Cause Of Nemaline Myopathy Research Articles

Generation of an induced pluripotent stem cell line from a 3-month-old nemaline myopathy patient with a heterozygous dominant c.515C > A (p.Ala172Glu) variant in the ACTA1 gene

Variants in the ACTA1 gene are a common cause of nemaline myopathy (NM); a muscle disease that typically presents at birth or early childhood with hypotonia and muscle weakness. Here, we generated an induced pluripotent stem cell line (iPSC) from lymphoblastoid cells of a 3-month-old female patient with intermediate NM caused by a dominant ACTA1 variant (c.515C > A (p.Ala172Glu)). iPSCs showed typical morphology, expressed pluripotency markers, demonstrated trilineage differentiation potential, and had a normal karyotype. This line complements our previously published ACTA1 iPSC lines derived from patients with typical and severe NM.

Lmod3 promotes myoblast differentiation and proliferation via the AKT and ERK pathways

Mutations in the Lmod3 gene have been identified as a genetic cause of nemaline myopathy. However, the mechanism underlying this disease and the function of Lmod3 remain largely unknown. In this study, we found that Lmod3 knockdown in C2C12 cells impaired myoblast differentiation, whereas enforced Lmod3 expression enhanced such differentiation. We also discovered that myoblast proliferation was promoted by Lmod3 overexpression but impeded by its knockdown. Additionally, knockdown of Lmod3 led to apoptosis in myoblasts. Concurrently, forced Lmod3 expression in C2C12 cells contributed to activation of the AKT and ERK pathways during myoblast differentiation and proliferation, respectively. Conversely, knockdown of Lmod3 in C2C12 cells produced the opposite results. Furthermore, administration of IGF-1, a booster of both AKT and ERK pathways, partially rescued the inhibitory effect of Lmod3 knockdown on both differentiation and proliferation of C2C12 cells. These results suggest that Lmod3 promotes differentiation and proliferation of myoblasts through the AKT and ERK pathways, respectively.

Novel Recessive TNNT1 Congenital Core-Rod Myopathy in French Canadians.

Recessive null variants of the slow skeletal muscle troponin T1 (TNNT1) gene are a rare cause of nemaline myopathy that is fatal in infancy due to respiratory insufficiency. Muscle biopsy shows rods and fiber type disproportion. We report on 4 French Canadians with a novel form of recessive congenital TNNT1 core-rod myopathy. Patients underwent full clinical characterization, lower limb magnetic resonance imaging (MRI), muscle biopsy, and genetic testing. A zebrafish loss-of-function model using morpholinos was created to assess the pathogenicity of the identified variant. Wild-type or mutated human TNNT1 mRNAs were coinjected with morpholinos to assess their abilities to rescue the morphant phenotype. Three adults and 1 child shared a novel missense homozygous variant in the TNNT1 gene (NM_003283.6: c.287T > C; p.Leu96Pro). They developed from childhood very slowly progressive limb-girdle weakness with rigid spine and disabling contractures. They suffered from restrictive lung disease requiring noninvasive mechanical ventilation in 3 patients, as well as recurrent episodes of rhabdomyolysis triggered by infections, which were relieved by dantrolene in 1 patient. Older patients remained ambulatory into their 60s. MRI of the leg muscles showed fibrofatty infiltration predominating in the posterior thigh and the deep posterior leg compartments. Muscle biopsies showed multiminicores and lobulated fibers, rods in half the patients, and no fiber type disproportion. Wild-type TNNT1 mRNA rescued the zebrafish morphants, but mutant transcripts failed to do so. This study expands the phenotypic spectrum of TNNT1 myopathy and provides functional evidence for the pathogenicity of the newly identified missense mutation. ANN NEUROL 2020;87:568-583.

