Becker Muscular Dystrophy Patients Research Articles

P.28 Introduction of 12 novel pathogenic DMD variants, associated phenotypes and studies of dystrophin and MAST1 abundances

Duchenne muscular dystrophy (DMD) affects one in 3,500-5,600 of male newborns, making it the most common inherited muscular dystrophy. Becker muscular dystrophy (BMD), its milder form, is much rarer, affecting only one in about 18,000 male newborns. Both are caused by different types of mutations in the <i>DMD</i> gene. The encoded protein dystrophin stabilises the plasma membrane of the muscle cell as part of the dystrophin-glycoprotein-complex. Current research into DMD and BMD can explain about 92% of the phenotypes based on their genotypes through the frame-shift hypothesis. However, the remaining 8% are not adequately explained by this hypothesis, so that there is a need for further research to explain - not only - intermediate courses. We screened the cohort of DMD and BMD patients treated at the department of pediatric neurology of the University Hospital Essen for novel pathogenic <i>DMD</i> variants and corresponding phenotypes. We analysed the protein signatures (including post-translational modifications), measured dystrophin levels and studied the interactions of dystrophin and MAST1 in different biomaterials. Here, we report on 12 novel pathogenic variants, which have not yet been described in literature and link though to several several atypical phenotypes. Clinical findings were correlated with abundances of dystrophin and MAST1 toward an attempt of patient stratification. Knowing about novel genotypes and its corresponding (evolving) phenotype is very important in terms of counselling of affected families. Analysing the protein signatures and dystrophin levels as well as interaction partners is crucial to better understand the pathophysiology of dystrophin expression, especially in view of the development of new therapeutic approaches.

Neuropsychological and behavioral profile in a cohort of Becker muscular dystrophy pediatric patients

Cognitive and executive function impairment as well as the association between executive functions and dystrophin gene mutation position have been widely investigated in individuals with Duchenne muscular dystrophy, whereas few studies explored these functions in Becker muscular dystrophy patients. The aim of this study is to investigate the neuropsychological and behavioral profile in a cohort of Becker muscular dystrophy patients and whether there is any correlation with site of dystrophin gene mutation. This is a single-center, observational, cross-sectional study in which a full neuropsychological assessment, including intellectual functioning, executive functions, and language abilities, was performed in children and adolescents without cognitive impairment. A comparison between groups based on site of mutation or Intelligence Quotient level was attempted. 22 patients were enrolled. Overall, the patients in our cohort did not perform well in tests investigating the executive functions. No statistically significant difference was found in groups stratified by site of mutation or cognitive level. This study confirms that these patients have a risk of impairment of the executive functions, despite having a normal Intelligence Quotient in most cases (mean 94). This is a very important aspect, as it puts them at risk of developing learning disabilities.

MicroRNAs as serum biomarkers in Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is an X‐linked neuromuscular disorder due to mutation in the DMD gene, encoding dystrophin. Despite a wide clinical variability, BMD is characterized by progressive muscle degeneration and proximal muscle weakness. Interestingly, a dysregulated expression of muscle‐specific microRNAs (miRNAs), called myomirs, has been found in patients affected with muscular dystrophies, although few studies have been conducted in BMD. We analysed the serum expression levels of a subset of myomirs in a cohort of 29 ambulant individuals affected by BMD and further classified according to the degree of alterations at muscle biopsy and in 11 age‐matched healthy controls. We found a significant upregulation of serum miR‐1, miR‐133a, miR‐133b and miR‐206 in our cohort of BMD patients, supporting the role of these miRNAs in the pathophysiology of the disease, and we identified serum cut‐off levels discriminating patients from healthy controls, confiming the potential of circulating miRNAs as promising noninvasive biomarkers. Moreover, serum levels of miR‐133b were found to be associated with fibrosis at muscle biopsy and with patients' motor performances, suggesting that miR‐133b might be a useful prognostic marker for BMD patients. Taken together, our data showed that these serum myomirs may represent an effective tool that may support stratification of BMD patients, providing the opportunity of both monitoring disease progression and assessing the treatment efficacy in the context of clinical trials.

Inducible Heat Shock Protein 70 Levels in Patients and the mdx Mouse Affirm Regulation during Skeletal Muscle Regeneration in Muscular Dystrophy.

