Nemaline Myopathy Research Articles

A pathogenic mechanism associated with myopathies and structural birth defects involves TPM2-directed myogenesis.

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.

Neonatal multiple long bone fractures: A case presentation due to nemaline myopathy and review of other potential causes

Nemaline myopathy is a rare genetic disorder caused by a mutation in genes encoding skeletal muscle proteins resulting in generalized hypotonia. It can be associated with neonatal multiple long bone fractures. We present a female neonate who had bilateral humerus and left femur fractures. Due to fetal distress, her delivery was at 37 weeks gestation by emergency cesarean section. The child was splinted and followed up for 8 weeks. She had a good union of fractured bones, callus formation, and no deformity. Muscle biopsy showed nemaline myopathy. Other biochemical and genetic tests were normal. The aim of this case report was to describe the presentation of multiple long bone fractures in neonates as an obstetric complication. Therefore, identifying the potential risk factors and planning the mode of delivery in future pregnancies, is critical in their management.

A review of major causative genes in congenital myopathies.

In this review, we focus on congenital myopathies, which are a genetically heterogeneous group of hereditary muscle diseases with slow or minimal progression. They are mainly defined and classified according to pathological features, with the major subtypes being core myopathy (central core disease), nemaline myopathy, myotubular/centronuclear myopathy, and congenital fiber-type disproportion myopathy. Recent advances in molecular genetics, especially next-generation sequencing technology, have rapidly increased the number of known causative genes for congenital myopathies; however, most of the diseases related to the novel causative genes are extremely rare. There remains no cure for congenital myopathies. However, there have been recent promising findings that could inform the development of therapy for several types of congenital myopathies, including myotubular myopathy, which indicates the importance of prompt and correct diagnosis. This review discusses the major causative genes (NEB, ACTA1, ADSSL1, RYR1, SELENON, MTM1, DNM2, and TPM3) for each subtype of congenital myopathies and the relevant latest findings.

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.

Generation of two isogenic induced pluripotent stem cell lines from a 1-month-old nemaline myopathy patient harbouring a homozygous recessive c.121C > T (p.Arg39Ter) variant in the ACTA1 gene

Nemaline myopathy (NM) is a congenital skeletal muscle disorder that typically results in muscle weakness and the presence of rod-like structures (nemaline bodies) in the sarcoplasma and/or in the nuclei of myofibres. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 1-month-old male with severe NM caused by a homozygous recessive mutation in the ACTA1 gene (c.121C > T, p.Arg39Ter). The iPSC lines demonstrated typical morphology, expressed pluripotency markers, exhibited trilineage differentiation potential and displayed a normal karyotype. These isogenic lines represent a potential resource to investigate and model recessive ACTA1 disease in a human context.

Pediatric Nemaline Myopathy: A Systematic Review Using Individual Patient Data.

Nemaline myopathy is a skeletal muscle disease that affects 1 in 50 000 live births. The objective of this study was to develop a narrative synthesis of the findings of a systematic review of the latest case descriptions of patients with NM. A systematic search of MEDLINE, Embase, CINAHL, Web of Science, and Scopus was performed using Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines using the keywords pediatric, child, NM, nemaline rod, and rod myopathy. Case studies focused on pediatric NM and published in English between January 1, 2010, and December 31, 2020, in order to represent the most recent findings. Information was collected about the age of first signs, earliest presenting neuromuscular signs and symptoms, systems affected, progression, death, pathologic description, and genetic changes. Of a total of 385 records, 55 case reports or series were reviewed, covering 101 pediatric patients from 23 countries. We review varying presentations in children ranging in severity despite being caused by the same mutation, in addition to current and future clinical considerations relevant to the care of patients with NM. This review synthesizes genetic, histopathologic, and disease presentation findings from pediatric NM case reports. These data strengthen our understanding of the wide spectrum of disease seen in NM. Future studies are needed to identify the underlying molecular mechanism of pathology, to improve diagnostics, and to develop better methods to improve the quality of life for these patients.

