The severe diaphragm weakness likely contributes to the development of respiratory failure in NebΔExon55 mice and might explain their early, postnatal death.

Nemaline myopathy is one of the most common non-dystrophic congenital myopathies. Individuals affected by this condition experience muscle weakness and muscle smallness, often requiring supportive measures like wheelchairs or respiratory support. A significant proportion of patients, approximately one-third, exhibit compound heterozygous nebulin mutations, which usually give rise to the typical form of the disease. Currently, there are no approved treatments available for nemaline myopathy. Our research explored the modulation of myostatin, a negative regulator of muscle mass, in combating the muscle smallness associated with the disease. To investigate the effect of myostatin inhibition, we employed a mouse model with compound heterozygous nebulin mutations that mimic the typical form of the disease. The mice were treated with mRK35, a myostatin antibody, through weekly intraperitoneal injections of 10 mg/kg mRK35, commencing at two weeks of age and continuing until the mice reached four months of age. The treatment resulted in an increase in body weight and an approximate 20% muscle weight gain across most skeletal muscles, without affecting the heart. The minimum Feret diameter of type IIA and IIB fibers exhibited an increase in compound heterozygous mice, while only type IIB fibers demonstrated an increase in wild-type mice. In vitro mechanical experiments conducted on intact extensor digitorum longus muscle revealed that mRK35 augmented the physiological cross-sectional area of muscle fibers and enhanced absolute tetanic force in both wild-type and compound heterozygous mice. Furthermore, mRK35 administration improved grip strength in treated mice. Collectively, these findings indicate that inhibiting myostatin can mitigate the muscle deficits in nebulin-based typical nemaline myopathy, potentially serving as a much-needed therapeutic option.

Nemaline myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle-specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as cap myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found 4 variants significantly affected muscle development and muscle function. Transient overexpression of the 4 variants also disrupted the morphogenesis of mouse myotubes in vitro and negatively affected zebrafish muscle development in vivo. We used transient overexpression assays in zebrafish to characterize 2 potentially novel TPM2 variants and 1 recurring variant that we identified in patients with DA (V129A, E139K, A155T, respectively) and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in our assays correlated with the severity of clinical phenotypes observed in our patients with DA, suggesting disrupted myogenesis is a potentially novel pathomechanism of TPM2 disorders and that our myogenic assays can predict the clinical severity of TPM2 variants.

Nemaline myopathy is an inherited muscle disease that is mainly diagnosed by the presence of nemaline rods in muscle biopsies. Of the nine genes associated with the disease, five encode components of striated muscle sarcomeres. In a genetic zebrafish screen, the mutant träge (trg) was isolated based on its reduction in muscle birefringence, indicating muscle damage. Myofibres in trg appeared disorganised and showed inhomogeneous cytoplasmic eosin staining alongside malformed nuclei. Linkage analysis of trg combined with sequencing identified a nonsense mutation in tropomodulin4 (tmod4), a regulator of thin filament length and stability. Accordingly, although actin monomers polymerize to form thin filaments in the skeletal muscle of tmod4(trg) mutants, thin filaments often appeared to be dispersed throughout myofibres. Organised myofibrils with the typical striation rarely assemble, leading to severe muscle weakness, impaired locomotion and early death. Myofibrils of tmod4(trg) mutants often featured thin filaments of various lengths, widened Z-disks, undefined H-zones and electron-dense aggregations of various shapes and sizes. Importantly, Gomori trichrome staining and the lattice pattern of the detected cytoplasmic rods, together with the reactivity of rods with phalloidin and an antibody against actinin, is reminiscent of nemaline rods found in nemaline myopathy, suggesting that misregulation of thin filament length causes cytoplasmic rod formation in tmod4(trg) mutants. Although Tropomodulin4 has not been associated with myopathy, the results presented here implicateTMOD4 as a novel candidate for unresolved nemaline myopathies and suggest that the tmod4(trg) mutant will be a valuable tool to study human muscle disorders.

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ~30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.

Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, NebY2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, NebY2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.

Nemaline myopathy, a disease of the actin-based thin filament, is one of the most frequent congenital myopathies. To date, no specific therapy is available to treat muscle weakness in nemaline myopathy. We tested the ability of tirasemtiv, a fast skeletal troponin activator that targets the thin filament, to augment muscle force-both in vivo and in vitro-in a nemaline myopathy mouse model with a mutation (H40Y) in Acta1. In Acta1H40Y mice, treatment with tirasemtiv increased the force response of muscles to submaximal stimulation frequencies. This resulted in a reduced energetic cost of force generation, which increases the force production during a fatigue protocol. The inotropic effects of tirasemtiv were present in locomotor muscles and, albeit to a lesser extent, in respiratory muscles, and they persisted during chronic treatment, an important finding as respiratory failure is the main cause of death in patients with congenital myopathy. Finally, translational studies on permeabilized muscle fibers isolated from a biopsy of a patient with the ACTA1H40Y mutation revealed that at physiological Ca2+ concentrations, tirasemtiv increased force generation to values that were close to those generated in muscle fibers of healthy subjects. These findings indicate the therapeutic potential of fast skeletal muscle troponin activators to improve muscle function in nemaline myopathy due to the ACTA1H40Y mutation, and future studies should assess their merit for other forms of nemaline myopathy and for other congenital myopathies.

Nemaline myopathies (NMs) are a group of congenital muscle diseases caused by mutations in at least 10 genes and associated with a range of clinical symptoms. NM is defined on muscle biopsy by the presence of cytoplasmic rod-like structures (nemaline rods) composed of cytoskeletal material. Myofiber smallness is also found in many cases of NM and may represent a cause of weakness that can be counteracted by treatment. We have used i.p. injection of activin type IIB receptor (ActRIIB)-mFc (an inhibitor of myostatin signaling) to promote hypertrophy and increase strength in our prior murine work; we therefore tested whether ActRIIB-mFc could improve weakness in NM mice through myofiber hypertrophy. We report a study of ActRIIB-mFc treatment in the Acta1 H40Y mouse model of NM. Treatment of Acta1 H40Y mice produced significant increases in body mass, muscle mass, quadriceps myofiber size, and survival, but other measurements of strength (forelimb grip strength, ex vivo measurements of contractile function) did not improve. Our studies also identified that the complications of urethral obstruction are associated with mortality in male hemizygote Acta1 H40Y mice. The incidence of urethral obstruction and histologic evidence of chronic obstruction (inflammation) were significantly lower in Acta1 H40Y mice that had been treated with ActRIIB-mFc. ActRIIB-mFc treatment produces a mild benefit to the disease phenotype in Acta1 H40Y mice.

Nemaline myopathy (NM) constitutes a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of recessively inherited NM. NEB is one of the most largest genes in human. To date, 68 NEB mutations, mainly small deletions or point mutations have been published. The only large mutation characterized is the 2.5 kb deletion of exon 55 in the Ashkenazi Jewish population. To investigate any copy number variations in this enormous gene, we designed a novel custom comparative genomic hybridization microarray, NM-CGH, targeted towards the seven known genes causative for NM. During the validation of the NM-CGH array we identified two novel deletions in two different families. The first is the largest deletion characterized in NEB to date, (∼53 kb) encompassing 24 exons. The second deletion (1 kb) covers two exons. In both families, the copy number change was the second mutation to be characterized and shown to have been inherited from one of the healthy carrier parents. In addition to these novel mutations, copy number variation was identified in four samples in three families in the triplicate region of NEB. We conclude that this method appears promising for the detection of copy number variations in NEB.