Becker Muscular Dystrophy Patients Research Articles

Muscular dystrophy patients show low exercise‐induced blood flow in muscles with normal strength

AbstractObjectiveNeuromuscular evaluation increasingly employs muscle ultrasonography to determine muscle thickness, mean grayscale echointensity, and visual semiquantitative echotexture attenuation. However, these measures provide low sensitivity for detection of mild muscle abnormality. Exercise‐induced intramuscular blood flow is a physiologic phenomenon, which may be impaired in mildly affected muscles, particularly in dystrophinopathies, and may indicate functional muscle ischemia. We aimed to determine if muscle blood flow is reduced in patients with neuromuscular disorders and preserved muscle strength, and if it correlates with echointensity and digital echotexture measurements.MethodsPeak exercise‐induced blood flow, echointensity, and echotexture were quantified in the elbow flexor muscles of 15 adult patients with Becker muscular dystrophy (BMD) and 13 patients with other muscular dystrophies (OMD). These were compared to 17 patients with Charcot–Marie–Tooth type 1 (CMT1) neuropathy and 21 healthy adults from a previous study.ResultsMuscle blood flow was reduced in all patient groups compared to controls, most prominently in BMD patients (p < 0.0001). Echointensity was similarly increased in all patient groups (p < 0.05), while echotexture was reduced only in muscular dystrophy patients (p ≤ 0.002). In BMD, blood flow correlated with echotexture (Pearson r = 0.6098, p = 0.0158) and strength (Spearman r = 0.5471; p = 0.0370). In patients with normal muscle strength, reduced muscle blood flow was evident in all patient groups (p < 0.001), echotexture was reduced in BMD and OMD (p < 0.01), and echointensity was increased in CMT (p < 0.05).InterpretationMuscle blood flow is a sensitive measure to detect abnormality, even in muscles with normal strength. Increased echointensity may indicate a neurogenic disorder when strength is preserved, while low echotexture suggests a dystrophic disease.

Single-Nucleus RNA Sequencing Unravels Early Mechanisms of Human Becker Muscular Dystrophy.

The transcriptional heterogeneity at a single-nucleus level in human Becker muscular dystrophy (BMD) dystrophic muscle has not been explored. Here, we aimed to understand the transcriptional heterogeneity associated with myonuclei, as well as other mononucleated cell types that underly BMD pathogenesis by performing single-nucleus RNA sequencing. We profiled single-nucleus transcriptional profiles of skeletal muscle samples from 7 BMD patients and 3 normal controls. A total of 17,216 nuclei (12,879 from BMD patients and 4,337 from controls) were classified into 13 known cell types, including 9 myogenic lineages and 4 non-myogenic lineages, and 1 unclassified nuclear type according to their cell identities. Among them, type IIx myonuclei were the first to degenerate in response to dystrophin reduction. Differential expression analysis revealed that the fibro-adipogenic progenitors (FAPs) population had the largest transcriptional changes among all cell types. Sub-clustering analysis identified a significantly compositional increase in the activated FAPs (aFAPs) subpopulation in BMD muscles. Pseudotime analysis, regulon inference, and deconvolution analysis of bulk RNA-sequencing data derived from 29 BMD patients revealed that the aFAPs subpopulation, a distinctive and previously unrecognized mononuclear subtype, was profibrogenic and expanded in BMD patients. Muscle quantitative real-time polymerase chain reaction and immunofluorescence analysis confirmed that the mRNA and protein levels of the aFAPs markers including LUM, DCN, and COL1A1 in BMD patients were significantly higher than those in controls, respectively. Our results provide insights into the transcriptional diversity of human BMD muscle at a single-nucleus resolution and new potential targets for anti-fibrosis therapies in BMD. ANN NEUROL 2024.