Stress-inducible heat shock protein 70 (HSP70) is both a protective chaperone involved in protein homeostasis and an immune regulator. In both capacities, HSP70 has been implicated in muscle disorders, yet with fragmented and differing results. In this study we aimed to compare results obtained in the mouse model for the severest form of muscular dystrophy (MD) equivalent to Duchenne MD, termed the mdx mouse, with results obtained in human MD. Skeletal muscle and serum samples were obtained from 11 healthy controls, 11 fully characterized patients diagnosed with Becker MD and limb girdle MD (LGMD), and six muscle disease controls. In addition, muscle extracts were prepared from tibialis anterior of mdx and control mice at ages 4, 8 and 12 weeks. The HSP70 levels were quantified using RT-PCR, western blotting and protein arrays, and localized in muscle tissue sections using double immunofluorescence. We found selective and significant 2.2-fold upregulation of HSP70 protein in mdx tibialis muscle at the earliest disease phase only. In LGMD and Becker MD patients, HSP70 protein levels were not significantly different from those of healthy muscle and serum. HSP70 was localized to regenerating muscle fibers both in mouse and human MD skeletal muscle tissues. Toll-like receptor (TLR) 2 and TLR4 expression was moderately increased on the sarcolemma in MD muscle, yet protein levels were not significantly different from normal controls. HSP70 upregulation in MD appears disease stage-dependent, marking the phase of most active muscle regeneration in the mdx mouse. We postulate that well-timed supportive therapeutic interventions with HSP70 agonists could potentially improve muscle tissue's regenerative capacities in MD, attenuating loss of muscle mass while we await gene therapies to become more widely available.

Stable-Isotope Dilution GC–MS Measurement of Metformin in Human Serum and Urine after Derivatization with Pentafluoropropionic Anhydride and Its Application in Becker Muscular Dystrophy Patients Administered with Metformin, l-Citrulline, or Their Combination

Metformin (N,N-dimethylguanylguanidine) is one of the most prescribed drugs with pleiotropic, exerted in part by not fully elucidated mechanisms of action. We developed and validated a gas chromatography–mass spectrometry (GC–MS) method for the quantitative analysis of metformin (metformin-d0) in 10-µL aliquots of human serum and urine using N,N-[dimethylo-2H6]guanylguanidine (metformin-d6) as the internal standard. The method involves evaporation of the samples to dryness, derivatization with pentafluoropropionic (PFP) anhydride in ethyl acetate (30 min, 65 °C), and extraction into toluene. The negative-ion chemical ionization GC–MS spectra of the PFP derivatives contain a single intense ion with mass-to-charge (m/z) ratios of m/z 383 for metformin-d0 and m/z 389 for metformin-d6. Our results suggest that all amine/imine groups of metformin-d0 and metformin-d6 are converted to their N,N,N-tripentafluoropropionyl derivatives, which cyclize to form a symmetric triazine derivative, of which the non-ring amine group is amidated. Quantification was performed by selected-ion monitoring (SIM) of m/z 383 and m/z 389. Upon validation, the method was applied to determine serum and urine metformin concentrations in 19 patients with Becker muscular dystrophy (BMD). Serum and urine samples were collected at baseline (Visit I), after six weeks of supplementation (Visit II) with metformin (3 × 500 mg/d; metformin group; n = 10) or l-citrulline (3 × 1500 mg/d; citrulline group; n = 9) followed by a six-week supplementation with 3 × 500 mg/d of metformin plus 3 × 1500 mg/d l-citrulline. At Visit I, the metformin concentration in the serum and urine was very low in both groups. The metformin concentrations in the serum and urine of the patients who first took metformin (MET group) were higher at Visit II and Visit III. The metformin concentration in the serum and urine samples of the patients who first took l-citrulline (CITR group) were higher at Visit III. The serum and urine concentrations of metformin were insignificantly lower in the CITR group at Visit III. The mean fractional excretion (FE) rate of metformin was 307% (Visit II) and 322% (Visit III) in the MET group, and 290% in the CITR group (Visit III). This observation suggests the accumulation of metformin in the kidney and its secretion in the urine. The GC–MS is suitable to measure reliably circulating and excretory metformin in clinical settings.

Body composition and myokines in a cohort of patients with Becker muscular dystrophy.

