Spinal Muscular Atrophy Muscle Research Articles

Machine learning-enhanced electrical impedance myography to diagnose and track spinal muscular atrophy progression

Objective.To evaluate electrical impedance myography (EIM) in conjunction with machine learning (ML) to detect infantile spinal muscular atrophy (SMA) and disease progression.Approach. Twenty-six infants with SMA and twenty-seven healthy infants had been enrolled and assessed with EIM as part of the NeuroNEXT SMA biomarker study. We applied a variety of modern, supervised ML approaches to this data, first seeking to differentiate healthy from SMA muscle, and then, using the best method, to track SMA progression.Main Results.Several of the ML algorithms worked well, but linear discriminant analysis (LDA) achieved 88.6% accuracy on subject muscles studied. This contrasts with a maximum of 60% accuracy that could be achieved using the single or multifrequency assessment approaches available at the time. LDA scores were also able to track progression effectively, although a multifrequency reactance-based measure also performed very well in this context.Significance.EIM enhanced with ML promises to be effective for providing effective diagnosis and tracking children and adults with SMA treated with currently available therapies. The normative trends identified here may also inform future applications of the technology in very young children. The basic analyses applied here could also likely be applied to other neuromuscular disorders characterized by muscle atrophy.

A novel CARM1-HuR axis involved in muscle differentiation and plasticity misregulated in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is characterized by the loss of alpha motor neurons in the spinal cord and a progressive muscle weakness and atrophy. SMA is caused by loss-of-function mutations and/or deletions in the survival of motor neuron (SMN) gene. The role of SMN in motor neurons has been extensively studied, but its function and the consequences of its loss in muscle have also emerged as a key aspect of SMA pathology. In this study, we explore the molecular mechanisms involved in muscle defects in SMA. First, we show in C2C12 myoblasts, that arginine methylation by CARM1 controls myogenic differentiation. More specifically, the methylation of HuR on K217 regulates HuR levels and subcellular localization during myogenic differentiation, and the formation of myotubes. Furthermore, we demonstrate that SMN and HuR interact in C2C12 myoblasts. Interestingly, the SMA-causing E134K point mutation within the SMN Tudor domain, and CARM1 depletion, modulate the SMN-HuR interaction. In addition, using the Smn2B/- mouse model, we report that CARM1 levels are markedly increased in SMA muscles and that HuR fails to properly respond to muscle denervation, thereby affecting the regulation of its mRNA targets. Altogether, our results show a novel CARM1-HuR axis in the regulation of muscle differentiation and plasticity as well as in the aberrant regulation of this axis caused by the absence of SMN in SMA muscle. With the recent developments of therapeutics targeting motor neurons, this study further indicates the need for more global therapeutic approaches for SMA.

SMA-miRs (miR-181a-5p, -324-5p, and -451a) are overexpressed in spinal muscular atrophy skeletal muscle and serum samples.

Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2-4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs. We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score). Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p<10-5). miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients. Telethon Italia (grant #GGP12116).

Spinal Muscular Atrophy autophagy profile is tissue-dependent: differential regulation between muscle and motoneurons

Spinal muscular atrophy (SMA) is a neuromuscular genetic disease caused by reduced survival motor neuron (SMN) protein. SMN is ubiquitous and deficient levels cause spinal cord motoneurons (MNs) degeneration and muscle atrophy. Nevertheless, the mechanism by which SMN reduction in muscle contributes to SMA disease is not fully understood. Therefore, studies evaluating atrophy mechanisms in SMA muscles will contribute to strengthening current knowledge of the pathology. Here we propose to evaluate autophagy in SMA muscle, a pathway altered in myotube atrophy. We analized autophagy proteins and mTOR in muscle biopsies, fibroblasts, and lymphoblast cell lines from SMA patients and in gastrocnemius muscles from a severe SMA mouse model. Human MNs differentiated from SMA and unaffected control iPSCs were also included in the analysis of the autophagy. Muscle biopsies, fibroblasts, and lymphoblast cell lines from SMA patients showed reduction of the autophagy marker LC3-II. In SMA mouse gastrocnemius, we observed lower levels of LC3-II, Beclin 1, and p62/SQSTM1 proteins at pre-symptomatic stage. mTOR phosphorylation at Ser2448 was decreased in SMA muscle cells. However, in mouse and human cultured SMA MNs mTOR phosphorylation and LC3-II levels were increased. These results suggest a differential regulation in SMA of the autophagy process in muscle cells and MNs. Opposite changes in autophagy proteins and mTOR phosphorylation between muscle cells and neurons were observed. These differences may reflect a specific response to SMN reduction, which could imply diverse tissue-dependent reactions to therapies that should be taken into account when treating SMA patients.

