X-linked Dystrophy Research Articles

Development of a novel startle response task in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), an X-linked childhood-onset muscular dystrophy caused by loss of the protein dystrophin, can be associated with neurodevelopmental, emotional and behavioural problems. A DMD mouse model also displays a neuropsychiatric phenotype, including increased startle responses to threat which normalise when dystrophin is restored in the brain. We hypothesised that startle responses may also be increased in humans with DMD, which would have potential translational therapeutic implications. To investigate this, we first designed a novel discrimination fear-conditioning task and tested it in six healthy volunteers, followed by male DMD (n = 11) and Control (n = 9) participants aged 7-12 years. The aims of this methodological task development study were to: i) confirm the task efficacy; ii) optimise data processing procedures; iii) determine the most appropriate outcome measures. In the task, two neutral visual stimuli were presented: one 'safe' cue presented alone; one 'threat' cue paired with a threat stimulus (aversive noise) to enable conditioning of physiological startle responses (skin conductance response, SCR, and heart rate). Outcomes were the unconditioned physiological startle responses to the initial threat, and retention of conditioned responses in the absence of the threat stimulus. We present the protocol development and optimisation of data processing methods based on empirical data. We found that the task was effective in producing significantly higher physiological startle SCR in reinforced 'threat' trials compared to 'safe' trials (P < .001). Different data extraction methods were compared and optimised, and the optimal sampling window was derived empirically. SCR amplitude was the most effective physiological outcome measure when compared to SCR area and change in heart rate, with the best profile on data processing, the least variance, successful conditioned response retention (P = .01) and reliability assessment in test-retest analysis (rho = .86). The definition of this novel outcome will allow us to study this response in a DMD population.

Emerin self-assembly and nucleoskeletal coupling regulate nuclear envelope mechanics against stress.

Emerin is an integral nuclear envelope protein that participates in the maintenance of nuclear shape. When mutated or absent, emerin causes X-linked Emery-Dreifuss muscular dystrophy (EDMD). To understand how emerin takes part in molecular --scaffolding at the nuclear envelope and helps protect the nucleus against mechanical stress, we established its nanoscale organization using single-molecule tracking and super-resolution microscopy. We show that emerin monomers form localized oligomeric nanoclusters stabilized by both lamin A/C and the SUN1-containing linker of nucleoskeleton and cytoskeleton (LINC) complex. Interactions of emerin with nuclear actin and BAF (also known as BANF1) additionally modulate its membrane mobility and its ability to oligomerize. In nuclei subjected to mechanical challenges, the mechanotransduction functions of emerin are coupled to changes in its oligomeric state, and the incremental self-assembly of emerin determines nuclear shape adaptation against mechanical forces. We also show that the abnormal nuclear envelope deformations induced by EDMD emerin mutants stem from improper formation of lamin A/C and LINC complex-stabilized emerin oligomers. These findings place emerin at the center of the molecular processes that regulate nuclear shape remodeling in response to mechanical challenges.

Combined growth hormone and insulin-like growth factor-1 rescues growth retardation in glucocorticoid-treated mdxmice but does not prevent osteopenia.

Short stature and osteoporosis are common in Duchenne muscular dystrophy (DMD) and its pathophysiology may include an abnormality of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis, which is further exacerbated by long-term glucocorticoid (GC) treatment. Hence, an agent that has anabolic properties and may improve linear growth would be beneficial in this setting and therefore requires further exploration. A 5-week-old x-linked muscular dystrophy (mdx) mice were used as a model of DMD. They were treated with prednisolone ± GH + IGF-1 for 4 weeks and then compared to control mdx mice to allow the study of both growth and skeletal structure. GC reduced cortical bone area, bone fraction, tissue area and volume and cortical bone volume, as assessed by micro computed tomography (CT) In addition, GC caused somatic and skeletal growth retardation but improved grip strength. The addition of GH + IGF-1 therapy rescued the somatic growth retardation and induced additional improvements in grip strength (16.9% increase, P < 0.05 compared to control). There was no improvement in bone microarchitecture (assessed by micro-CT and static histomorphometry) or biomechanical properties (assessed by three-point bending). Serum bone turnover markers (Serum procollagen 1 intact N-terminal propeptide (P1NP), alpha C-terminal telopeptide (αCTX)) also remained unaffected. Further work is needed to maximise these gains before proceeding to clinical trials in boys with DMD.