Comprehensive analysis of 2097 patients with dystrophinopathy based on a database from 2011 to 2021

BackgroundAn increasing number of clinical trials for new therapeutic strategies are underway or being considered for dystrophinopathy. Having detailed data on the natural progression of this condition is crucial for assessing the effectiveness of new drugs. However, there’s a lack of data regarding the long-term data on the natural course and how it’s managed in China. In this study, we offer a comprehensive overview of clinical and molecular findings, as well as treatment outcomes in the Chinese population.MethodsInstitutional data on all patients with dystrophinopathy from August 2011 to August 2021 were retrospectively reviewed. The data included geographic distribution, age at diagnosis, molecular findings, and treatment options, such as corticosteroids, cardiac interventions, and clinical outcomes.ResultsIn total, 2097 patients with dystrophinopathy, including 1703 cases of Duchenne muscular dystrophy (DMD), 311 cases of Becker muscular dystrophy (BMD), 46 cases of intermediate muscular dystrophy (IMD), and 37 cases categorized as “pending” (individuals with an undetermined phenotype), were registered in the Children’s Hospital of Fudan University database for dystrophinopathy from August 2011 to August 2021. The spectrum of identified variants included exonic deletions (66.6%), exonic duplications (10.7%), nonsense variants (10.3%), splice-site variants (4.5%), small deletions (3.5%), small insertions/duplications (1.8%), and missense variants (0.9%). Four deep intronic variants and two inversion variants were identified. Regarding treatment, glucocorticoids were administered to 54.4% of DMD patients and 39.1% of IMD patients. The median age at loss of ambulation was 2.5 years later in DMD patients who received glucocorticoid treatment. Overall, one cardiac medicine at least was prescribed to 7.4% of DMD patients, 8.3% of IMD patients, and 2.6% of BMD patients. Additionally, ventilator support was required by four DMD patients. Eligibility for exon skipping therapy was found in 55.3% of DMD patients, with 12.9%, 10%, and 9.6% of these patients being eligible for skipping exons 51, 53, and 45, respectively.ConclusionsThis is one of the largest studies to have evaluated the natural history of dystrophinopathy in China, which is particularly conducive to the recruitment of eligible patients for clinical trials and the provision of real-world data to support drug development.

Acute hepatotoxicity of intravenous amiodarone in a Becker muscular dystrophy patient with decompensated heart failing and ABCB4 gene mutation: as assessed for causality using the updated RUCAM

BackgroundCardiac dysfunction, including arrhythmias, may be one of the main clinical manifestations of Becker muscular dystrophy (BMD). Amiodarone is widely used to treat arrhythmia. However, multi-systemic toxicity caused by amiodarone, especially hepatotoxicity, should not be neglected. Here, we introduce a novel case of multi-systemic amiodarone toxicity involving the liver, renal and coagulation in BDM patient with ABCB4 gene mutation.Case presentationWe present a case of a 16-year-old boy admitted with heart failure and atrial fibrillation (AF). He was diagnosed with Becker muscular dystrophy (BMD) and gene testing showed comorbid mutations in gene DMD, ABCB4 and DSC2. Amiodarone was prescribed to control the paroxysmal atrial fibrillation intravenously. However, his liver enzyme levels were sharply elevated, along with cardiac shock, renal failure and coagulation disorders. After bedside continuous renal replacement therapy, the patient’s liver function and clinical status rehabilitated.ConclusionsABCB4 gene mutation might be involved in amiodarone-induced hepatotoxicity. Studies in a cohort might help to prove this hypothesis in the future.

Classification of Muscular Dystrophies from MR Images Improves Using the Swin Transformer Deep Learning Model.

Muscular dystrophies present diagnostic challenges, requiring accurate classification for effective diagnosis and treatment. This study investigates the efficacy of deep learning methodologies in classifying these disorders using skeletal muscle MRI scans. Specifically, we assess the performance of the Swin Transformer (SwinT) architecture against traditional convolutional neural networks (CNNs) in distinguishing between healthy individuals, Becker muscular dystrophy (BMD), and limb-girdle muscular Dystrophy type 2 (LGMD2) patients. Moreover, 3T MRI scans from a retrospective dataset of 75 scans (from 54 subjects) were utilized, with multiparametric protocols capturing various MRI contrasts, including T1-weighted and Dixon sequences. The dataset included 17 scans from healthy volunteers, 27 from BMD patients, and 31 from LGMD2 patients. SwinT and CNNs were trained and validated using a subset of the dataset, with the performance evaluated based on accuracy and F-score. Results indicate the superior accuracy of SwinT (0.96), particularly when employing fat fraction (FF) images as input; it served as a valuable parameter for enhancing classification accuracy. Despite limitations, including a modest cohort size, this study provides valuable insights into the application of AI-driven approaches for precise neuromuscular disorder classification, with potential implications for improving patient care.