Mislocalization of SMN from the I-band and M-band in human skeletal myofibers in spinal muscular atrophy associates with primary structural alterations of the sarcomere.

Spinal muscular atrophy (SMA) is caused by a deletion or mutation of the survival motor neuron 1 (SMN1) gene. Reduced SMN levels lead to motor neuron degeneration and muscular atrophy. SMN protein localizes to the cytoplasm and Cajal bodies. Moreover, in myofibrils from Drosophila and mice, SMN is a sarcomeric protein localized to the Z-disc. Although SMN participates in multiple functions, including the biogenesis of spliceosomal small nuclear ribonucleoproteins, its role in the sarcomere is unclear. Here, we analyzed the sarcomeric organization of SMN in human control and type I SMA skeletal myofibers. In control sarcomeres, we demonstrate that human SMN is localized to the titin-positive M-band and actin-positive I-band, and to SMN-positive granules that flanked the Z-discs. Co-immunoprecipitation assays revealed that SMN interacts with the sarcomeric protein actin, α-actinin, titin, and profilin2. In the type I SMA muscle, SMN levels were reduced, and atrophic (denervated) and hypertrophic (nondenervated) myofibers coexisted. The hypertrophied myofibers, which are potential primary targets of SMN deficiency, exhibited sites of focal or segmental alterations of the actin cytoskeleton, where the SMN immunostaining pattern was altered. Moreover, SMN was relocalized to the Z-disc in overcontracted minisarcomeres from hypertrophic myofibers. We propose that SMN could have an integrating role in the molecular components of the sarcomere. Consequently, low SMN levels might impact the normal sarcomeric architecture, resulting in the disruption of myofibrils found in SMA muscle. This primary effect might be independent of the neurogenic myopathy produced by denervation and contribute to pathophysiology of the SMA myopathy.

Muscle-specific SMN reduction reveals motor neuron-independent disease in spinal muscular atrophy models.

Paucity of the survival motor neuron (SMN) protein triggers the oft-fatal infantile-onset motor neuron disorder, spinal muscular atrophy (SMA). Augmenting the protein is one means of treating SMA and recently led to FDA approval of an intrathecally delivered SMN-enhancing oligonucleotide currently in use. Notwithstanding the advent of this and other therapies for SMA, it is unclear whether the paralysis associated with the disease derives solely from dysfunctional motor neurons that may be efficiently targeted by restricted delivery of SMN-enhancing agents to the nervous system, or stems from broader defects of the motor unit, arguing for systemic SMN repletion. We investigated the disease-contributing effects of low SMN in one relevant peripheral organ - skeletal muscle - by selectively depleting the protein in only this tissue. We found that muscle deprived of SMN was profoundly damaged. Although a disease phenotype was not immediately obvious, persistent low levels of the protein eventually resulted in muscle fiber defects, neuromuscular junction abnormalities, compromised motor performance, and premature death. Importantly, restoring SMN after the onset of muscle pathology reversed disease. Our results provide the most compelling evidence yet for a direct contributing role of muscle in SMA and argue that an optimal therapy for the disease must be designed to treat this aspect of the dysfunctional motor unit.

Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder, which causes dysfunction/loss of lower motor neurons and muscle weakness as well as atrophy. While SMA is primarily considered as a motor neuron disease, recent data suggests that survival motor neuron (SMN) deficiency in muscle causes intrinsic defects. We systematically profiled secreted proteins from control and SMN deficient muscle cells with two combined metabolic labeling methods and mass spectrometry. From the screening, we found lower levels of C1q/TNF-related protein 3 (CTRP3) in the SMA muscle secretome and confirmed that CTRP3 levels are indeed reduced in muscle tissues and serum of an SMA mouse model. We identified that CTRP3 regulates neuronal protein synthesis including SMN via mTOR pathway. Furthermore, CTRP3 enhances axonal outgrowth and protein synthesis rate, which are well-known impaired processes in SMA motor neurons. Our data revealed a new molecular mechanism by which muscles regulate the physiology of motor neurons via secreted molecules. Dysregulation of this mechanism contributes to the pathophysiology of SMA.

Impaired myogenic development, differentiation and function in hESC-derived SMA myoblasts and myotubes.