Histopathology of Duchenne muscular dystrophy in correlation with changes in proteomic biomarkers.

Duchenne muscular dystrophy is an inherited disorder of early childhood that affects multiple systems in the body. Besides late-onset cardio-respiratory syndrome and various body-wide pathophysiological changes, X-linked muscular dystrophy is primarily classified as a disorder of the skeletal musculature. This is reflected by severe histopathological alterations in voluntary contractile tissues, including progressive fibre degeneration, fat substitution, reactive myofibrosis and chronic inflammation. The underlying cause for dystrophinopathy are genetic abnormalities in the DMD gene, which can result in the almost complete loss of the membrane cytoskeletal protein dystrophin, which triggers the collapse of the dystrophin-associated glycoprotein complex and disintegration of sarcolemmal integrity. This in turn results in an increased frequency of membrane micro-rupturing and abnormal calcium ion fluxes through the impaired plasmalemma, which renders muscle fibres more susceptible to enhanced proteolytic degradation and necrosis. This review focuses on the complexity of skeletal muscle changes in X-linked muscular dystrophy and outlines cell biological and histological alterations in correlation to proteome-wide variations as judged by mass spectrometric analyses. This includes a general outline of sample handling, subcellular fraction protocols and modern proteomic approaches using gel electrophoretic and liquid chromatographic methods for efficient protein separation prior to mass spectrometry. The proteomic profiling of the dystrophic and highly fibrotic diaphragm muscle is described as an example to swiftly identify novel proteomic markers of complex histopathological changes during skeletal muscle degeneration. The potential usefulness of new protein markers is examined in relation to key histopathological hallmarks for establishing improved diagnostic, prognostic and therapy-monitoring approaches in the field of dystrophinopathy.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

The PPLD has advantages over conventional regression methods in application to moderately sized genome-wide association studies.

In earlier work, we have developed and evaluated an alternative approach to the analysis of GWAS data, based on a statistic called the PPLD. More recently, motivated by a GWAS for genetic modifiers of the X-linked Mendelian disorder Duchenne Muscular Dystrophy (DMD), we adapted the PPLD for application to time-to-event (TE) phenotypes. Because DMD itself is relatively rare, this is a setting in which the very large sample sizes generally assembled for GWAS are simply not attainable. For this reason, statistical methods specially adapted for use in small data sets are required. Here we explore the behavior of the TE-PPLD via simulations, comparing the TE-PPLD with Cox Proportional Hazards analysis in the context of small to moderate sample sizes. Our results will help to inform our approach to the DMD study going forward, and they illustrate several respects in which the TE-PPLD, and by extension the original PPLD, offer advantages over regression-based approaches to GWAS in this context.

DMD - TREATMENT: EP.146 scAAV.U7snRNA-mediated therapy: prolonged dystrophin expression and muscle function correction in adult Dup2 mice

Exon 2 duplications of the DMD gene, encoding the dystrophin protein, account for around 6-11% of all duplication mutations associated with X-linked Duchenne muscular dystrophy (DMD). As part of the preclinical development of a U7snRNA vector currently in a clinical trial (ClinicalTrials.gov NCT04240314), we have previously evaluated the therapeutic efficacy, absence of off-target splicing effects in AAV9.U7snRNA-mediated skipping of exon 2 in a murine Dmd model, and lack of toxicity in non-human primates. Here we report that 3-month-old Dup2 mice systemically injected with scAAV9.U7.ACCA vector, containing four copies of U7snRNA targeted to the exon 2 splice acceptor and splice donor sites, showed efficient exon 2 skipping, long-term dystrophin expression, and skeletal muscle function correction 18-months post vector administration. The RT-PCR data showed that a single vector injection (3E13 vg/kg) resulted in significant exon 2 skipping in tibialis anterior (TA), diaphragm (Dia) and heart tissues, showing an average of 46%, 32% and 73% total therapeutic transcripts, respectively. To determine the degree of functional rescue, in situ and in vitro physiology studies on TA and Dia muscles were performed. Both Dia and TA from 21-month-old control Dup2 mice exhibited a functional deficit with a significant reduction in specific force output (45-61%) compared with Bl6 mice. The significant force drop was also observed in those mice compared with Bl6 following a rigorous fatigue protocol. The single vector infusion resulted in a dramatic improvement in specific force output up to 64-76% in Dia and TA, and better protection of the TA muscle (up to 73%) from repeated fatigue. Overall, our results confirm that scAAV9.U7.ACCA provides long-term protection by restoring the disrupted dystrophin reading frame in straight muscles from Dup2 mice and functional recovery of TA and Dia muscles 18-month post vector administration.