Enhancing Sarcolemmal Membrane Repair Using a Novel Protein Therapeutic Approach can Improve Membrane Integrity in Duchenne Muscular Dystrophy Cells and Mouse Models

Duchenne Muscular Dystrophy (DMD) is a fatal neuromuscular disease involving progressive cardiac and skeletal myocyte cell death and subsequent muscle degeneration. DMD and Becker muscular dystrophy (BMD) result from mutations in the dystrophin gene that compromise the structural integrity of the sarcolemma. While there has been important recent progress in the treatment of DMD, the currently available therapeutics have significant limitations. Thus, novel therapies that address the compromised sarcolemmal membrane integrity are an unmet medical need. Tripartite motif protein 72, or mitsugumin 53 (TRIM72/MG53), facilitates the sarcolemma repair response after disruption of the membrane. Loss of TRIM72/MG53 function in mice leads to a progressive myopathy. Exogenous delivery of recombinant human MG53 protein (rhMG53) can increase sarcolemmal membrane repair in many different cell types and ameliorate pathology in models of DMD and multiple limb girdle muscular dystrophies. Despite the promise of rhMG53 as a therapeutic invention the development of this protein to treat DMD has been complicated by findings showing that high levels of the protein correlate with metabolic dysfunction in animal models and some human patients. Deletion analysis of the major domains of the rhMG53 protein allowed us to design an improved version of rhMG53 called MyoTRIM. MyoTRIM contains amino acid replacements to eliminate metabolic effects and improve solubility while maintaining membrane repair effects. One set of modifications inactivate the E3 ligase activity of the protein, which revealed this function is dispensable for subcellular localization of the protein. TRIM72/MG53 is reported to mediate this effect by binding phosphatidylserine (PS) at membrane injury sites, so we demonstrate that MyoTRIM can bind PS-coated beads to illustrate that carboxy-terminal domains are required for effcient PS binding rather than the E3 ligase function. External application of MyoTRIM can increase membrane repair capacity in a variety of cell-based assays using DMD and BMD patient myoblasts and myotubes. We also establish that MyoTRIM can increase membrane repair and prevent eccentric contraction induced injury in a dystrophin deficient mouse model. Taken together, these results provide mechanistic insight into rhMG53 function and indicate that MyoTRIM can recapitulate the therapeutic effects on sarcolemmal membrane repair while eliminating E3 ligase activity and associated side effects. This work was supported by the Offce of the Assistant Secretary of Defense for Health Affairs through the Duchenne Muscular Dystrophy Research Program (DMDRP) under Translational Research Award HT9425-23-1-0940. The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diffusion tensor imaging reveals subclinical alterations in muscles of patients with Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is a relatively less investigated neuromuscular disease, partially overlapping the phenotype of Duchenne dystrophy (DMD). Physiopathological and anatomical patterns are still not comprehensively known, despite recent effort in the search of early biomarkers. Aim of this study was to selectively compare normal appearing muscles of BMD with healthy controls. Among a pool of 40 BMD patients and 20 healthy controls, Sartorius and gracilis muscles were selected on the basis of a blinded clinical quantitative/qualitative evaluation, if classified as normal (0 or 1 on Mercuri scale) and subsequently segmented on diffusion tensor MRI scans with a tractographic approach. Diffusion derived parameters were extracted. Non-parametric testing revealed significant differences between normal and normal appearing BMD derived parameters in both muscles, the difference being more evident in sartorius. Bonferroni-corrected P-values (<.05) of Mann-Whitney test could discriminate between BMD and controls for standard deviation of all diffusion parameters (mean diffusivity, fractional anisotropy, axial and radial diffusivity) in both sartorius and gracilis, while in sartorius the significant difference was found also in the average values of the same parameters (with exception of RD). This method could identify microstructural alterations in BMD normal appearing sartorius and gracilis. Diffusion based MRI could be able to identify possible early or subclinical microstructural alterations in dystrophic patients with BMD.