Nemaline myopathy in a six-month-old Pomeranian dog

Nemaline myopathy - a clinically and genetically complex heterogenous group of disorders - is described uncommonly in humans and rarely in animals, and is characterised by progressive muscle weakness. The diagnosis is confirmed by histological and/or ultrastructural identification of subsarcolemmal, thread-like, rod-shaped structures called nemaline rod bodies within more than 40% of skeletal muscle fibres. These rods contain the Z-line protein, α-actinin, that can be effectively stained in skeletal muscles using Gomori or Masson trichrome and negatively stained with periodic acid-Schiff. Similar rod-like bodies have been found in smaller numbers in dogs with endocrine disorders and occasionally in other conditions in humans. This report is of a six-monthold Pomeranian dog which had progressive exercise intolerance over a two-month period associated with severe disuse muscle atrophy of the thoracic limbs, as well as gradual pelvic limb weakness and regurgitation of food. Baseline diagnostics ruled out endocrinopathies and after histological and ultrastructural evaluation of thoracic limb muscles and nerve biopsies confirmed nemaline myopathy. The clinical course, diagnostic test results, ultrastructure of skeletal muscle and peripheral nerve, gross necropsy findings and histopathology using various stains are described and illustrated.

A.3 A novel recessive TNNT1 congenital core-rod myopathy in French Canadians

Background: Mutations in the slow skeletal muscle troponin T (TNNT1) gene cause a congenital nemaline myopathy resulting in death from respiratory insufficiency in early infancy. We report on four French Canadians with a novel congenital TNNT1 myopathy. Methods: Patients underwent lower extremity and paraspinal MRI, quadriceps biopsy and genetic testing. TNNT1 expression in muscle was assessed by quantitative PCR and immunoblotting. Wild type or mutated TNNT1 mRNAs were co-injected with morpholinos in a zebrafish knockdown model to assess for rescue of the morphant phenotype. Results: Four patients shared a novel missense homozygous mutation in TNNT1. They developed from childhood slowly progressive limb-girdle weakness with spinal rigidity and contractures. They suffered from restrictive lung disease and recurrent episodes of rhabdomyolysis. Older patients remained ambulatory into their sixties. Lower extremity MRI showed symmetrical myopathic changes. Paraspinal MRI showed diffuse fibro-fatty involution. Biopsies showed multi-minicores. Nemaline rods were seen in half the patients. TNNT1 mRNA expression was similar in controls and patients, while levels of TNNT1 protein were reduced in patients. Wild type TNNT1 mRNA rescued the zebrafish morphants but mutant transcripts failed to do so. Conclusions: This study expands the spectrum of TNNT1-related myopathy to include a milder clinical phenotype caused by a functionally-confirmed novel mutation.

Clinicopathologic Profiles of Sporadic Late-Onset Nemaline Myopathy: Practical Importance of Anti-α-Actinin Immunostaining.

Background and ObjectivesSporadic late-onset nemaline myopathy (SLONM) is a treatable or otherwise fatal myopathy. Diagnosis of SLONM is still challenging, and no therapeutic consensus has been achieved. Here, we reported the clinicopathologic features and long-term follow-up data of SLONM in a Chinese cohort.MethodsWe performed a retrospective evaluation of clinical, pathologic, and treatment outcomes of 17 patients with SLONM diagnosed between March 1986 and April 2021 at our neuromuscular center. Immunohistochemistry (IHC) with antibodies against 5 Z-disc–associated proteins was performed in the muscle biopsies of SLONM to identify a potential pathologic marker in aid of diagnosis. In comparison, we also performed muscle IHC in patients with selective type II fiber atrophy (n = 22), neurogenic atrophy (n = 22), mitochondrial myopathy (n = 5), immune-mediated necrotizing myopathy (n = 5), and normal controls (n = 5).ResultsMost of the patients exhibited asymmetric limb muscles weakness (71%, 12/17) and neck extensor weakness (53%, 9/17). Immunofixation electrophoresis was performed in 11 patients, and 4 of them were identified with monoclonal gammopathy of undetermined significance (MGUS). EMG from 16 patients demonstrated a myopathic pattern with spontaneous activities in 69% (11/16) of them. Muscle MRI showed preferential involvement of paraspinal, gluteus minimus and medius, semimembranosus, and soleus muscles. Suspected nemaline bodies on modified Gomori trichrome were confirmed by IHC using anti–α-actinin antibody (100%, 17/17), anti-myotilin antibody (94%, 16/17), anti-desmin antibody (94%, 16/17), anti–α-B crystallin antibody (65%, 11/17), and anti-telethonin antibody (18%, 3/17) with various positive rates. Notably, anti–α-actinin IHC showed the highest percentage of strongly positive staining (77%, 13/17), being the only one without negative results. Moderate improvement following autologous stem cell transplantation (ASCT) was noted in 3/4 patients with MGUS; favorable outcomes were also achieved in 6/7 patients without MGUS, including 3 patients with complete recovery who were given a combined treatment of prednisone and another immunosuppressant.DiscussionSLONM is a treatable myopathy with ASCT or traditional immunotherapy, especially when combined with steroids and immunosuppressants. Anti–α-actinin immunostaining is the most reliable pathologic marker to identify rod-bearing fibers, and it should be performed routinely in adult patients with undiagnosed nonnecrotic myopathies.