Abstract P343: Physiologic Mechanical Stress In An Induced Pluripotent Stem Cell Derived Cardiomyocyte Model Of Duchene Muscular Dystrophy-related Cardiomyopathy Treated With A Membrane Resealant

Heart failure is leading cause of morbidity and mortality in the X-linked disease Duchenne muscular dystrophy (DMD). DMD is due to mutations the gene encoding dystrophin. Dystrophin localizes to the costamere in skeletal and cardiac muscle and is part of the larger dystrophin complex, which forms a critical connection linking the sarcomere to extracellular matrix. Disruptions in this complex lead to membrane fragility and multiple forms of muscular dystrophy, most of which have significant cardiac involvement. Therapeutic strategies for DMD include FDA-approved agents for exon skipping, as well as micro-dystrophins gene therapy, which is currently in clinical trials. Despite this progress, there is inadequate information as to how these and other novel agents will affect the DMD heart. Given the critical importance of cardiac muscle efficacy for any therapeutic for DMD and importance of membrane fragility in the disease phenotype, we assessed the susceptibility of patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring an out-of-frame exon 46-47 DMD deletion. iPSC-CMs were reprogrammed via a standard approach and differentiated, expanded, and purified by published methods. Troponin T flow cytometry was performed to and a minimum troponin T positivity &gt;85% positivity was set for inclusion. DMD and control cells were plated onto flexible silicone membranes and subjected to equibiaxial strain as physiologic mechanical stressor, consistent with the inciting pathologic insult in DMD. Troponin and LDH release were assessed as clinically-relevant biomarkers of injury. Physiologic stress parameters were defined using troponin and LDH release relative to unstressed conditions. A membrane resealant being developed for treating DMD was shown to reduce troponin and LDH release in DMD iPSC-CMs, and also showed benefit in control cells. This work provides a ready platform for assessing therapeutics that target not only DMD-related cardiomyopathy, but other forms of cardiomyopathy and myocardial injury.

Duchenne Muscular Dystrophy: Genetic and Clinical Profile in the Population of Rajasthan, India.

Background:Duchenne Muscular Dystrophy (DMD) is an X-linked recessive muscular dystrophy that affects young boys and is caused by mutation of the dystrophin gene located over X chromosome.Materials and Methods:In this prospective study, 120 clinically diagnosed DMD patients were tested for exon deletions, duplication or point mutation.Results:Of the 120 clinically suspected DMD patients, the diagnosis of DMD was confirmed by the genetic study or muscle biopsy in 116 patients. The mean age of onset was 3.2 years and the mean age at presentation was 7.2 years. 110/120 cases were confirmed by genetic testing and six were by absence of staining for dystrophin on muscle biopsy. DMD gene deletion was present in 78.5%, duplication in 5.3% and point mutation in 11.2% cases. 70.3% of patients had deletion located at a distal hot spot region. Single exon deletion was found in 16.5%. Distal hotspot exons 47, 48 and 50 were the commonly deleted exons.Conclusions:In our study, 94.8% cases showed genetic change in the DMD gene. Muscle biopsy was the choice of investigation in earlier days. Detection of DMD by DNA based method eliminates the need to do an invasive procedure for diagnosis. Hence the genetic testing should be the investigation of choice in suspected cases of DMD. The pattern of deletion, obtained in the population of Rajasthan was similar when compared with other ethnic groups of the Indian population. It would be helpful for researchers to develop drugs specific to exons or for ongoing mutation-specific therapies.

Rehabilitation in Duchenne Muscular Dystrophy: A Case Report

Background: Duchenne muscular dystrophy (DMD) seems to be the most common X-linked muscular dystrophy condition in children marked by proximal muscle weakness and calf hypertrophy and affected mostly in boys. In more than 20 muscular dystrophies the most prevalent form is Duchenne muscular dystrophy. Each 1/3500 male births, the presence of DMD worldwide, indicating that in the UK alone there are approximately 2400 people living with DMD. Assessing improvements in severity, range of motion and functional mobility, implementation of therapies and guidance on positioning are important key factors.&#x0D; Clinical Finding: Mostly in proximal muscles around the hips and shoulders, which are those around the trunk of the body, muscle weakness occurs mainly. In the lower extremities, weakness usually begins proximally, and then passes distally. Symptoms typically begin about the age of 1-3 years. To see the child run and get up off the floor while searching for signs of DMD is necessary.&#x0D; Diagnosis: A very common physical finding in boys with Duchenne's is Gower's Sign. In order to get up, it requires using their hands to 'climb' their legs. It is because of a weakness in the boy's hip muscles, which was seen in this patient in progressive in nature. The proper examination and evaluation the symptoms the child presented to the physiotherapy clinic played an important in reaching to the diagnosis of the condition.&#x0D; Conclusion: Prevent the child from developing contracture, correct posture of the child and maintain quality of life these are important criteria in for the child suffered from Duchenne muscular dystrophy.