P.166Retrospective longitudinal study of patients with NEB-related nemaline myopathy in the United Kingdom

Recessive mutations in the NEB gene encoding nebulin are the most common known cause of nemaline myopathy (NEB-NM). Natural history studies on NEB-NM are limited and mostly focused on cross sectional data. We present a retrospective longitudinal study of 24 NEB-NM patients seen at the Dubowitz Neuromuscular Centre and the Evelina Children's Hospital, London. Age range was 2.93-27 years (median 17.34 yrs), follow up range 1.75-12.25 years (median 4.75 yrs), with a median of 5 yearly follow-up/patients (2-13 yrs). All patients were homozygous or compound heterozygous for 2 NEB variants. Altogether, 26 variants were identified, 4 missense, 17 frameshift, 5 splice site. Longitudinal motor function scores on the Hammersmith Functional Motor Scale were available for 13 patients, with a median of 4 assessments/child. Two patients with severe NEB-NM never sat independently, 2 with the typical form lost ambulation at 6 and 12 years, respectively. All other patients were ambulant at last follow-up. 17 patients (70%) developed scoliosis and/or kyphosis, 7 had spinal surgery at a median age of 15 years (11-16.42 yrs). Forced vital capacity % trends were available for 12 patients, with a median of 4 yearly measurements/child. 9/24 (37.5%) patients required nocturnal non-invasive ventilation at a median age of 8 years (2-18.5 yrs). Two patients with severe NEB-NM required tracheostomy before age 6 months and one, with typical form, after spinal surgery at 11 years. One patient died aged 19 years due to respiratory complications. Weight trends were available for 16 patients, with a median of 4.5 measurements/child. Gastrostomy was needed in 11/24 (46%) at a median age of 4 years (0-15.75 yrs). Statistical analysis is now in progress to describe trajectories and calculate estimated change/year of motor abilities, FVC and weight. This study expands knowledge on clinical course of NEB-NM and further emphasizes the importance of long-term multidisciplinary assessment and management.

P.028 A milder congenital myopathy in the french canadians caused by a novel TNNT1 homozygous missense mutation

Background: Mutations of the slow skeletal muscle troponin-T1 (TNNT1) gene are a rare cause of nemaline myopathy. The phenotype is characterized by severe amyotrophy and contractures. Death from respiratory insufficiency occurs in infancy. We report on four French Canadians with a novel congenital TNNT1-related myopathy. Methods: Patients underwent MRI of leg muscles, quadriceps biopsy and genetic testing. Wild type or mutated human TNNT1 mRNAs were co-injected with morpholinos in a zebrafish knockdown model to assess their relative abilities to rescue the morphant phenotype. Results: Three adults and one child shared a novel missense homozygous pathogenic variant in the TNNT1 gene. They developed from childhood slowly progressive limb-girdle weakness with spinal rigidity and contractures. They suffered from restrictive lung disease and recurrent episodes of infection-triggered rhabdomyolysis, which were relieved by dantrolene in one patient. Older patients remained ambulatory into their sixties. MRI of leg muscles showed symmetrical atrophy and fatty infiltration in a proximal-to-distal gradient. Biopsies showed multi-minicores, while nemaline rods were seen in half the patients. Wild type TNNT1 mRNA rescued the zebrafish morphants but mutant transcripts failed to rescue the morphants. Conclusions: This study expands the spectrum of TNNT1-related myopathy to include a milder clinical phenotype caused by a functionally-confirmed novel missense mutation.

Two alternatively-spliced human nebulin isoforms with either exon 143 or exon 144 and their developmental regulation