GC–MS analysis of 4-hydroxyproline: elevated proline hydroxylation in metformin-associated lactic acidosis and metformin-treated Becker muscular dystrophy patients

Metformin (N,N-dimethylbiguanide), an inhibitor of gluconeogenesis and insulin sensitizer, is widely used for the treatment of type 2 diabetes. In some patients with renal insufficiency, metformin can accumulate and cause lactic acidosis, known as metformin-associated lactic acidosis (MALA, defined as lactate ≥ 5 mM, pH < 7.35, and metformin concentration > 38.7 µM). Here, we report on the post-translational modification (PTM) of proline (Pro) to 4-hydroxyproline (OH-Pro) in metformin-associated lactic acidosis and in metformin-treated patients with Becker muscular dystrophy (BMD). Pro and OH-Pro were measured simultaneously by gas chromatography–mass spectrometry before, during, and after renal replacement therapy in a patient admitted to the intensive care unit (ICU) because of MALA. At admission to the ICU, plasma metformin concentration was 175 µM, with a corresponding lactate concentration of 20 mM and a blood pH of 7.1. Throughout ICU admission, the Pro concentration was lower compared to healthy controls. Renal excretion of OH-Pro was initially high and decreased over time. Moreover, during the first 12 h of ICU admission, OH-Pro seems to be renally secreted while thereafter, it was reabsorbed. Our results suggest that MALA is associated with hyper-hydroxyprolinuria due to elevated PTM of Pro to OH-Pro by prolyl-hydroxylase and/or inhibition of OH-Pro metabolism in the kidneys. In BMD patients, metformin, at the therapeutic dose of 3 × 500 mg per day for 6 weeks, increased the urinary excretion of OH-Pro suggesting elevation of Pro hydroxylation to OH-Pro. Our study suggests that metformin induces specifically the expression/activity of prolyl-hydroxylase in metformin intoxication and BMD.

Generation of human induced pluripotent stem cell line derived from Becker muscular dystrophy patient with CRISPR/Cas9-mediated correction of DMD gene mutation

Becker muscular dystrophy (BMD) is an X-linked recessive disorder caused by in-frame deletions in the dystrophin gene (DMD), leading to progressive muscle degeneration and weakness. We generated a human induced pluripotent stem cell (hiPSC) line from a BMD patient. BMD hiPSCs were then engineered by CRISPR/Cas9-mediated knock-in of missing exons 3–9 of DMD gene. Obtained hiPSC line may be a valuable tool for investigating the mechanisms underlying BMD pathogenesis.

Diverse Cardiac Phenotype of Becker Muscular Dystrophy: Under-Recognized Subclinical Cardiomyopathy Due to Partial Dystrophin Deficiency in a Contemporary Era.

Becker muscular dystrophy (BMD) is an X-linked recessive disorder responsible for mild skeletal muscle involvement and variable degree of cardiomyopathy. The characteristics of cardiac phenotype of BMD in childhood remain elusive. Clinical manifestations, genotype, serum biomarkers, and echocardiogram were retrospectively reviewed in BMD patients. Cardiac phenotype was classified into acute progressive (AP), chronic persistent (CP), and latent (L) groups based upon symptoms and echocardiographic findings. Twenty-five BMD patients were studied over 9.5 ± 2.5years. Sixteen patients presented initially with variable degree of muscle weakness whereas 9 were asymptomatic. Three patients developed medically refractory heart failure by age 18 with progressive dilated cardiomyopathy (DCM) (AP). Six patients developed mild to moderate left ventricular (LV) systolic dysfunction with LV dilatation but remained asymptomatic (CP). Although 16 patients continued to show normal LV function (L), they demonstrated variable degrees of skeletal muscle involvement. The AP groups presented with significantly larger LV size and LV mass index (LVMI) at the initial encounter than groups CP or L, suggesting early myocardial remodeling predicts rapid disease progression. None presented with atrophic myocardial phenotype commonly observed in Duchenne muscular dystrophy (DMD). Wide availability of genetic testing has changed the scope of clinical presentation of BMD. Cardiomyopathy in BMD presents with a diverse clinical spectrum with variable progression of DCM where larger LV dimension and mass at the time of diagnosis may predict the progressiveness of cardiomyopathy.