Leadless intracardiac transcatheter pacing system: 20 months follow up in adult Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked muscular dystrophy in relation with dystrophin deficient. Heart and respiratory function are classically involved and affect long-term prognosis. Significant atrio-ventricular block may occur in some patients. Implantation of traditional pacemaker may be challenging in patients with tracheotomy and on permanent home mechanical ventilation. We report the first case of a successful leadless intra-cardiac trans-catheter pacing system implantation in an adult DMD on wheelchair and on permanent home mechanical ventilation.

Early Myocardial Dysfunction and Benefits of Cardiac Treatment in Young X-Linked Duchenne Muscular Dystrophy Mice.

Duchenne muscular dystrophy (DMD) is associated with a progressive alteration in cardiac function. The aim of this study was to detect early cardiac dysfunction using the high sensitive two-dimensional speckle-tracking echocardiography (2D strain) in mdx mouse model and to investigate the potential preventive effects of the S107 ryanodine receptor (RyR2) stabilizer on early onset of DMD-related cardiomyopathy. Conventional echocardiography and global and segmental left ventricle (LV) 2D strains were assessed in male mdx mice and control C57/BL10 mice from 2 to 12months of age. Up to 12months of age, mdx mice showed preserved myocardial function as assessed by conventional echocardiography. However, global longitudinal, radial, and circumferential LV 2D strains significantly declined in mdx mice compared to controls from the 9months of age. Segmental 2D strain analysis found a predominant alteration in posterior, inferior, and lateral LV segments, with a more marked impairment with aging. Then, mdx mice were treated with S107 in the drinking water at a dose of 250mg/L using two different protocols: earlier therapy from 2 to 6months of age and later therapy from 6 to 9months of age. The treatment with S107 was efficient only when administered earlier in very young animals (from 2 to 6months of age) and prevented the segmental alterations seen in non-treated mdx mice. This is the first animal study to evaluate the therapeutic effect of a drug targeting early onset of DMD-related cardiomyopathy, using 2D strain echocardiography. Speckle-tracking analyses revealed early alterations of LV posterior segments that could be prevented by 4months of RyR2 stabilization.

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.

Enhanced cell survival and therapeutic benefits of IL-10-expressing multipotent mesenchymal stromal cells for muscular dystrophy

BackgroundMultipotent mesenchymal stromal cells (MSCs) are potentially therapeutic for muscle disease because they can accumulate at the sites of injury and act as immunosuppressants. MSCs are attractive candidates for cell-based strategies that target diseases with chronic inflammation, such as Duchenne muscular disease (DMD). We focused on the anti-inflammatory properties of IL-10 and hypothesized that IL-10 could increase the typically low survival of MSCs by exerting a paracrine effect after transplantation.MethodsWe developed a continuous IL-10 expression system of MSCs using an adeno-associated virus (AAV) vector. To investigate the potential benefits of IL-10 expressing AAV vector-transduced MSCs (IL-10-MSCs), we examined the cell survival rates in the skeletal muscles after intramuscular injection into mice and dogs. Systemic treatment with IL-10-MSCs derived from dental pulp (DPSCs) was comprehensively analyzed using the canine X-linked muscular dystrophy model in Japan (CXMDJ), which has a severe phenotype similar to that of DMD patients.ResultsIn vivo bioluminescence imaging analysis revealed higher retention of IL-10-MSCs injected into the hindlimb muscle of mice. In the muscles of dogs, myofiber-like tissue was formed after the stable engraftment of IL-10-MSCs. Repeated systemic administration of IL-10-DPSCs into the CXMDJ model resulted in long-term engraftment of cells and slightly increased the serum levels of IL-10. IL-10-hDPSCs showed significantly reduced expression of pro-inflammatory MCP-1 and upregulation of stromal-derived factor-1 (SDF-1). MRI and histopathology of the hDPSC-treated CXMDJ indicated the regulation of inflammation in the muscles, but not myogenic differentiation from treated cells. hDPSC-treated CXMDJ showed improved running capability and recovery in tetanic force with concomitant increase in physical activity. Serum creatine kinase levels, which increased immediately after exercise, were suppressed in IL-10-hDPSC-treated CXMDJ.ConclusionsIn case of local injection, IL-10-MSCs could maintain the long-term engraftment status and facilitate associated tissue repair. In case of repeated systemic administration, IL-10-MSCs facilitated the long-term retention of the cells in the skeletal muscle and also protected muscles from physical damage-induced injury, which improved muscle dysfunction in DMD. We can conclude that the local and systemic administration of IL-10-producing MSCs offers potential benefits for DMD therapy through the beneficial paracrine effects of IL-10 involving SDF-1.