Nebulin is a very large protein required for assembly of the contractile machinery in muscle. Mutations in the nebulin gene NEB are a common cause of nemaline myopathy. Nebulin mRNA is alternatively-spliced so that each mRNA contains either exon 143 or exon 144. We have produced monoclonal antibodies specific for the regions of nebulin encoded by these two exons, enabling analysis of expression of isoforms at the protein level for the first time. All antibodies recognized a protein of the expected size (600–900 kD) and stained cross-striations of sarcomeres in muscle sections. Expression of exon 143 is developmentally-regulated since newly-formed myotubes in cell culture expressed nebulin with exon 144 only; this was confirmed at the mRNA level by qPCR. In fetal muscle, nebulin with exon 143 was expressed in some myotubes by 12-weeks of gestation and strongly-expressed in most myotubes by 17-weeks. In mature human muscle, the exon 144 antibody stained all fibres, but the exon 143 antibody staining varied from very strong in some fibres to almost-undetectable in other fibres. The results show that nebulin containing exon 144 is the default isoform early in myogenesis, while regulated expression of nebulin containing exon 143 occurs at later stages of muscle development.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.230Clinical, genetic and pathological characterization of a wide cohort of UK patients with NEB gene related nemaline myopathy

Recessive NEB gene mutations are the most common cause of nemaline myopathy (NM), characterized by early onset generalized weakness and nemaline rods on muscle histopathology. Here we report a clinical, pathological, and genetic evaluation of 46 patients (42 families) with NEB-related NM referred for diagnosis to the Dubowitz Neuromuscular Centre. NEB gene analysis was performed by next generation sequencing. Review of available clinical data suggests a typical course in 26/38 patients, with onset at birth/first year of life, hypotonia and/or developmental delay. All 26 achieved independent ambulation and showed prominent axial weakness, 7 with severe foot drop. Scoliosis and/or kyphosis was observed in 13/26 patients. 9/25 needed overnight respiratory support, 10/24 had dysphagia (9 needing a PEG), and 10/20 variable speech involvement. Seven/38 patients showed more severe prenatal/neonatal onset, with early termination of pregnancy in one case and death < 6 months in 2. The remaining patients showed severe skeletal and respiratory weakness. Finally, 5/38 individuals had a later onset (>2 years of age) and followed a milder disease course. 32 patients were homozygous or compound heterozygotes for 2 NEB variants. Phasing of variants is in progress in 14 patients with 2 NEB variants. Altogether, we identified 53 variants, 16 frameshift, 10 stop and 10 splice site. Muscle biopsies were available in 16 patients. Frequent nemaline rods (14/16) and slow fibre predominance (9/16) were common findings. In 2 cases no rods were seen on light microscopy. The pathological spectrum also included increased central nuclei (2 cases), frequent cytoplasmic bodies (2 cases), and features of rod-core myopathy (2 cases). Although further deep phenotypic analysis is in progress, the available results confirm the wide clinical and pathological spectrum of NEB-related NM and further expand current knowledge on this rare condition.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.229Genetics and modeling of TNNT1 genetic variants in nemaline myopathy

TNNT1-related nemaline myopathy (NEM5) comprises a small but increasingly significant cause of nemaline myopathy. Initially described as a severe autosomal recessive condition confined to the Old Order Amish of Pennsylvania in the US, subsequent patients with severe phenotypes have been described throughout the world. The prototypic clinical features include congenital hypotonia and weakness of proximal, bulbar, facial, and neck flexor muscles that may result in death secondary to respiratory insufficiency. This condition appears to exhibit allelic heterogeneity with multiple mutations within the same gene causing the severe recessive form of the disease. We recently described the first autosomal dominant c.311A>T, p.E104V pathogenic variant of TNNT1 gene occurring in an Ashkenazi Jewish family in the United States. This family had an overall milder phenotype than the recessive cases with marked clinical heterogeneity ranging from a Gower's maneuver in childhood to mild difficulty weight training late into adulthood. Recent whole exome sequencing studies in a large cohort of congenital myopathy cases have identified two additional unrelated cases with potentially pathogenic dominant TNNT1 mutations as well as two new instances of possible recessive variants. To assess pathogenicity of these variants we are establishing zebrafish models of both dominant gain of function and recessive loss of function for this gene. Morpholino-based knock down studies lead to significant neuromuscular abnormalities, including poor swimming ability, truncal curvature, and loss of birefringence, all indicative of defective muscle structure and function within the first week of life. Studies to model the dominant variants are in progress.

Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3).

Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.

Nebulin Deficiency in Adult Muscle Causes Sarcomeric Defects and Muscle-Type Dependent Changes in Trophicity -- Novel Insights in Nemaline Myopathy

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods, and undergo fiber-type switching towards oxidative types. Nebulin deficiency causes a large deficit in specific-force and mechanistic studies provide evidence that a reduced fraction of force-generating crossbridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas, worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of nemaline myopathy (muscle weakness, oxidative fiber type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy.

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods and undergo fiber-type switching toward oxidative types. Nebulin deficiency causes a large deficit in specific force, and mechanistic studies provide evidence that a reduced fraction of force-generating cross-bridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas (CSAs), worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even have hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of NEM (muscle weakness, oxidative fiber-type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

Effect of levosimendan on the contractility of muscle fibers from nemaline myopathy patients with mutations in the nebulin gene

BackgroundNemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM.MethodsTo test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured.ResultsNo effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested.ConclusionsThese findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0037-7) contains supplementary material, which is available to authorized users.

Mutation update: the spectra of nebulin variants and associated myopathies.

A mutation update on the nebulin gene (NEB) is necessary because of recent developments in analysis methodology, the identification of increasing numbers and novel types of variants, and a widening in the spectrum of clinical and histological phenotypes associated with this gigantic, 183 exons containing gene. Recessive pathogenic variants in NEB are the major cause of nemaline myopathy (NM), one of the most common congenital myopathies. Moreover, pathogenic NEB variants have been identified in core-rod myopathy and in distal myopathies. In this update, we present the disease-causing variants in NEB in 159 families, 143 families with NM, and 16 families with NM-related myopathies. Eighty-eight families are presented here for the first time. We summarize 86 previously published and 126 unpublished variants identified in NEB. Furthermore, we have analyzed the NEB variants deposited in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), identifying that pathogenic variants are a minor fraction of all coding variants (∼7%). This indicates that nebulin tolerates substantial changes in its amino acid sequence, providing an explanation as to why variants in such a large gene result in relatively rare disorders. Lastly, we discuss the difficulties of drawing reliable genotype-phenotype correlations in NEB-associated disease.

G.O.2: Mutations in LMOD3 cause severe nemaline myopathy by disrupting thin filament organisation in skeletal muscle

Nemaline myopathy (NM) is a disorder of the skeletal muscle thin filament characterised by muscle dysfunction and electron-dense protein accumulations (nemaline bodies). Pathogenic mutations have been described in nine genes to date, but the genetic basis remains unknown in many cases. We used whole exome sequencing (WES) in two families with NM and subsequent gene sequencing in over 540 additional genetically unresolved NM patients to identify and characterise a new genetic cause of NM. We developed a knock-down zebrafish model of this condition and used immunohistochemistry, western blotting, single-fibre contractility studies and recombinant protein studies to characterise the expression, localisation and biochemical functions of the new disease-related protein. We identified homozygous or compound heterozygous variants in LMOD3, which encodes leiomodin-3 (Lmod3) in 21 patients from 14 families. Affected individuals had severe generalised weakness and hypotonia, and most affected individuals died in the neonatal period. We demonstrated that Lmod3 is expressed from early muscle differentiation, localises to thin filaments with enrichment at the pointed ends, and has strong actin nucleating activity. Loss of Lmod3 in patient muscle results in shortening and disorganisation of thin filaments. Knockdown of lmod3 in the zebrafish replicates this phenotype. These findings define a new genetic subtype of congenital myopathy and demonstrate an essential, previously unrecognised role for Lmod3 in the regulation of sarcomeric thin filaments in skeletal muscle.

The nebulin repeat protein Lasp regulates I-band architecture and filament spacing in myofibrils.