A custom ddPCR method for the detection of copy number variations in the nebulin triplicate region.

The human genome contains repetitive regions, such as segmental duplications, known to be prone to copy number variation. Segmental duplications are highly identical and homologous sequences, posing a specific challenge for most mutation detection methods. The giant nebulin gene is expressed in skeletal muscle. It harbors a large segmental duplication region composed of eight exons repeated three times, the so-called triplicate region. Mutations in nebulin are known to cause nemaline myopathy and other congenital myopathies. Using our custom targeted Comparative Genomic Hybridization arrays, we have previously shown that copy number variations in the nebulin triplicate region are pathogenic when the copy number of the segmental duplication block deviates two or more copies from the normal number, which is three per allele. To complement our Comparative Genomic Hybridization arrays, we have established a custom Droplet Digital PCR method for the detection of copy number variations within the nebulin triplicate region. The custom Droplet Digital PCR assays allow sensitive, rapid, high-throughput, and cost-effective detection of copy number variations within this region and is ready for implementation a screening method for disease-causing copy number variations of the nebulin triplicate region. We suggest that Droplet Digital PCR may also be used in the study and diagnostics of other segmental duplication regions of the genome.

The Mechanisms of Thin Filament Assembly and Length Regulation in Muscles.

The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial components of the contractile apparatus in muscles. The thin filaments are organized into densely packed lattices interdigitated with myosin-based thick filaments. The crossbridge interactions between these myofilaments drive muscle contraction, and the degree of myofilament overlap is a key factor of contractile force determination. As such, the optimal length of the thin filaments is critical for efficient activity, therefore, this parameter is precisely controlled according to the workload of a given muscle. Thin filament length is thought to be regulated by two major, but only partially understood mechanisms: it is set by (i) factors that mediate the assembly of filaments from monomers and catalyze their elongation, and (ii) by factors that specify their length and uniformity. Mutations affecting these factors can alter the length of thin filaments, and in human cases, many of them are linked to debilitating diseases such as nemaline myopathy and dilated cardiomyopathy.

Congenital myopathies: The current status.

Within the history of neuromuscular diseases (NMD), congenital myopathies (CM) represent a relatively new category introduced in the mid-nineteen hundreds upon advent and subsequent application of enzyme histochemistry and electron microscopy by establishing the three major CM, central core disease, nemaline myopathy, and centronuclear myopathy which later pluralized each when the molecular era began at the end of last century. Quickly, during the following 5 decades, many new CM entities were described, based on muscle biopsies and their CM-characteristic myopathology, the former a prerequisite to recognizing an individual CM, the latter of the nosological hallmark of the individual CM. When the molecular era ushered in immunohistochemistry the spectrum and nosography of CM altered in that some CM became allelic to other cohorts of NMD, e.g., congenital muscular dystrophies, other muscular dystrophies, distal myopathies based on different or identical mutations in the same gene. The nosological spectrum of a defective gene also enlarged by recognizing several entities with mutations in the same gene, and same or similar nosological conditions originated from mutations in different genes. Lately, however, CM were reported which lacked any individual myopathological hallmarks, but were clearly based on molecular defects, a fair number of them being newly identified ones. Few CM still remain without any molecular clarification. This nosographic development rendered the original definition of such new CM questionable and brought uncertainty to their classification and nomenclature.

Electron microscopy in the diagnosis of skeletal muscle disorders: Its utility and limitations.

Electron microscopy (EM) has a substantial role in the diagnosis of skeletal muscle disorders. The ultrastructural changes can be observed in muscle fibers and other components of the muscle tissue. EM serves as a confirmatory tool where the diagnosis is already established by enzyme histochemistry staining. Although it is indispensable in the diagnosis of rare forms of congenital myopathies not appreciated by light microscope, such as cylindrical spiral myopathy, zebra body myopathy, fingerprint body myopathy, and intranuclear rod myopathy, in cases not subjected to histochemical staining, it is required for definitive diagnosis in certain groups of muscle disorders, which includes congenital myopathies, metabolic myopathies in particular mitochondrial myopathies and glycogenosis, and in vacuolar myopathies. It does not have diagnostic implications in muscular dystrophies and neurogenic disorders. In the recent past, despite the availability of advanced diagnostic techniques, electron microscopy continues to play a vital role in the diagnosis of skeletal muscle disorders. This review gives an account of ultrastructural features of skeletal muscle disorders, the role of EM in the diagnosis, and its limitations.