Introduction/AimsBecker muscular dystrophy (BMD) is an X‐linked disease leading to muscle wasting and weakness. The decrease in lean body mass (LBM) in Duchenne muscular dystrophy, has shown correlation with loss of muscle function and bone density (BD). Myokines (including irisin) are hormones secreted by skeletal muscle that allow crosstalk between muscle and bone. The present study analyzed body composition and circulating myokine levels in a cohort of BMD patients; moreover, the association between dual energy X‐ray absorptiometry (DXA) parameters, functional motor assessments, and myokine levels was investigated.MethodsAll patients underwent DXA, blood samples for myokine assays, and functional motor assessments. A group of healthy controls (HCs) was also included.ResultsThirty BMD patients, median age at evaluation 36.0 y [26.0–41.0], were included. Twenty‐nine patients underwent whole‐body DXA. Median value of total body Z‐score was −0.70. The prevalence of low skeletal muscle mass defined as appendicular skeletal muscle mass index (ASMMI) < 7.59 kg/m2 was 83%. Irisin levels were significantly lower in BMD compared to HCs (p = .03). All DXA parameters showed significant correlation with the functional motor assessments, in particular the h2‐standardized lean mass lower limb index (p = .0006); h2‐standardized total fat mass showed negative correlations with North Star Ambulatory Assessment and 6 min walk test (p = .03).DiscussionDXA is a useful tool to evaluate body composition in BMD patients; the decrease in BD and LBM is associated with a reduction of motor function in BMD.

Muscle histological changes in a large cohort of patients affected with Becker muscular dystrophy

Becker muscular dystrophy (BMD) is a severe X-linked muscle disease. Age of onset, clinical variability, speed of progression and affected tissues display wide variability, making a clinical trial design for drug development very complex. The histopathological changes in skeletal muscle tissue are central to the pathogenesis, but they have not been thoroughly elucidated yet. Here we analysed muscle biopsies from a large cohort of BMD patients, focusing our attention on the histopathological muscle parameters, as fibrosis, fatty replacement, fibre cross sectional area, necrosis, regenerating fibres, splitting fibres, internalized nuclei and dystrophy evaluation. We correlated histological parameters with both demographic features and clinical functional evaluations. The most interesting results of our study are the accurate quantification of fibroadipose tissue replacement and the identification of some histopathological aspects that well correlate with clinical performances. Through correlation analysis, we divided our patients into three clusters with well-defined histological and clinical features. In conclusion, this is the first study that analyses in detail the histological characteristics of muscle biopsies in a large cohort of BMD patients, correlating them to a functional impairment. The collection of these data help to better understand the histopathological progression of the disease and can be useful to validate any pharmacological trial in which the modification of muscle biopsy is utilized as outcome measure.

A Case of Muscular Dystrophy with Dilated Cardiomyopathy: Do Not Forget Your Basics

<h3>Introduction</h3> Becker muscular dystrophy (BMD) is an X-linked recessive disorder with dystrophin mutation. Dilated Cardiomyopathy (DCM) is a leading cause of death in BMD patients. Herein, we are presenting a patient with BMD that initially sought medical attention for acute onset of systolic heart failure that highlights the importance of careful clinical assessment and appropriate work up. <h3>Case Report</h3> A 29-year-old male with medical history of asthma presented to the hospital with progressive dyspnea and leg swelling. He was diagnosed with DCM with an LVIDD of 6.5 cm and LV ejection fraction of 20-25% by echocardiogram. Coronary angiogram revealed no coronary artery disease. Initial blood work and electrocardiogram are below (Figure). Cardiac MRI showed severely reduced biventricular systolic function with near circumferential, sub-epicardial to mid-myocardial delayed gadolinium enhancement (Figure). Initial differential diagnosis included prior myocarditis vs. burnt out sarcoidosis. It was subsequently noted that patient began recurrently falling with muscle weakness from age 20 years with chronically elevated AST and CK. His exam was notable for atrophy of the bilateral quadriceps muscles, decreased muscular strength and bilateral calves hypertrophy. Electromyography showed evidence of chronic proximal and distal myopathy, predominantly affecting the lower extremity. Skeletal muscle biopsy showed fascicular atrophy and hypertrophy, focal endomyosial fibrosis and an increase of central nuclei without evidence of inflammation or granuloma which was most suggestive of a muscular dystrophy. Genetic testing was then completed and showed hemizygous dystrophin mutation confirming diagnosis of BMD. <h3>Summary</h3> BMD has a diffuse phenotype and should be considered in young patients with cardiomyopathy and chronically elevated CK and AST. A thorough clinical history, exam, and CMR can assist in directing need for skeletal muscle biopsy and subsequent genetic testing.

Patterns of cardiac involvement in different muscular dystrophies assessed by magnetic resonance imaging.

