Congenital Neuromuscular Disorders Research Articles

Comprehensive phenotypic characterization of an allelic series of zebrafish models of NEB-related nemaline myopathy.

Nemaline myopathy (NM) is a rare congenital neuromuscular disorder characterized by muscle weakness and hypotonia, slow gross motor development, and decreased respiratory function. Mutations in at least twelve genes, all of each encode proteins that are either components of the muscle thin filament or regulate its length and stability, have been associated with NM. Mutations in Nebulin (NEB), a giant filamentous protein localized in the sarcomere, account for more than 50% of NM cases. At present, there remains a lack of understanding of whether NEB genotype influences nebulin function and NM-patient phenotypes. In addition, there is a lack of therapeutically tractable models that can enable drug discovery and address the current unmet treatment needs of patients. To begin to address these gaps, here we have characterized five new zebrafish models of NEB-related NM. These mutants recapitulate most aspects of NEB-based NM, showing drastically reduced survival, defective muscle structure, reduced contraction force, shorter thin filaments, presence of electron-dense structures in myofibers, and thickening of the Z-disks. This study represents the first extensive investigation of an allelic series of nebulin mutants, and thus provides an initial examination in pre-clinical models of potential genotype-phenotype correlations in human NEB patients. It also represents the first utilization of a set of comprehensive outcome measures in zebrafish, including correlation between molecular analyses, structural and biophysical investigations, and phenotypic outcomes. Therefore, it provides a rich source of data for future studies exploring the NM pathomechanisms, and an ideal springboard for therapy identification and development for NEB-related NM.

A centronuclear myopathy-causing mutation in dynamin-2 disrupts neuronal morphology and excitatory synaptic transmission in a murine model of the disease.

Dynamin-2 (DNM2) is a large GTPase, a member of the dynamin superfamily that regulates membrane remodelling and cytoskeleton dynamics. Mutations in DNM2 cause autosomal dominant centronuclear myopathy (CNM), a congenital neuromuscular disorder characterized by progressive weakness and atrophy of the skeletal muscles. Cognitive defects have been reported in some DNM2-linked CNM patients suggesting that these mutations can also affect the central nervous system (CNS). Here we studied how a DNM2 CNM-causing mutation influences the CNS function. Heterozygous mice harbouring the p.R465W mutation in Dnm2 (HTZ), the most common causing autosomal dominant CNM, were used as the disease model. We evaluated dendritic arborization and spine density in hippocampal cultured neurons, analysed excitatory synaptic transmission by electrophysiological field recordings in hippocampal slices, and evaluated cognitive function by performing behavioural tests. HTZ hippocampal neurons exhibited reduced dendritic arborization and lower spine density than WT neurons, which was reversed by transfecting an interference RNA against the Dnm2 mutant allele. Additionally, HTZ mice showed defective hippocampal excitatory synaptic transmission and reduced recognition memory compared to the WT condition. Our findings suggest that the Dnm2 p.R465W mutation perturbs the synaptic and cognitive function in a CNM mouse model and support the idea that Dnm2 plays a key role in regulating neuronal morphology and excitatory synaptic transmission in the hippocampus.

MEGF10 gene mutations causing a recessive congenital multiminicore myopathy (P9-9.010)