Dental pulp stem cells can improve muscle dysfunction in animal models of Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is an inherited progressive disorder that causes skeletal and cardiac muscle deterioration with chronic inflammation. Dental pulp stem cells (DPSCs) are attractive candidates for cell-based strategies for DMD because of their immunosuppressive properties. Therefore, we hypothesized that systemic treatment with DPSCs might show therapeutic benefits as an anti-inflammatory therapy.MethodsTo investigate the potential benefits of DPSC transplantation for DMD, we examined disease progression in a DMD animal model, mdx mice, by comparing them with different systemic treatment conditions. The DPSC-treated model, a canine X-linked muscular dystrophy model in Japan (CXMDJ), which has a severe phenotype similar to that of DMD patients, also underwent comprehensive analysis, including histopathological findings, muscle function, and locomotor activity.ResultsWe demonstrated a therapeutic strategy for long-term functional recovery in DMD using repeated DPSC administration. DPSC-treated mdx mice and CXMDJ showed no serious adverse events. MRI findings and muscle histology suggested that DPSC treatment downregulated severe inflammation in DMD muscles and demonstrated a milder phenotype after DPSC treatment. DPSC-treated models showed increased recovery in grip-hand strength and improved tetanic force and home cage activity. Interestingly, maintenance of long-term running capability and stabilized cardiac function was also observed in 1-year-old DPSC-treated CXMDJ.ConclusionsWe developed a novel strategy for the safe and effective transplantation of DPSCs for DMD recovery, which included repeated systemic injection to regulate inflammation at a young age. This is the first report on the efficacy of a systemic DPSC treatment, from which we can propose that DPSCs may play an important role in delaying the DMD disease phenotype.

Linked-Read Whole Genome Sequencing Solves a Double DMD Gene Rearrangement.

Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16–29 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a “non-carrier” status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1–15 and 30–34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16–29 deletion, while the second allele showed a 1–34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16–29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.

C–X–C motif chemokine ligand 12: a potential therapeutic target in Duchenne muscular dystrophy

ABSTRACT Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by a mutant dystrophin protein. DMD patients undergo gradual progressive paralysis until death. Chronic glucocorticoid therapy remains one of the main treatments for DMD, despite the significant side effects. However, its mechanisms of action remain largely unknown. We used bioinformatics tools to identify pathogenic genes involved in DMD and glucocorticoid target genes. Two gene expression profiles containing data from DMD patients and healthy controls (GSE38417 and GSE109178) were downloaded for further analysis. Differentially expressed genes (DEGs) between DMD patients and controls were identified using GEO2R, and glucocorticoid target genes were predicted from the Pharmacogenetics and Pharmacogenomics Knowledge Base. Surprisingly, only one gene, CXCL12 (C–X–C motif chemokine ligand 12), was both a glucocorticoid target and a DEG. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, Gene Ontology term enrichment analysis, and gene set enrichment analysis were performed. A protein–protein interaction network was constructed and hub genes identified using the Search Tool for the Retrieval of Interacting Genes (STRING) database and Cytoscape. Enriched pathways involving the DEGs, including CXCL12, were associated with the immune response and inflammation. Levels of CXCL12 and its receptor CXCR4 (C–X–C motif chemokine receptor 4) were increased in X-linked muscular dystrophy (mdx) mice (DMD models) but became significantly reduced after prednisone treatment. Metformin also reduced the expression of CXCL12 and CXCR4 in mdx mice. In conclusion, the CXCL12–CXCR4 pathway may be a potential target for DMD therapy.