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting

BackgroundThe phenotype of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients is determined by the type of DMD gene variation, its location, effect on reading frame, and its size. The primary objective of this investigation was to determine the frequency and distribution of DMD gene variants (deletions/duplications) in Sri Lanka through the utilization of a combined approach involving multiplex polymerase chain reaction (mPCR) followed by Multiplex Ligation Dependent Probe Amplification (MLPA) and compare to the international literature. The current consensus is that MLPA is a labor efficient yet expensive technique for identifying deletions and duplications in the DMD gene.MethodologyGenetic analysis was performed in a cohort of 236 clinically suspected pediatric and adult myopathy patients in Sri Lanka, using mPCR and MLPA. A comparative analysis was conducted between our findings and literature data.ResultsIn the entire patient cohort (n = 236), mPCR solely was able to identify deletions in the DMD gene in 131/236 patients (DMD-120, BMD-11). In the same cohort, MLPA confirmed deletions in 149/236 patients [DMD-138, BMD -11]. These findings suggest that mPCR has a detection rate of 95% (131/138) among all patients who received a diagnosis. The distal and proximal deletion hotspots for DMD were exons 45–55 and 6–15. Exon 45–60 identified as a novel in-frame variation hotspot. Exon 45–59 was a hotspot for BMD deletions. Comparisons with the international literature show significant variations observed in deletion and duplication frequencies in DMD gene across different populations.ConclusionDMD gene deletions and duplications are concentrated in exons 45–55 and 2–20 respectively, which match global variation hotspots. Disparities in deletion and duplication frequencies were observed when comparing our data to other Asian and Western populations. Identified a 95% deletion detection rate for mPCR, making it a viable initial molecular diagnostic approach for low-resource countries where MLPA could be used to evaluate negative mPCR cases and cases with ambiguous mutation borders. Our findings may have important implications in the early identification of DMD with limited resources in Sri Lanka and to develop tailored molecular diagnostic algorithms that are regional and population specific and easily implemented in resource limited settings.

Social cognition in two brothers with Becker muscular dystrophy: an exploratory study revealing divergent behavioral phenotypes.

Only few studies investigated social cognition in Becker muscular dystrophy (BMD). However, brain dystrophin deficiency could be a neural substrate for cognitive, emotional, and neuropsychological features in BMD. We compared interoceptive accuracy and interpersonal comfort distance in two brothers with BMD presenting with the same genetic deletion and a healthy control. When possible, we collected neuropsychological and psychopathological assessments. Our BMD patients were significantly different in interoceptive accuracy, with patient 1 being extremely accurate and patient 2 being significantly less accurate than his brother but more accurate than the control. Interestingly, they presented opposite patterns of interpersonal distance. Patient 1 was comfortable with very short interpersonal distance (≤50 cm from the confederate/object) vs the control and patient 2. By contrast, patient 2 preferred larger distance vs the control and patient 1. Patient 1 also presented difficulties in social and emotional skills on the psychopathological assessment. We are aware this is a small sample; nonetheless, this is also the first description of such aspects in BMD and the first report ever of such divergent behavioral pattern. As impaired social cognition affects the quality of life and social relationship, further studies are needed for a closer understanding of involved mechanisms.

A novel biomarker of fibrofatty replacement in dystrophinopathies identified by integrating transcriptome, magnetic resonance imaging, and pathology data.