Spinal muscular atrophy (SMA) is a severe genetic disorder that manifests in progressive neuromuscular degeneration. SMA originates from loss-of-function mutations of the SMN1 (Survival of Motor Neuron 1) gene. Recent evidence has implicated peripheral deficits, especially in skeletal muscle, as key contributors to disease progression in SMA. In this study we generated myogenic cells from two SMA-affected human embryonic stem cell (hESC) lines with deletion of SMN1 bearing two copies of the SMN2 gene and recapitulating the molecular phenotype of Type 1 SMA. We characterized myoblasts and myotubes by comparing them to two unaffected, control hESC lines and demonstrate that SMA myoblasts and myotubes showed altered expression of various myogenic markers, which translated into an impaired in vitro myogenic maturation and development process. Additionally, we provide evidence that these SMN1 deficient cells display functional deficits in cholinergic calcium signaling response, glycolysis and oxidative phosphorylation. Our data describe a novel human myogenic SMA model that might be used for interrogating the effect of SMN depletion during skeletal muscle development, and as model to investigate biological mechanisms targeting myogenic differentiation, mitochondrial respiration and calcium signaling processes in SMA muscle cells.

Bulbar muscle MRI changes in patients with SMA with reduced mouth opening and dysphagia.

We performed a study in patients with proximal spinal muscular atrophy (SMA) to determine the prevalence of reduced maximal mouth opening (MMO) and its association with dysphagia as a reflection of bulbar dysfunction and visualized the underlying mechanisms using MRI. We performed a cross-sectional study of MMO in 145 patients with SMA types 1-4 and 119 healthy controls and used MRI in 12 patients to visualize mandibular condylar shape and sliding and the anatomy of muscle groups relevant for mouth opening and closing. We analyzed associations of reduced MMO with SMA severity and complaints of dysphagia. Reduced MMO was defined as an interincisal distance ≤ 35 mm and was found in none of the healthy controls and in 100%, 79%, 50%, and 7% of patients with SMA types 1, 2, 3a, and 3b/4, respectively. MRI showed severe fatty degeneration of the lateral pterygoid muscles that mediate mouth opening by allowing mandibular condylar sliding but relatively mild involvement of the mouth closing muscles in patients with reduced MMO. Reduced MMO was associated with SMA type, age, muscle weakness, and dysphagia (p < 0.05). Reduced MMO is common in SMA types 1-3a and is mainly caused by fatty degeneration of specific mouth opening muscles. Reduced MMO is a sign of bulbar dysfunction in SMA.

Supraphysiological expression of survival motor neuron protein from an adenovirus vector does not adversely affect cell function

Spinal muscular atrophy (SMA) is the most common inherited neurodegenerative disease that leads to infant mortality. It is caused by mutations in the survival motor neuron (SMN) protein resulting in death of alpha motor neurons. Increasing evidence suggests that several other tissues are also affected in SMA, including skeletal and cardiac muscle, liver, and pancreas, indicating that systemic delivery of therapeutics may be necessary for true disease correction. Due to the natural biodistribution of therapeutics, a level of SMN several-fold above physiological levels can be achieved in some tissues. In this study, we address whether supraphysiological levels of SMN adversely affects cell function. Infection of a variety of cell types with an adenovirus (Ad) vector encoding SMN leads to very high expression, but the resulting protein correctly localizes within the cell, and associates with normal cellular partners. Although SMN affects transcription of certain target genes and can alter the splicing pattern of others, we did not observe any difference in select target gene splicing or expression in cells overexpressing SMN. However, normal human fibroblasts treated with Ad-SMN showed a slight reduction in growth rate, suggesting that certain cell types may be differently impacted by high levels of SMN.

Alterations of signaling pathways in muscle tissues of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a degenerative disorder of the central nervous system, manifests as progressive weakening of muscles. The diagnosis and prognosis of ALS are often unclear, so useful biomarkers are needed. Total proteins were extracted from muscle samples from 36 ALS, 17 spinal muscular atrophy (SMA), and 36 normal individuals. The expression levels of 134 proteins and phosphoproteins were assessed using protein pathway array analysis. Seventeen proteins were differentially expressed between ALS and normal muscle, and 9 proteins were differentially expressed between ALS and SMA muscle. The low-level expression of Akt and Factor XIIIB correlates with unfavorable survival, and the risk score calculated based on these proteins predicts the survival of each individual patient. Some proteins could be selected as clinically useful biomarkers. Specifically, Akt and Factor XIIIB were found to be promising biomarkers for estimating prognosis in ALS.

Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

ObjectivesGenetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA.MethodsA sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed.ResultsThe assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA.ConclusionsThis SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts.

Reversible molecular pathology of skeletal muscle in spinal muscular atrophy

Low levels of full-length survival motor neuron (SMN) protein cause the motor neuron disease, spinal muscular atrophy (SMA). Although motor neurons undoubtedly contribute directly to SMA pathogenesis, the role of muscle is less clear. We demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic severe SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomic data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. Robust upregulation of voltage-dependent anion-selective channel protein 2 (Vdac2) and downregulation of parvalbumin in severe SMA mice was confirmed in a milder SMA mouse model and in human patient muscle biopsies. Molecular pathology of skeletal muscle was ameliorated in mice treated with the FDA-approved histone deacetylase inhibitor, suberoylanilide hydroxamic acid. We conclude that intrinsic pathology of skeletal muscle is an important and reversible event in SMA and also suggest that muscle proteins have the potential to act as novel biomarkers in SMA.

The Developmental Pattern of Myotubes in Spinal Muscular Atrophy Indicates Prenatal Delay of Muscle Maturation

The loss and degeneration of spinal cord motor neurons result in muscle denervation in spinal muscular atrophy (SMA), but whether there are primary pathogenetic abnormalities of muscle in SMA is not known. We previously detected increased DNA fragmentation and downregulation of Bcl-2 and Bcl-X(L) expression but no morphological changes in spinal motor neurons of SMA fetuses. Here, we performed histological and morphometric analysis of myotubes and assessed DNA fragmentation and Bcl-2/Bcl-X(L) expression in skeletal muscle from fetuses with type I SMA (at approximately 12 and 15 weeks' gestational ages, n = 4) and controls (at approximately 10-15 weeks' gestational ages, n = 7). Myotubes were smaller in the SMA than in control samples at all ages analyzed (p < 0.001) and were often arranged in clusters close to isolated and larger myotubes. Numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells in control and SMA fetuses were similar, and no differences in Bcl-2 or Bcl-X(L) immunostaining between control and SMA muscle were identified. Areas with smaller myotubes and the morphometric analysis suggested a delay in growth and maturation in SMA muscle. These results suggest that spinal motor neurons and skeletal muscle undergo different pathogenetic processes in SMA during development; they imply that muscle as well as motor neurons may be targets for early therapeutic intervention in SMA.

Implication of Fetal SMN2 Expression in Type I SMA Pathogenesis: Protection or Pathological Gain of Function?

Spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron gene 1 (SMN1). The SMN2 gene, which is the highly homologous SMN1 copy that is present in all the patients, is unable to prevent the disease. Most of the SMN1 transcript is full-length, whereas a substantial proportion of the SMN2 transcript lacks exon 7 (delta7). We characterized the developmental expression of SMN2 by comparing control and SMA fetuses. The control spinal cord revealed the highest amount of FL SMN, most of which was of SMN1 origin. When analyzing the SMA spinal cord transcripts, we detected a considerable reduction in the FL/delta7 ratios due to a decrease in the FL and an increase in delta7 isoform. After immunoblot and immunohistochemistry analyses, we found that the amount of SMN2 protein in the SMA spinal cord and muscle was lower than in the controls. However, the results of the expression of SMN2 in intestine, lung, adrenal gland, kidney, and eye, which are unaffected by the disease, were the same in controls and SMA samples. In these tissues, SMN2 may compensate for the absence of SMN1, whereas in SMA motor neurons, a cell-specific dysregulation of the SMN2 expression could favor the onset of the acute form of the disease.

Severe depletion of mitochondrial DNA in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disorder in childhood leading to a dramatic loss of muscle strength. Functional investigations with high-resolution polarography and enzyme measurements of the respiratory chain revealed lowered activities in muscle tissue of SMA patients. To gain a better understanding of this low energy supply we analyzed the amount of mitochondrial DNA (mtDNA) in skeletal muscle of 20 unrelated children with genetically proven SMA and 31 controls. Quantitative Southern blot analysis revealed a severe and homogeneous decrease in the content of muscle mtDNA in relation to nuclear DNA in SMA patients (90.3+/-7.8%), whereas by immunofluorescence no decrease in the number of mitochondria was detected. In addition, a two- to threefold reduction of the nuclear-encoded complex II (succinate dehydrogenase) activity was detected in SMA muscle tissue. Western blot analysis showed a significant reduction of both mitochondrial- and nuclear-encoded cytochrome c oxidase subunits. Our results indicate that mtDNA depletion in SMA is a consequence of severe atrophy, and has to be differentiated by measurement of complex II from an isolated reduction of mtDNA as found in patients with mitochondriocytopathies and the so-called mtDNA depletion syndrome.