<h3>Objective:</h3> Congenital myopathies are classified into five main types: core myopathies, nemaline myopathies, centronuclear myopathies, congenital fiber-type disproportion, and myosin storage myopathies. The most common forms are core myopathies. Despite their phenotypic diversity, patients demonstrate common symptoms including hypotonia, muscle weakness, dysmorphic features, and respiratory problems. There are several mutations in <i>MEGF10</i> that have been reported to cause autosomal recessive congenital myopathy, areflexia, respiratory distress, muscle weakness, dysphagia with early or late-onset syndrome, minicore myopathy and limb girdle muscular dystrophy. Affected individuals frequently become ventilator dependent or die secondary to respiratory failure. <h3>Background:</h3> A 5-year-old girl from a consanguineous couple was referred to our service due to weakness and hypotonia. After birth, she developed respiratory failure. Severe motor delay was already evident in the first months of life: at 6 months she had no cervical control and at 12 months she could not sit without support. At age 3 she developed respiratory problems with apnea and hypercapnia, requiring ventilation with two levels of positive airway pressure. She was indicated for gastrostomy at age 4 after aspiration pneumonia. On her evaluation, she had axial and proximal muscle weakness, facial weakness, scoliosis, and nasal speech. Despite presenting hypotonia and gait difficulties, she was able to walk independently and had no cognitive changes. A muscle biopsy was performed which suggested multiminicore myopathy. Genetic investigation resulted in a homozygous mutation of the <i>MEGF10</i> gene. <h3>Design/Methods:</h3> NA <h3>Results:</h3> NA <h3>Conclusions:</h3> <i>MEGF10</i> mutations should be considered in the differential diagnosis of individuals with respiratory insufficiency and myopathy, particularly when accompanied by facial weakness, scoliosis or dysphagia. The phenotypic similarities with other congenital neuromuscular disorders may cause difficulties in reaching a definite diagnosis. Treatment with a multidisciplinary team is important and family counseling is essential since consanguineous unions play a role in recessive genetic mutations manifestations. <b>Disclosure:</b> Miss Faria has nothing to disclose. Dr. Barbosa has nothing to disclose. Mrs. Ribeiro Torres has nothing to disclose. Mr. E Souza Filho has received research support from Universidade Federal de Minas Gerais. Mrs. Thiersch has nothing to disclose. Mrs. Oliveira has nothing to disclose.

Characterization of a novel zebrafish model of SPEG-related centronuclear myopathy.

ABSTRACTCentronuclear myopathy (CNM) is a congenital neuromuscular disorder caused by pathogenic variation in genes associated with membrane trafficking and excitation–contraction coupling (ECC). Bi-allelic autosomal-recessive mutations in striated muscle enriched protein kinase (SPEG) account for a subset of CNM patients. Previous research has been limited by the perinatal lethality of constitutive Speg knockout mice. Thus, the precise biological role of SPEG in developing skeletal muscle remains unknown. To address this issue, we generated zebrafish spega, spegb and spega;spegb (speg-DKO) mutant lines. We demonstrated that speg-DKO zebrafish faithfully recapitulate multiple phenotypes associated with CNM, including disruption of the ECC machinery, dysregulation of calcium homeostasis during ECC and impairment of muscle performance. Taking advantage of zebrafish models of multiple CNM genetic subtypes, we compared novel and known disease markers in speg-DKO with mtm1-KO and DNM2-S619L transgenic zebrafish. We observed Desmin accumulation common to all CNM subtypes, and Dnm2 upregulation in muscle of both speg-DKO and mtm1-KO zebrafish. In all, we establish a new model of SPEG-related CNM, and identify abnormalities in this model suitable for defining disease pathomechanisms and evaluating potential therapies. This article has an associated First Person interview with the joint first authors of the paper.

Oxidative Stress, Inflammation and Connexin Hemichannels in Muscular Dystrophies

Muscular dystrophies (MDs) are a heterogeneous group of congenital neuromuscular disorders whose clinical signs include myalgia, skeletal muscle weakness, hypotonia, and atrophy that leads to progressive muscle disability and loss of ambulation. MDs can also affect cardiac and respiratory muscles, impairing life-expectancy. MDs in clude Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy and limb-girdle muscular dystrophy. These and other MDs are caused by mutations in genes that encode proteins responsible for the structure and function of skeletal muscles, such as components of the dystrophin-glycoprotein-complex that connect the sarcomeric-actin with the extracellular matrix, allowing contractile force transmission and providing stability during muscle contraction. Consequently, in dystrophic conditions in which such proteins are affected, muscle integrity is disrupted, leading to local inflammatory responses, oxidative stress, Ca2+-dyshomeostasis and muscle degeneration. In this scenario, dysregulation of connexin hemichannels seem to be an early disruptor of the homeostasis that further plays a relevant role in these processes. The interaction between all these elements constitutes a positive feedback loop that contributes to the worsening of the diseases. Thus, we discuss here the interplay between inflammation, oxidative stress and connexin hemichannels in the progression of MDs and their potential as therapeutic targets.

CONGENITAL MYOPATHIES 2: P.97 Valproic acid improves survival rate and skeletal muscle phenotype in mtm1 knockout mice

X-linked myotubular myopathy (XLMTM) is a rare congenital neuromuscular disorder affecting boys at a rate of 1 in 50,000 live births. The majority (∼80%) of the affected children are born with serious and diffuse skeletal muscle weakness and hypotonia that necessitate immediate and sustained extensive care including tracheostomy and feeding by gastric intubation. Considerable proportions (∼25%) of the cases succumb to death within their first year of life and the majority do so later due mainly to respiratory failure.