In muscular dystrophies, it is not only skeletal muscles that can be affected, but also the myocardium. This cardiac involvement can represent a major cause of morbidity and mortality. To investigate cardiac involvement in Duchenne (DMD), Becker (BMD), and limb girdle muscular dystrophy (LGMD) patients, and carriers of DMD/BMD by cardiac magnetic resonance (CMR) imaging and to search for differences in the pattern of cardiac involvement. All patients with genetically or histologically proven DMD, BMD, and LGMD, or confirmed carriers of DMD/BMD who had undergone CMR at our clinic between January 2008 and November 2018 were retrospectively included and re-evaluated for regional and global left ventricular function, increased trabecularization, and late enhancement. A total of 26 DMD, 10 BMD, 11 LGMD, and seven DMD/BMD carriers were included. Only one carrier of DMD presented with normal CMR results; all other participants showed cardiac abnormalities. Regional wall motion abnormalities (RWMA; prevalence in LGMD patients: 55%) and late enhancement (prevalence in LGMD patients: 82%) were frequent. RWMA were accentuated basal inferolateral in DMD/BMD carriers, while in LGMD they were accentuated apical. In all groups late enhancement was located mainly subepicardial/midmyocardial with a basal inferolateral accentuation. Apart from the different RWMA distribution, no further group-specific differences were found. We found a high rate of cardiac involvement not only in DMD/BMD, but also in LGMD and DMD/BMD carriers with a different RWMA accentuation (apical in LGMD and basal inferolateral in DMD/BMD) as a single group-specific difference.

Baseline fat fraction is a strong predictor of disease progression in Becker muscular dystrophy.

In Becker muscular dystrophy (BMD), muscle weakness progresses relatively slowly, with a highly variable rate among patients. This complicates clinical trials, as clinically relevant changes are difficult to capture within the typical duration of a trial. Therefore, predictors for disease progression are needed. We assessed if temporal increase of fat fraction (FF) in BMD follows a sigmoidal trajectory and whether fat fraction at baseline (FFbase) could therefore predict FF increase after 2 years (ΔFF). Thereafter, for two different MR‐based parameters, we tested the additional predictive value to FFbase. We used 3‐T Dixon data from the upper and lower leg, and multiecho spin‐echo MRI and 7‐T 31P MRS datasets from the lower leg, acquired in 24 BMD patients (age: 41.4 [SD 12.8] years). We assessed the pattern of increase in FF using mixed‐effects modelling. Subsequently, we tested if indicators of muscle damage like standard deviation in water T2 (stdT2) and the phosphodiester (PDE) over ATP ratio at baseline had additional value to FFbase for predicting ∆FF. The association between FFbase and ΔFF was described by the derivative of a sigmoid function and resulted in a peak ΔFF around 0.45 FFbase (fourth‐order polynomial term: t = 3.7, p < .001). StdT2 and PDE/ATP were not significantly associated with ∆FF if FFbase was included in the model. The relationship between FFbase and ∆FF suggests a sigmoidal trajectory of the increase in FF over time in BMD, similar to that described for Duchenne muscular dystrophy. Our results can be used to identify muscles (or patients) that are in the fast progressing stage of the disease, thereby facilitating the conduct of clinical trials.

Hybrid CMR- and FDG-PET-Imaging Gives New Insights Into the Relationship of Myocardial Metabolic Activity and Fibrosis in Patients With Becker Muscular Dystrophy.