Possible pathogenic role of muscle cell dysfunction in motor neuron death in spinal muscular atrophy.

We have previously shown that myofibers formed by fusion of muscle satellite cells from spinal muscular atrophy (SMA) I or II undergo degeneration 1 to 3 weeks after innervation by rat embryonic spinal cord explants, whereas normal myofibers survive for several months. In the "muscle component" of the coculture, the only cells responsible for the degeneration are the SMA muscle satellite cells. Moreover, SMA muscle satellite cells do not fuse as rapidly as do normal muscle satellite cells. To determine whether death of muscle cells precedes that of motor neurons, we studied the origin and kinetics of release of apoptotic microparticles. In SMA cocultures, motor neuron apoptosis occurred before myofiber degeneration becomes visible, indicating that SMA myofibers were unable to sustain survival of motor neurons. In normal cocultures, motor neuron apoptosis occurred 4 days after innervation. However, it did not continue beyond 2 days. These results strengthen the hypothesis that SMA is due to a defect in neurotrophic muscle cell function.

Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy.

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons of the spinal cord associated with muscle paralysis and caused by mutations of the survival motor neuron gene (SMN). To determine whether SMN gene defect in skeletal muscle might have a role in SMA pathogenesis, deletion of murine SMN exon 7, the most frequent mutation found in SMA, has been restricted to skeletal muscle by using the Cre-loxP system. Mutant mice display ongoing muscle necrosis with a dystrophic phenotype leading to muscle paralysis and death. The dystrophic phenotype is associated with elevated levels of creatine kinase activity, Evans blue dye uptake into muscle fibers, reduced amount of dystrophin and upregulation of utrophin expression suggesting a destabilization of the sarcolemma components. The mutant mice will be a valuable model for elucidating the underlying mechanism. Moreover, our results suggest a primary involvement of skeletal muscle in human SMA, which may contribute to motor defect in addition to muscle denervation caused by the motor neuron degeneration. These data may have important implications for the development of therapeutic strategies in SMA.

Cell Types Required to Efficiently Innervate Human Muscle Cells in Vitro

Previous studies carried out in our laboratory have shown that myofibers formed by fusion of muscle satellite cells from donors with spinal muscular atrophy (SMA) type I or II undergo a characteristic degeneration 1.5–3 weeks after innervation with rat embryonic spinal cord explants. The only cells responsible for degeneration of innervated cocultures are SMA muscle satellite cells. In order to study the kinetics of nerve and muscle cell degeneration in nerve–muscle cocultures implicating SMA muscle cells, we attempted to simplify the nervous component of the coculture and identify the nerve cell types necessary for a successful innervation. We demonstrate here that motoneurons alone were unable to innervate myotubes. However, when three cell types (motoneurons, sensory neurons, and Schwann cells) were added onto a reconstituted muscular component consisting of cloned muscle satellite cells and cloned muscular fibroblasts, myotubes contracted, indicating that functional neuromuscular junctions were formed. We concluded that the three cell types were required for a successful innervation. Moreover, we studied the effects of culture medium conditioned by different combinations of nerve cells on innervation; we observed that physical contacts among sensory neurons, motoneurons, and myotubes are required for a successful innervation; in contrast Schwann cells can be replaced by a Schwann-cell-conditioned medium, indicating that these cells produce a putative soluble “innervation-promoting factor.” Obviously such a reconstituted system does not reflect the in vivo situation but it allows the formation of functional motor synapses and could therefore allow us to elucidate neuromuscular disease pathogenesis, especially that of spinal muscular atrophy.

Ultrastructural changes in muscle in spinal muscular atrophy. Werding-Hoffmann's disease.

An ultrastructural study was carried out on muscle biopsies of 7 children with Werdnig-Hoffmann's disease. The muscle fibres were classified into 3 categories: 1. Muscle cells of normal diameter without any distinct or only slight changes. 2. Small muscle cells with a large central nucleus with the appearance of myoblast at a later stage of development. 3. Cells resembling myotubes containing in a common basement membrane two to four cells at different stages of maturation.