New and Developing Therapies in Spinal Muscular Atrophy: From Genotype to Phenotype to Treatment and Where Do We Stand?

Spinal muscular atrophy (SMA) is a congenital neuromuscular disorder characterized by motor neuron loss, resulting in progressive weakness. SMA is notable in the health care community because it accounts for the most common cause of infant death resulting from a genetic defect. SMA is caused by low levels of the survival motor neuron protein (SMN) resulting from SMN1 gene mutations or deletions. However, patients always harbor various copies of SMN2, an almost identical but functionally deficient copy of the gene. A genotype–phenotype correlation suggests that SMN2 is a potent disease modifier for SMA, which also represents the primary target for potential therapies. Increasing comprehension of SMA pathophysiology, including the characterization of SMN1 and SMN2 genes and SMN protein functions, has led to the development of multiple therapeutic approaches. Until the end of 2016, no cure was available for SMA, and management consisted of supportive measures. Two breakthrough SMN-targeted treatments, either using antisense oligonucleotides (ASOs) or virus-mediated gene therapy, have recently been approved. These two novel therapeutics have a common objective: to increase the production of SMN protein in MNs and thereby improve motor function and survival. However, neither therapy currently provides a complete cure. Treating patients with SMA brings new responsibilities and unique dilemmas. As SMA is such a devastating disease, it is reasonable to assume that a unique therapeutic solution may not be sufficient. Current approaches under clinical investigation differ in administration routes, frequency of dosing, intrathecal versus systemic delivery, and mechanisms of action. Besides, emerging clinical trials evaluating the efficacy of either SMN-dependent or SMN-independent approaches are ongoing. This review aims to address the different knowledge gaps between genotype, phenotypes, and potential therapeutics.

Joint hypermobility in children: a neglected sign needing more attention.

Joint hypermobility refers to the ability that a joint has to move beyond its normal range of motion. It is common in the general population, particularly in children. While many individuals manifesting joint hypermobility are healthy, this feature can accompany a wide range of symptoms and systemic disorders, whose management can be influenced by their prompt recognition. Given the increasing attention that joint hypermobility is attracting in various fields of medicine, many practitioners are asked to approach more carefully joint hypermobility, in order to avoid over- and under-diagnosis of related disorders. Among the most common conditions featuring joint hypermobility there are hypermobility spectrum disorders and hereditary connective tissue disorders, in particular, the Ehlers-Danlos syndromes. In children, joint hypermobility also accompany a variety of disorders affecting neurodevelopment. The nature of such an association is protean, as joint hypermobility may occur in selected congenital neuromuscular disorders, monogenic multiple malformation/intellectual disability syndromes, and well-known and emerging genomic syndromes. In addition, joint hypermobility seems strongly associated with developmental coordination disorders. This review offers an overview on definitions, assessment procedures, patterns of associated manifestations and disorders related to joint hypermobility, as well as treatment principles of associated musculoskeletal pain for practitioners that are not familial with this issue but encounter people featuring this physical attribute in their daily activity.

Omecamtiv mecarbil lowers the contractile deficit in a mouse model of nebulin-based nemaline myopathy.

Nemaline myopathy (NEM) is a congenital neuromuscular disorder primarily caused by nebulin gene (NEB) mutations. NEM is characterized by muscle weakness for which currently no treatments exist. In NEM patients a predominance of type I fibers has been found. Thus, therapeutic options targeting type I fibers could be highly beneficial for NEM patients. Because type I muscle fibers express the same myosin isoform as cardiac muscle (Myh7), the effect of omecamtiv mecarbil (OM), a small molecule activator of Myh7, was studied in a nebulin-based NEM mouse model (Neb cKO). Skinned single fibers were activated by exogenous calcium and force was measured at a wide range of calcium concentrations. Maximal specific force of type I fibers was much less in fibers from Neb cKO animals and calcium sensitivity of permeabilized single fibers was reduced (pCa50 6.12 ±0.08 (cKO) vs 6.36 ±0.08 (CON)). OM increased the calcium sensitivity of type I single muscle fibers. The greatest effect occurred in type I fibers from Neb cKO muscle where OM restored the calcium sensitivity to that of the control type I fibers. Forces at submaximal activation levels (pCa 6.0-6.5) were significantly increased in Neb cKO fibers (~50%) but remained below that of control fibers. OM also increased isometric force and power during isotonic shortening of intact whole soleus muscle of Neb cKO mice, with the largest effects at physiological stimulation frequencies. We conclude that OM has the potential to improve the quality of life of NEM patients by increasing the force of type I fibers at submaximal activation levels.