Clinical and molecular analysis of four unrelated Chinese families with pathogenic KLHL40 variants causing nemaline myopathy 8

BackgroundHomozygous or compound heterozygous variants in the KLHL40 gene cause nemaline myopathy 8 (NEM8), a severe autosomal recessive muscle disorder characterized by prenatal polyhydramnios, fetal akinesia or hypokinesia, joint contractures, fractures, respiratory failure and dysphagia. Currently, 46 individuals with NEM8 have been described in the literature, and 30 variants in KLHL40 have been identified.ResultsHere, we reported five individuals from four unrelated Chinese families who presented common features of nemaline myopathy and infrequent clinical characteristics. Whole-exome sequencing (WES) was used to identify the causative gene. WES identified a recurrent missense variant c.1516A>C (p.Thr506Pro) and a novel frameshift variant c.543del (p.Ser182Profs*17) in KLHL40 in patient 1, a nonsense variant c.602G>A (p.Trp201*) and a missense variant c.1516A>C (p.Thr506Pro) in KLHL40 in patient 2, and homozygous variant c.1516A>C (p.Thr506Pro) in KLHL40 in patient 3 and both siblings (patients 4 and 5), all of which were confirmed by Sanger sequencing. Next, we estimated the incidence of this disorder in the southern and northern Chinese population to be 4.59/106 and 2.95/106, respectively, based on the cumulative allele frequency of pathogenic variants in internal database.ConclusionThe results of our study expand the mutation spectrum of KLHL40 and enrich our understanding of the clinical characteristics of NEM8. Genetic counseling was provided for the four families involved in this study. Given the severity and the relatively high incidence of this condition, we strongly suggest that KLHL40 be incorporated into a carrier screening panel for the Chinese population.

Progressing nemaline myopathy in a patient repeatedly operated on the spine: clinical case and literature review

Objective. To present a clinical case of a patient with spinal deformity associated with myopathy, which was initially undiagnosed and assessed as secondary myopathy after performed spinal surgeries.Material and Methods. Study design: clinical observation and review of the literature. A 51-year-old female patient has been repeatedly operated on for scoliotic deformity of the spine with progressing neurological disorders. Clinical and complex radiological assessment (spinal telemetry in sitting position, CT, MRI) of the pathology was performed. In connection with the atypical course of the disease, a pathohistological study of muscle tissue fragments was performed with their fixation in a solution of 10 % neutral formalin. After histological processing, the fragments were embedded in paraffin and stained with hematoxylin-eosin by the Masson trichromic method and by the PTAH method. The preparations were examined using the Pannoramic MIDI II BF hardware and software complex to obtain digital images using the Whole slide imaging technology in the Single layer and Extended focus modes and an AxioScope.A1 stereomicroscope with a digital camera.Results. Two patterns of distribution of nemaline rods of various density and configuration stained dark blue (by PTAH) were identified in sarcoplasm of parts of muscular tissues: diffusely throughout the myocyte and forming clusters of various sizes in transverse sections, and as filamentous structures – in longitudinal ones. Extensive fields of fatty degeneration, transformation of fibers into lipocytes, contracturely changed fibers, pictures of myophaga and areas of fibrosis of the interstitial space associated with residual myocytes were noted. Intramuscular nervous conductors were subjected to complete involution, singular nervous fibers were deformed, internal space was fibrotic. Neuromuscular spindles had unusually increased connective tissue capsule filled with lipocytes. Arterial vessels had fibrosis of t. adventicia and t. media, narrowed or obliterated lumen, and venous vessels were thin-walled and tortuous, which causes their permeability and can cause hemorrhage. The revealed changes in myocytes with the presence of nemaline rods are characteristic of primary nemaline myopathy.Conclusion. The atypicality of clinical manifestations, primarily of the neurological status in scoliosis, requires excluding neuromuscular disease by conducting a histopathological examination of the surgical material.