Mutations in nebulin, a giant muscle protein with 185 actin-binding nebulin repeats, are the major cause of nemaline myopathy in humans. Nebulin sets actin thin filament length in sarcomeres, potentially by stabilizing thin filaments in the I-band, where nebulin and thin filaments coalign. However, the precise role of nebulin in setting thin filament length and its other functions in regulating power output are unknown. Here, we show that Lasp, the only member of the nebulin family in Drosophila melanogaster, acts at two distinct sites in the sarcomere and controls thin filament length with just two nebulin repeats. We found that Lasp localizes to the Z-disc edges to control I-band architecture and also localizes at the A-band, where it interacts with both actin and myosin to set proper filament spacing. Furthermore, introducing a single amino acid change into the two nebulin repeats of Lasp demonstrated different roles for each domain and established Lasp as a suitable system for studying nebulin repeat function.

KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy

Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40-/- mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients.

Nemaline myopathy caused by TNNT1 mutations in a Dutch pedigree

Nemaline myopathy (NM) is genetically heterogeneous disorder characterized by early onset muscular weakness and sarcoplasmatic or intranuclear inclusions of rod-shaped Z-disk material in muscle fibers. Thus far, mutations in seven genes have been identified as cause of NM. Only one singleTNNT1 nonsense mutation has been previously described that causes autosomal recessive NM in the old order Amish with a very specific clinical phenotype including rapidly progressive contractures. Here, we report a patient who is compound heterozygous for a c.309+1G>A mutation and an exon 14 deletion in theTNNT1 gene. This report confirms the specific clinical phenotype ofTNNT1 NM and documents two newTNNT1 mutations outside the old order Amish.

P.9.10 Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy

Nebulin – a giant sarcomeric protein – plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb Δex55 ) to replicate a founder mutation seen frequently in NM patients with Ashkenazi Jewish heritage. Neb Δex55 mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies – a hallmark feature of NM – in muscle fibers from Neb Δex55 mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb ΔExon55 mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibers and single myofibrils is reduced in Neb Δex55 mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb Δex55 mice the regulation of contraction is impaired, as evidenced by marked changes in cross bridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based NM model, which recapitulates important features of the phenotype observed in patients harboring this particular mutation, and which has severe muscle weakness caused by thin filament dysfunction.

Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy

Nebulin--a giant sarcomeric protein--plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb(ΔExon55)) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. Neb(ΔExon55) mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies--a hallmark feature of nemaline myopathy--in muscle fibres from Neb(ΔExon55) mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb(ΔExon55) mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in Neb(ΔExon55) mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb(ΔExon55) mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by thin filament dysfunction.

Nebulin (NEB) mutations in a childhood onset distal myopathy with rods and cores uncovered by next generation sequencing

Recessive nebulin (NEB) mutations are a common cause of nemaline myopathy (NM), typically characterized by generalized weakness of early-onset and nemaline rods on muscle biopsy. Exceptional adult cases with additional cores and an isolated distal weakness have been reported. The large NEB gene with 183 exons has been an obstacle for the genetic work-up. Here we report a childhood-onset case with distal weakness and a core-rod myopathy, associated with recessive NEB mutations identified by next generation sequencing (NGS). This 6-year-old boy presented with a history of gross-motor difficulties following a normal early development. He had distal leg weakness with bilateral foot drop, as well as axial muscle weakness, scoliosis and spinal rigidity; additionally he required nocturnal respiratory support. Muscle magnetic resonance (MR) imaging showed distal involvement in the medial and anterior compartment of the lower leg. A muscle biopsy featured both rods and cores. Initial targeted testing identified a heterozygous Nebulin exon 55 deletion. Further analysis using NGS revealed a frameshifting 4 bp duplication, c.24372_24375dup (P.Val8126fs), on the opposite allele. This case illustrates that NEB mutations can cause childhood onset distal NM, with additional cores on muscle biopsy and proves the diagnostic utility of NGS for myopathies, particularly when large genes are implicated.