BackgroundCardiac involvement in patients with Becker muscular dystrophy (BMD) is an important predictor of mortality. The cardiac phenotype of BMD patients is characterized by slowly progressive myocardial fibrosis that starts in the left ventricular (LV) free wall segments and extends into the septal wall during the disease course.PurposeSince the reason for this characteristic cardiac phenotype is unknown and comprehensive approaches using e.g. hybrid imaging combining cardiovascular magnetic resonance (CMR) with 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) are limited, the present study addressed this issue by a comprehensive non-invasive imaging approach.MethodsHybrid CMR- and FDG-PET-imaging was performed in N = 14 patients with BMD on a whole-body Biograph mMR system (Siemens, Erlangen, Germany). The CMR protocol comprised cine- and late-gadolinium-enhancement (LGE)-imaging. Metabolism was assessed with FDG-PET after oral glucose loading to effect myocardial carbohydrate uptake. PET was acquired for 65 min starting with tracer injection. Uptake values from 60 to 65 min p.i. were divided by the area under the blood activity curve and reported as percentages relative to the segment with maximal myocardial FDG uptake.ResultsA characteristic pattern of LGE in the LV lateral wall was observed in 13/14 patients whereas an additional septal LGE pattern was documented in 6/14 patients only. There was one patient without any LGE. Segmental FDG uptake was 88 ± 6% in the LV lateral wall vs. 77 ± 10% in the septal wall (p < 0.001). There was an inverse relationship between segmental FDG activity compared to segmental LGE extent (r = −0.33, p = 0.089). There were N = 6 LGE-positive patients with a segmental difference in FDG uptake of >15% in the LV lateral wall compared to the septal wall = ΔFDG-high group (lateral FDG = 91±3% vs. septal FDG = 69±8%; p < 0.001) while the remaining N = 7 LGE-positive patients showed a segmental difference in FDG uptake of ≤ 15% = ΔFDG-low group (lateral FDG = 85±7% vs. septal FDG = 83 ± 5%; p = 0.37). Patients in the ΔFDG-high group showed only a minor difference in the LGE extent between the LV lateral wall vs. septal wall (p = 0.09) whereas large differences were observed in the ΔFDG-low group (p < 0.004).ConclusionsSegmental FDG uptake—reflecting myocardial metabolic activity—is higher in the LV free wall of BMD patients—possibly due to a higher segmental work load. However, segmental metabolic activity seems to be dependent on and limited by the respective segmental extent of myocardial fibrosis as depicted by LGE-imaging.

Diagnostic Value of Dystrophin Immunostaining in the Diagnosis of Duchenne and Becker Muscular Dystrophy Patients

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive muscular disorders caused by the absence or reduction of the muscle cytoskeletal protein dystrophin. Standard procedures to detect deletion and duplication of the DMD gene use Multiplex Ligation-Dependent Probe Amplification (MLPA). However, genetic testing, such as MLPA, is not covered by the national insurance scheme in Indonesia. Immunohistochemical (IHC) staining of dystrophin from muscle biopsy in the form of Formalin-Fixed Paraffin-Embedded (FFPE) specimens can be an alternative method to detect dystrophin expression in protein levels to establish the diagnosis of DMD or BMD.&#x0D; Objectives: To determinate sensitivity, specificity and accuracy of IHC analysis of dystrophin in DMD/BMD patient in comparison with the standard genetic testing, MLPA.&#x0D; Methods: Twenty-six patients enrolled in this study were clinically diagnosed as DMD/BMD in Dr. Sardjito Hospital and Universitas Gadjah Mada Academic Hospital. Genomic DNA was isolated from 3 mL of EDTA-peripheral whole blood samples. The deletion and duplication of DMD genes were detected by MLPA. IHC examination was performed using a specific antibody dystrophin (DYS2). Complete loss of dystrophin staining indicated DMD, while partial loss of dystrophin staining indicated BMD. MLPA result was used as the gold standard to determine sensitivity, specificity, and accuracy of IHC technique using a 2x2 table.&#x0D; Results: MLPA results revealed 18 (18/26; 69.3%) patients with deletion and 3 (3/26; 11.5%) patients with duplication. Five (5/26; 19.2%) patients who showed no deletion nor duplication were excluded from the analysis. Among 21 patients with deletion or duplication, 18 (18/21; 85.7%) patients were out-of-frame (DMD) and 3 (3/21; 14.3%) patients were in-frame (BMD). Six patients showed a discrepancy between the IHC and MLPA results with 9.5% (2/21) false positive and 19% (4/21) false negative. The sensitivity of dystrophin IHC was 77.78%, specificity 33.33%, positive predictive value 87.5%, negative predictive value 20%, and accuracy 71.43%.&#x0D; Conclusion: Muscle biopsy followed by IHC can be one of the diagnostic tools to diagnose BMD or DMD, with high sensitivity. The protein-based strategy is probably the most efficient way to approach the diagnosis of Duchenne and Becker muscular dystrophy in limited health care settings.

Assessing Cognitive Function in Neuromuscular Diseases: A Pilot Study in a Sample of Children and Adolescents.