Cullin-3 dependent deregulation of ACTN1 represents a new pathogenic mechanism in nemaline myopathy.

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

Nemaline myopathy diagnosed in two young Border collies in formalin-fixed paraffin-embedded muscle samples using conventional stains

Nemaline myopathy (NM) is a congenital neuromuscular disorder with heterogeneous clinical signs such as muscle weakness, affecting humans, seldom dogs and cats. For human NM, several causative gene mutations have...

CONGENITAL MYOPATHIES (CNM): P.138The CHOP INTEND scale (Children's Hospital of Philadelphia infant test of neuromuscular disorders) in X-linked myotubular myopathy: content validity and psychometric performance

XLMTM is a rare, congenital neuromuscular disorder characterized by extreme hypotonia, respiratory failure, and premature death. The CHOP INTEND is a measurement tool developed to assess motor function in patients with spinal muscular atrophy type I (SMAI), and successfully used in clinical trials to assess treatment response. As SMA I patients have a similar phenotype to infants with XLMTM, this study evaluated the CHOP INTEND as a measure of motor function in children with XLMTM. A literature review and qualitative interviews were completed (11 clinical experts, 5 caregivers); findings were used to determine content validity and develop a conceptual model of XLMTM, which were also assessed in a Delphi panel (7 clinical experts). Inter-rater reliability was evaluated among 15 physical therapists, with agreement between the ratings assessed based on independent review of videotaped assessments. The conceptual model of XLMTM includes body function and structural impairments (neuromuscular, pulmonary, musculoskeletal, gastrointestinal), as well as disease impacts (development skills, activities of daily living, caregiver burden). Concepts related to motor function map to most CHOP INTEND items. Caregiver descriptions of their child's function also align with items evaluated in the CHOP INTEND. The Delphi panel achieved consensus on the conceptual model of XLMTM and the appropriateness of the CHOP INTEND to assess motor function in XLMTM. Finally, the inter-rater reliability was supported with an intraclass correlation coefficient of 0.938 (95% CI: .902-.965). Results from this study suggest that the CHOP INTEND is a valid and reliable assessment tool for children with XLMTM, and support the appropriateness of its use in the ASPIRO gene therapy clinical trial as a co-primary endpoint. Next steps include evaluation of the psychometric performance of the CHOP INTEND in this population.

P.102 - Registry of congenital neuromuscular disorders in Japan: establishment and implementation

Congenital neuromuscular disorders are rare, making clinical studies and recruitment of patients for clinical trials difficult. To facilitate research, we established a Japanese registry of congenital neuromuscular disorders. The registry included patients with congenital neuromuscular disorder whose clinical presentation was consistent with the known pathology, genetic diagnosis, or clinical diagnosis. Patients with dystrophinopathy, myotonic dystrophy, Fukuyama-type congenital muscular dystrophy, or spinal muscular atrophy were excluded because separate registries are available for these disorders. Patients to be included in this registry should have undergone genetic diagnosis and at least one muscular pathological examination. However, patients with only a clinical diagnosis were also included in the registry. To assure the accuracy of diagnosis prior to registration, patients were stratified into 3 levels: 1) diagnostic category A (definitive diagnosis based on neuromuscular pathological examination after muscle biopsy and/or identification of the responsible gene by genetic analysis, without any discrepancy from the known clinical course); 2) category B (no discrepancy from the known clinical symptoms or test findings but failure to identify the cause with muscle biopsy or genetic analysis); and 3) category C (patients who had not undergone diagnostic or genetic analysis based on muscle biopsy, but were clinically diagnosed by experts as not contradicting the known clinical symptoms or test findings of the disease). The registry was established in September 2016. To date, 13 patients have been registered, including 3 with muscular dystrophy, 7 with congenital myopathy, 1 with congenital myasthenia, and 2 with myofibrillar myopathy. This registry should stimulate research on congenital neuromuscular disorders. An important task of this registry will be rapid dissemination of data/materials on clinical trials, treatment, and education.