We aimed to analyse genome-wide transcriptome differences between Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients and identify biomarkers that correlate well with muscle magnetic resonance imaging (MRI) and histological fibrofatty replacement in both patients, which have not been reported. One hundred and one male patients with dystrophinopathies (55 DMD and 46 BMD) were enrolled. Muscle-derived genome-wide RNA-sequencing was performed in 31 DMD patients, 29 BMD patients, and 11 normal controls. Fibrofatty replacement was scored on muscle MRI and histological levels in all patients. A unique pipeline, single-sample gene set enrichment analysis combined with Spearman's rank correlations (ssGSEA-Cor) was developed to identify the most correlated gene signature for fibrofatty replacement. Quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, and single-nucleus RNA-sequencing (snRNA-seq) were performed in the remaining patients to validate the most correlated gene signature. Comparative transcriptomic analysis revealed that 31 DMD muscles were characterized by a significant increase of inflammation/immune response and extracellular matrix remodelling compared with 29 BMD muscles (P<0.05). The ssGSEA-Cor pipeline revealed that the gene set of CDKN2A and CDKN2B was the most correlated gene signature for fibrofatty replacement (histological rs =0.744, P<0.001; MRI rs =0.718, P<0.001). Muscle qRT-PCR confirmed that CDKN2A mRNA expression in both 15 DMD (median=25.007, P<0.001) and 12 BMD (median=5.654, P<0.001) patients were significantly higher than that in controls (median=1.101), while no significant difference in CDKN2B mRNA expression was found among DMD, BMD, and control groups. In the 27 patients, muscle CDKN2A mRNA expression respectively correlated with muscle MRI (rs =0.883, P<0.001) and histological fibrofatty replacement (rs =0.804, P<0.001) and disease duration (rs =0.645, P<0.001) and North Star Ambulatory Assessment total scores (rs =-0.698, P<0.001). Muscle western blot analysis confirmed that both four DMD (median=2.958, P<0.05) and four BMD (median=1.959, P<0.01) patients had a significantly higher level of CDKN2A protein expression than controls (median=1.068). The snRNA-seq analysis of two DMD muscles revealed that CDKN2A was mainly expressed in fibro-adipogenic progenitors, satellite cells, and myoblasts. We identify CDKN2A expression as a novel biomarker of fibrofatty replacement, which might be a new target for antifibrotic therapy in dystrophinopathies.

Cognitive abnormalities in Becker muscular dystrophy: a mysterious link between dystrophin deficiency and executive functions

BackgroundDistrophinopathies are a heterogeneous group of neuromuscular disorders due to mutations in the DMD gene. Different isoforms of dystrophin are also expressed in the cerebral cortex and Purkinje cells. Despite cognitive abnormalities in Duchenne muscular dystrophy subjects that have been described in the literature, little is known about a comprehensive cognitive profile in Becker muscular dystrophy patients.AimThe aim of this study was to assess cognitive functioning in Becker muscular dystrophy patients by using an extensive neuropsychological battery. Our hypothesis is that the most impaired functions are the highly intentional and conscious ones, such as working memory functions, which require a prolonged state of cellular activation.MethodsWe performed an extensive neuropsychological assessment on 28 Becker muscular dystrophy patients from 18 to 65 years old. As control subjects, we selected 20 patients with limb-girdle muscular dystrophy, whose clinical picture was similar except for cognitive integrity. The evaluation, although extended to all areas, was focused on prefrontal control skills, with a distinction between inhibitory processes of selective attention and activating processes of working memory.Results and conclusionsSignificant underperformances were found exclusively in the Dual Task and PASAT tests, to demonstrate a selective impairment of working memory that, while not causing intellectual disability, reduces the intellectual potential of patients with Becker muscular dystrophy.