68-year old man with progressive weakness and ventilator dependent respiratory failure: a case report of sporadic late onset nemaline myopathy

BackgroundNeuromuscular pathologies must be considered when caring for patients with persistent or progressive respiratory failure. Pertinent disease states may involve skeletal muscles of respiration or associated neurologic structures including motor neurons, peripheral neurons and the neuromuscular junction. Diagnosis may require pulmonary function testing, neurophysiologic studies, imaging, and/or muscle biopsy.Case presentationA 68-year-old male was transferred to our intensive care unit (ICU) for management of ventilator dependent respiratory failure. Upon further historical review, he described gradually worsening gait instability and muscle weakness, which was previously attributed to vascular Parkinsonism in the setting of known cerebrovascular disease. Upon arrival to our hospital, he was found to have elevated muscle specific enzymes, prompting evaluation for neuromuscular causes of respiratory failure. He was also found to have elevated HMG-CoA Reductase (HMGCR) antibodies. Ultimately, a right quadriceps muscle biopsy was performed and electron microscopy identified nemaline bodies within skeletal myofibers. Given the clinical course and other histopathologic findings, he was diagnosed with Sporadic late-onset nemaline myopathy (SLONM).ConclusionThe diagnosis of neuromuscular disease in patients with ventilator dependent respiratory failure is challenging. A detailed history of a patient’s clinical course prior to hospitalization is key and may raise suspicion for underlying neuromuscular pathology. Further evaluation in non-critically ill patients may include pulmonary function, electromyography and confirmatory muscle biopsy. Sporadic late onset nemaline myopathy remains a rare disease entity which rarely presents with respiratory failure and lacks effective treatment.

Nemaline myopathy in newly diagnosed systemic lupus erythematosus and Sj\xf6gren\u2019s overlap syndrome complicated by macrophage activation syndrome

BackgroundNemaline myopathies are congenital or acquired muscle disorders that typically present in childhood but can occasionally occur in adults with underlying malignant, infectious or autoimmune disorders. There is a great genetic heterogeneity as well as clinical variability among the disease.Case presentationHere, we present a case of nemaline myopathy in a young woman who was newly diagnosed with systemic lupus erythematosus (SLE) and Sjögren’s overlap syndrome complicated by macrophage activation syndrome (MAS). She had no personal or family history of myopathy and was reporting progressive thigh weakness. A muscle biopsy revealed type 1 myofiber predominance with granular material in atrophic myocytes consistent with nemaline myopathy. Her symptoms markedly improved with immunotherapy for her SLE and MAS supporting the diagnosis of sporadic late-onset nemaline myopathy (SLONM) associated with her autoimmune disease.ConclusionsSLONM is a type of nemaline myopathy that presents in adults and can occasionally be associated with autoimmune disease. In these cases, treatment of the underlying disorder with immunosuppression appears to improve symptoms of myopathy.

Proteomic profiling of sporadic late-onset nemaline myopathy.

ObjectiveTo define the proteomic profile of sporadic late‐onset nemaline myopathy (SLONM) and explore its pathogenesis.MethodsWe performed mass spectrometry on laser‐dissected frozen muscle samples from five patients with SLONM, three of whom with an associated monoclonal protein (MP), and four controls, to determine the proteomic profile of SLONM. Furthermore, we assessed the role of the MP by evaluating the expression of the immunoglobulin light chain variable regions (IGVL).ResultsThere were 294 differentially expressed proteins: 272 upregulated and 22 downregulated. Among the top 100 upregulated proteins, the most common categories were: nuclear or nucleic acid metabolism (24%), extracellular matrix and basal lamina (17%), immune response (13%), and actin dynamics (8%). Downregulated proteins consisted mostly of contractile proteins. Among upregulated proteins, there were 65 with a role related to the immune system, including eight proteins involved in major histocompatibility complex 1 (MHC1) and antigen processing, 15 in MHCII complex and phagocytosis, and 23 in B and/or T‐cell function. Among nine upregulated immunoglobulin proteins, there were two IGVL genes. However, these were also detected in SLONM cases without an MP, with no evidence of clonally dominant immunoglobulin deposition. In muscle sections from SLONM patients, nemaline rods tended to accumulate in atrophic fibers with marked rarefaction of the myofibrils. Increased MHC1 reactivity was present in fibers containing nemaline rods as well as adjacent nonatrophic fibers.ConclusionOur findings suggest that aberrant immune activation is present in SLONM, but do not support a direct causal relationship between the MP and SLONM.

Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin.

In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.

Congenital Nemaline Myopathy with Dense Protein Masses.

Congenital Nemaline Myopathy with Dense Protein Masses.