P.335 - Two different NGS approaches to address molecular diagnosis of congenital neuromuscular diseases

Congenital neuromuscular diseases are early onset muscle disorders encompassing great clinical and genetic heterogeneity so that reaching and accurate genetic diagnosis is still a challenge. We aim to evaluate the diagnostic advantage of different NGS approaches in a cohort of congenital neuromuscular disorders and to validate an efficient and cost-effective diagnostic strategy to be incorporated to the National health system. Sixty-two gDNA samples from patients with clinical suspicion of congenital neuromuscular diseases were analysed by NGS. A custom panel including 118 genes (Nextera Rapid Capture, Illumina) and TruSight One Panel (Clinical Exome, Illumina) were employed. Sequencing data were analysed by Variant Studio and DNA Nexus. All candidate variants were confirmed by sanger sequencing and pathogenicity was analysed by using alamut software (Interactive Biosoftwares) and LOVD database. The genetic cause of the disease was elucidated in 11/25 samples (44%) by the clinical exome and in 28/48 samples (58%) by the custom panel. Eleven samples from the second group (custom panel) had previously been analysed by the clinical exome without positive results. Mutations in RYR1 were the most common cause of disease in our cohort. Variants in genes related to Congenital myasthenic syndromes were identified in 8 patients. This was crucial to choose the appropriate treatment, which depends on the particular molecular defect. The custom panel design has proved to be a more useful tool than the clinical exome to provide a molecular diagnosis of congenital neuromuscular diseases.

Clinical course of growth in patients with congenital neuromuscular disease in a single multidisciplinary neuromuscular clinic.

To assess the effect of nutritional interventions on growth and on respiratory status in patients with congenital myopathy (CM), congenital muscular dystrophy (CMD), and congenital myasthenic syndrome (CMS). Retrospective cohort study based on case-note review of 18 patients affected by CM, CMD, and CMS, followed at a single pediatric neuromuscular center, between 2006 and 2014. Seventy-two percent of patients required placement of a gastrostomy tube for bulbar weakness or for growth failure. Of those patients, 10 had 1 year follow up anthropometric data and 6 had 2 year follow up anthropometric data. Height percentiles and z-scores were significantly improved in patients after 1 year, while weight and BMI percentiles and z-scores were not. Weight and height percentiles and z-scores were significantly improved in patients at 2 year follow up, while BMI percentiles and z-scores were not. The number of respiratory illnesses was not significantly different before or after placement of the feeding tube. Of the patients who did not have placement of a gastrostomy tube, 4 had 1 year follow up anthropometric data and 3 had 2 year follow up anthropometric data. Gastrostomy tube fed patients had significantly higher mean weight percentiles and z-scores compared to orally fed patients. There was no significant difference in height or BMI between the gastrostomy fed and orally fed groups. Individual growth curves highlight the effect of intervention on weight and height. This is a single multidisciplinary center experience describing the effect of nutritional interventions on growth in patients with congenital neuromuscular disorders. While the number of patients and their data in this report are limited, it highlights that the growth in this group of patients is unique but that the low weight and short stature respond to nutritional interventions with changes typically seen after 2 years of intervention.

Mutation spectrum of the MTM1 gene in XLMTM patients: 10 years of experience in prenatal and postnatal diagnosis.

X-linked myotubular myopathy (XLMTM) is a congenital neuromuscular disorder defined by severe hypotonia, respiratory failure and histopathologic changes in muscle biopsy. The objective of this report is to inform about our experience of genetic analysis on a group of 25 unrelated XLMTM patients, clinically diagnosed by several Italian and European Medical Institutes from 2006 to 2015. The molecular strategy used for genotyping involved Sanger sequencing of coding and intron/exon regions and the Multiplex Ligation Probe Amplification method. A total of 13 different point variants (6 nonsense, 5 missense, 1 splicing and 1 small deletion) were found in 15 patients (60%). Three were new missense variants: c.185G>T p.(Arg62Ile), c.719T>A p.(Val240Glu), and c.1262G>T p.(Arg421Leu). No large duplications/deletions have been identified. We performed carrier testing of at-risk female relatives. Only one mutation was de novo. Successively, we offered XLMTM prenatal testing for seven pregnancies in five unrelated families. In this context, the aim to propose an effective molecular diagnostic service is to confirm clinical XLMTM diagnosis, to monitor the cause-disease mutation segregation in the family and to offer genetic counseling to have correct information regarding offspring risks and the prenatal testing.