Diffusion-tensor magnetic resonance imaging captures increased skeletal muscle fibre diameters in Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is an X-linked disorder characterized by slow, progressive muscle damage and muscle weakness. Hallmarks include fibre-size variation and replacement of skeletal muscle with fibrous and adipose tissues, after repeated cycles of regeneration. Muscle histology can detect these features, but the required biopsies are invasive, are difficult to repeat and capture only small muscle volumes. Diffusion-tensor magnetic resonance imaging (DT-MRI) is a potential non-invasive alternative that can calculate muscle fibre diameters when applied with the novel random permeable barrier model (RPBM). In this study, we assessed muscle fibre diameters using DT-MRI in BMD patients and healthy controls and compared these with histology. We included 13 BMD patients and 9 age-matched controls, who underwent water-fat MRI and DT-MRI at multiple diffusion times, allowing RPBM parameter estimation in the lower leg muscles. Tibialis anterior muscle biopsies were taken from the contralateral leg in 6 BMD patients who underwent DT-MRI and from an additional 32 BMD patients and 15 healthy controls. Laminin and Sirius-red stainings were performed to evaluate muscle fibre morphology and fibrosis. Twelve ambulant patients from the MRI cohort underwent the North Star ambulatory assessment, and 6-min walk, rise-from-floor and 10-m run/walk functional tests. RPBM fibre diameter was significantly larger in BMD patients (P=0.015): mean (SD)=68.0 (25.3) μm versus 59.4 (19.2) μm in controls. Inter-muscle differences were also observed (P≤0.002). Both inter- and intra-individual RPBM fibre diameter variability were similar between groups. Laminin staining agreed with the RPBM, showing larger median fibre diameters in patients than in controls: 72.5 (7.9) versus 63.2 (6.9) μm, P=0.006. However, despite showing similar inter-individual variation, patients showed more intra-individual fibre diameter variability than controls-mean variance (SD)=34.2 (7.9) versus 21.4 (6.9) μm, P<0.001-and larger fibrosis areas: median (interquartile range)=21.7 (5.6)% versus 14.9 (3.4)%, P<0.001. Despite good overall agreement of RPBM and laminin fibre diameters, they were not associated in patients who underwent DT-MRI and muscle biopsy, perhaps due to lack of colocalization of DT-MRI with biopsy samples. DT-MRI RPBM metrics agree with histology and can quantify changes in muscle fibre size that are associated with regeneration without the need for biopsies. They therefore show promise as imaging biomarkers for muscular dystrophies.

Drugs for Genetic Myopathy

Specific therapy for genetic myopathy is still limited to enzyme replacement therapy for Pompe disease with alglucosidase alfa and avalglucosidase alfa, and exon skipping therapy with antisense nucleotide viltolarsen for about 7% of patients with Duchenne muscular dystrophy. Corticosteroid treatment (predonisolone 10-15mg/day) to Duchenne muscular dystrophy, from the age of 5-6 years old, regardless of mutations. Continuation of corticosteroid after loss of ambulation is controversial. Becker muscular dystrophy patients and manifesting female carriers of DMD mutation may also benefit from corticosteroid, but adverse effects should be avoided. In other types of muscular dystrophy, usefulness of corticosteroid has been reported but may be more limited. Drug therapy should be added on the basis of appropriate evaluation and fundamental symptomatic treatment including rehabilitation, in genetic myopathy.

The X-linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45-47.

Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy(DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P<0.0001), wire hang (P=0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P=0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P<0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P=0.0017) and centrally located nuclei indicating degeneration/regeneration (P<0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P<0.0001), collagen deposition (+1.4-fold, P=0.0217), and sarcolemmal damage measured by intracellular IgM (P=0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

Determination of equilibria constants of arginine:glycine amidinotransferase (AGAT)-catalyzed reactions using concentrations of circulating amino acids

Arginine:glycine amidinotransferase (AGAT) catalyzes mainly two reactions that generate 1) L-homoarginine (hArg) from L-arginine and L-lysine (Kharg) and 2) guanidinoacetate (GAA) and L-ornithine from L-arginine and glycine (Kgaa). Previously, we found that pharmacological treatment of Becker muscular dystrophy (BMD) patients with metformin or L-citrulline resulted in antidromic effects on serum hArg and GAA concentrations, seemingly acting as an inhibitor and effector of AGAT activity, respectively. Here, we used data of this study as a model to determine Kharg and Kgaa values by using the concentrations of the participating amino acids measured in serum samples of the BMD patients. The study aimed to prove the general utility of this approach to investigate effects of amino acids and drugs on AGAT-catalyzed reactions in vivo in humans.