Development of outcome measures according to dystrophic phenotypes in canine X-linked muscular dystrophy in Japan.

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder characterized by primary muscle degeneration. Therapeutic strategies for DMD have been extensively explored, and some are in the stage of human clinical trials. Along with the development of new therapies, sensitive outcome measures are needed to monitor the effects of new treatments. Therefore, we investigated outcome measures such as biomarkers and motor function evaluation in a dystrophic model of beagle dogs, canine X-linked muscular dystrophy in Japan (CXMDJ). Osteopontin (OPN), a myogenic inflammatory cytokine, was explored as a potential biomarker in dystrophic dogs over the disease course. The serum OPN levels of CXMDJ dystrophic dogs were elevated, even in the early disease phase, and this could be related to the presence of regenerating muscle fibers; as such, OPN would be a promising biomarker for muscle regeneration. Next, accelerometry, which is an efficient method to quantify performance in validated tasks, was used to evaluate motor function longitudinally in dystrophic dogs. We measured three-axis acceleration and angular velocity with wireless hybrid sensors during gait evaluations. Multiple parameters of acceleration and angular velocity showed notedly lower values in dystrophic dogs compared with wild-type dogs, even at the onset of muscle weakness. These parameters accordingly decreased with exacerbation of clinical manifestations along with the disease course. Multiple parameters also indicated gait abnormalities in dystrophic dogs, such as a waddling gait. These outcome measures could be applicable in clinical trials of patients with DMD or other muscle disorders.

A Case Report of Progressive Muscle Weakness Involving all the Limbs in a 8 yr Old Boy

Duchenne Muscular Dystrophy (DMD) is the most common X-linked muscular dystrophy in children, presenting in early child-hood and characterized by proximal muscle weakness and calf hypertrophy in affected boys. There is usually delay in motor development and eventually wheelchair confinement followed by premature death from cardiac or respiratory complications. Treatment modalities such as corticosteroid therapy and use of intermittent positive pressure ventilation have provided improve-ments in function, ambulation, quality of life and life expectancy, although novel therapies still aim to provide a cure for this devastating disorder. Here, we present a case of DMD in a 8 year-old male with remarkable clinical manifestations of Duchenne muscular dystrophy

DMD – BIOMARKERS & OUTCOME MEASURES: P.127 Identification of biallelic DMD variants during maternal carrier testing for Becker muscular dystrophy

Dystrophinopathies are allelic X-linked recessive muscular dystrophies caused by pathogenic variants in the dystrophin (DMD) gene. In males, out-of-frame DMD variants typically cause more severe Duchenne muscular dystrophy (DMD), while in-frame variants typically result in Becker muscular dystrophy (BMD). Females who carry a DMD variant are at increased risk for cardiomyopathy and may have skeletal muscle symptoms. We report a mother (proband) and three sons, the oldest (10-year-old) and youngest (8-year-old) of whom have a pathogenic in-frame deletion of exons 45-47 and diagnoses of Becker muscular dystrophy. Both have progressive muscle weakness, recurrent rhabdomyolysis, and elevated creatine phosphokinase (CPK) levels ranging from 2,500 – 18,000 unit/L (ref 22-250 unit/L). During confirmatory carrier testing, the mother was found to carry the familial exon 45-47 deletion in addition to an unexpected second pathogenic in-frame DMD deletion of exons 49-51, which was not present in her two previously tested sons. She reports muscle cramps and pain but no muscle weakness. Cardiac MRI shows no current evidence of cardiomyopathy. DMD testing for the family's 9-year-old son detected the exon 49-51 deletion, which supported a diagnosis of BMD for him and confirmed the two DMD changes are situated on opposite alleles in his mother. He had an existing diagnosis of Perthes disease, but had otherwise not come to clinical attention for BMD-related symptoms. The family had reported that CPK levels measured locally had been within normal limits. CPK levels measured subsequent to genetic testing were mildly elevated (up to 321 unit/L), and muscle pain and fatigue were noted during clinical visit, consistent with BMD. This case highlights unexpected outcomes of maternal dystrophinopathy carrier testing, namely the detection of a second variant not previously known in the family, and subsequent diagnosis of BMD in a son who had been presumed to be unaffected. These results underscore the importance of genetic counselling in this setting. This case also describes an additional example of mild BMD in the setting of exon 49-51 deletion, as well as a further example of biallelic DMD variants in a female, of which only a few cases have been described.