The phenotype of the musculocontractural type of Ehlers-Danlos syndrome due to CHST14 mutations.

The musculocontractural type of Ehlers-Danlos syndrome (MC-EDS) has been recently recognized as a clinical entity. MC-EDS represents a differential diagnosis within the congenital neuromuscular and connective tissue disorders spectrum. Thirty-one and three patients have been reported with MC-EDS so far with bi-allelic mutations identified in CHST14 and DSE, respectively, encoding two enzymes necessary for dermatan sulfate (DS) biosynthesis. We report seven additional patients with MC-EDS from four unrelated families, including the follow-up of a sib-pair originally reported with the kyphoscoliotic type of EDS in 1975. Brachycephaly, a characteristic facial appearance, an asthenic build, hyperextensible and bruisable skin, tapering fingers, instability of large joints, and recurrent formation of large subcutaneous hematomas are always present. Three of seven patients had mildly elevated serum creatine kinase. The oldest patient was blind due to retinal detachment at 45 years and died at 59 years from intracranial bleeding; her affected brother died at 28 years from fulminant endocarditis. All patients in this series harbored homozygous, predicted loss-of-function CHST14 mutations. Indeed, DS was not detectable in fibroblasts from two unrelated patients with homozygous mutations. Patient fibroblasts produced higher amounts of chondroitin sulfate, showed intracellular retention of collagen types I and III, and lacked decorin and thrombospondin fibrils compared with control. A great proportion of collagen fibrils were not integrated into fibers, and fiber bundles were dispersed into the ground substance in one patient, all of which is likely to contribute to the clinical phenotype. This report should increase awareness for MC-EDS.

Validity of a Neurological Scoring System for Canine X-Linked Myotubular Myopathy.

A simple clinical neurological test was developed to evaluate response to gene therapy in a preclinical canine model of X-linked myotubular myopathy (XLMTM). This devastating congenital myopathy is caused by mutation in the myotubularin (MTM1) gene. Clinical signs include muscle weakness, early respiratory failure, and ventilator dependence. A spontaneously occurring canine model has a similar clinical picture and histological abnormalities on muscle biopsy compared with patients. We developed a neuromuscular assessment score, graded on a scale from 10 (normal) to 1 (unable to maintain sternal recumbency). We hypothesize that this neurological assessment score correlates with genotype and established measures of disease severity and is reliable when performed by an independent observer. At 17 weeks of age, there was strong correlation between neurological assessment scores and established methods of severity testing. The neurological severity score correctly differentiated between XLMTM and wild-type dogs with good interobserver reliability, on the basis of strong agreement between neurological scores assigned by independent observers. Together, these data indicate that the neurological scoring system developed for this canine congenital neuromuscular disorder is reliable and valid. This scoring system may be helpful in evaluating response to therapy in preclinical testing in this disease model, such as response to gene therapy.

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies.

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.

Validity of a neurological scoring system for canine X-linked myotubular myopathy

A simple clinical neurological test was developed to evaluate response to gene therapy in a preclinical canine model of X-linked Myotubular Myopathy (XLMTM). This devastating congenital myopathy is caused by mutation in the myotubularin (MTM1) gene. Clinical signs include muscle weakness, early respiratory failure and ventilator dependence. A spontaneously occurring canine model has a similar clinical picture and histological abnormalities on muscle biopsy compared to patients. We developed a neuromuscular assessment score, graded on a scale from 10 (normal) to 1 (unable to maintain sternal recumbency). We hypothesize that this neurological assessment score correlates with genotype and established measures of disease severity and is reliable when performed by an independent observer. At 17 weeks of age, there was strong correlation between neurological assessment scores and established methods of severity testing. The neurological severity score correctly differentiated between XLMTM and wild type dogs with good interobserver reliability, on the basis of strong agreement between neurological scores assigned by independent observers. Together, these data indicate that the neurological scoring system developed for this canine congenital neuromuscular disorder is reliable and valid. This scoring system may be helpful in evaluating response to therapy in preclinical testing in this disease model, such as response to gene therapy.