P.62 Increased skeletal muscle extracellular volume fraction in patients with Becker muscular dystrophy assessed by quantitative magnetic resonance imaging

Quantitative magnetic resonance imaging (MRI) is increasingly proposed in clinical trials related to dystrophinopathies, including Becker muscular dystrophy (BMD). Extracellular volume fraction (ECV) is a candidate variable to characterize muscle tissues microstructure. Previous studies have demonstrated that the myocardium of BMD patients presented increased ECV. Given that cardiac muscle shares important structural features with skeletal muscles, we hypothesize that ECV could also be a sensitive biomarker to monitor pathological alterations of the skeletal muscle structure in BMD patients. We established the sensitivity of ECV quantification using magnetic resonance fingerprinting with water and fat separation (MRF T1-FF) as an imaging biomarker of skeletal muscle tissue alterations in BMD, by comparison with fat fraction (FF) and water relaxation time quantification. Data from the thighs of 28 BMD patients and 8 healthy volunteers were acquired. The MRI exam comprised FF mapping, water T2 mapping and water T1 mapping before and after an intravenous injection of a gadolinium-based contrast agent using MRF T1-FF from which ECV was calculated. Mann-Whitney tests, Kruskal-Wallis tests, ROC curves analysis and Spearman-rank correlation tests were performed. ECV was higher in dystrophic patients than in controls (0.20 [0.16 – 0.28] vs 0.07 [0.06 – 0.07], p<.001). In muscles of BMD patients with normal FF, ECV was also higher than in muscles of healthy controls (0.11 [0.10 – 0.13] vs 0.07 [0.06 – 0.09], p<.001). ECV classified dystrophic muscles with high sensitivity and specificity (AUC, 0.98; 95%CI: 0.96-0.99, p <.001), and correlated significantly with FF (ρ = .62, p < .001), water T2 (ρ = .56, p = .002), Walton score (ρ = .58, p = .001) and serum cardiac troponin-T level (ρ = .63, p < .001). In conclusion, muscle ECV measured with MRF represents a promising imaging biomarker for the early detection of skeletal muscle involvement in patients with Becker muscular dystrophy.

P.12 A case report of near normalization of serum creatine phosphokinase in a patient with Duchenne muscular dystrophy during acute pancreatitis

Creatine phosphokinase (CPK) is a muscle-specific protein that has served a vital role as a diagnostic biomarker for Duchenne Muscular Dystrophy (DMD). It is also often used to track disease progression in DMD. However, its role as a monitoring, or predictive biomarker is less clear. We describe a 6-year-old boy with DMD caused by duplication of exon 2 of the dystrophin gene as well at IgG deficiency. He presented acutely to the emergency department with brown colored urine in the setting of five days of non-bloody non-bilious emesis, abdominal pain and poor oral intake. His initial examination was notable for signs of moderate dehydration and a soft, non-tender abdomen. His initial laboratory evaluation was notable for anion gap metabolic acidosis, hyponatremia and hypoglycemia. He was ultimately diagnosed with acute pancreatitis after lipase was found to be 2201.0 unit/L (ref 48.0-199.0 unit/L) on day of his admission. He was placed on intravenous fluids and admitted for a total of 3 days with complete recovery. This individual had a baseline CPK of 11,000-21,000 unit/L at previous outpatient visits. Of note, CPK level was obtained on the day of his hospital admission and was found to be 284 unit/L (ref 30-250). The day after admission, CPK was rechecked twice and were 312 and 322 unit/L separately. Fourteen days after admission, CPK was rechecked and had returned to near his previous baseline at 15,496 unit/L. Pancreatitis and other acute inflammatory conditions in DMD are rare. Transient near normalization of CPK has been described previously in one patient with Becker Muscular Dystrophy patient during transient arthritis. To our knowledge, our patient is the first reported case of normalization of CPK level in DMD during acute inflammatory disease. We hypothesize the transient normalization of CPK was secondary to active systemic response during the acute inflammation. Studies to look deeper into reasons that may underlie such a phenomenon may shed light in finding protective mechanisms associated with the inflammation pathway.