Central nervous system (CNS) involvement has been variously studied in pediatric neuromuscular disorders (NMDs). The primary goal of this study was to assess cognitive functioning in NMDs, and secondary aims were to investigate possible associations of cognitive impairment with motor impairment, neurodevelopmental delay, and genotype. This was a cross-sectional study of 43 pediatric patients, affected by six NMDs. Myotonic dystrophy type 1 (DM1) and glycogen storage disease type 2 (GSD2) patients had a delay on the Bayley-III scales. On Wechsler scales, DMD and DM1 patients showed lower FSIQ scores, with an intellectual disability (ID) in 27% and 50%, respectively. FSIQ was normal in Becker muscular dystrophy (BMD), GSD2, and hereditary motor sensory neuropathy (HMSN) patients, while higher individual scores were found in the spinal muscular atrophy (SMA) group. In the DM1 cohort, lower FSIQ correlated with worse motor performance (ρ = 0.84, p < 0.05), and delayed speech acquisition was associated with ID (p = 0.048), with worse cognitive impairment in the congenital than in the infantile form (p = 0.04). This study provides further evidence of CNS in some NMDs and reinforces the need to include cognitive assessment in protocols of care of selected pediatric NMDs.

Electrocardiographic prediction of late gadolinium enhancement on cardiac magnetic resonance in Becker muscular dystrophy

Dystrophin deficiency results in cardiomyopathy and fibrosis with variable onset. Little is known about electrocardiographic abnormalities in Becker muscular dystrophy and their relationship to underlying cardiac pathology. We hypothesized QRS fragmentation is associated with myocardial fibrosis on cardiac magnetic resonance imaging in Becker muscular dystrophy patients. We retrospectively evaluated 44 patients, and extracted data from clinically obtained electrocardiogram and cardiac magnetic resonance. Ventricular function and presence or absence late gadolinium enhancement representing myocardial fibrosis were recorded from imaging. Nearly half (19/42, 45%) of patients interrogated had myocardial fibrosis on cardiac magnetic resonance. Total number of electrocardiogram leads with QRS fragmentation (median 1 vs 4, p < 0.001) and either right or left axis deviation from median was significantly increased (13.6 vs. 44.8°, p < 0.001) in patients with myocardial fibrosis. Decreased leftward voltage in V6 correlated to both increased fibrosis and decreased cardiac function (p < 0.01). The positive likelihood ratio for underlying myocardial fibrosis in patients with two of the three findings on electrocardiogram was 8.47 (p < 0.0001). QRS fragmentation and axis deviation on electrocardiography are strongly predictive of myocardial fibrosis in Becker muscular dystrophy and may inform the use of advanced imaging in evaluation of these patients.

Clinical and genetic profile of duchenne and becker muscular dystrophy patients in the far west of Algeria

Duchenne's (DMD) and Becker's (BMD) muscular dystrophy are the principals causes of myopathy in male children. It is linked to a mutation in the dystrophygene and isinheritedin an X-linked mode. The aims of our study are to Describe the clinical and genetic phenotype of BMD and DMD patients in the far west of Algeria.

DMD – CLINICAL CARE: EP.101 Appendicular lean mass index (ALMI) changes in patients with dystrophinopathies

The musculo-skeletal phenotype of patients with dystrophinopathies range from Severe DMD with loss of ambulation (LOA) before age 13 years, to Intermediate DMD with LOA between 13 to 16 years and asymptomatic patients with Becker muscular dystrophy (BMD). Compared to healthy controls, we found that males with DMD have progressive decline in Appendicular Lean Mass Index (ALMI) with age and with worsening functional mobility. The age-related trajectories of ALM/ALMI in pediatric patients with BMD and Intermediate DMD have not yet been characterized. We aimed to: (1) compare ALM/ALMI of pediatric male patients with DMD in different stages of disease with that of patients with BMD; (2) compare ALM trajectories of patients with Intermediate DMD and BMD with those of typical DMD; and (3) compare ALM trajectories by the 4 DMD genotypes – mutations in exons 1-30, 31-44, 45 – 62 and 63-79. We conducted a retrospective longitudinal study of 499 patients (ages 2 – 20 y) with DMD (466 typical DMD; 33 DMD Intermediate) and 46 patients with BMD and normal motor function. ALM and ALMI (kg/m2) were measured by dual energy X-ray absorptiometry (DXA). ALM and ALMI from 693 healthy male controls were used as a reference to calculate age-specific Z-scores. Results: Mean ALMI Z-scores of DMD patients fell below -2 around age 12y while that of BMD patients remained within ± 2 without decline. ALMI of DMD patients declined after age 10 y while BMD patients accrued ALM but at a slower rate than healthy controls. ALMI started to decline in the milder Intermediate DMD patients around age 14 y. DMD patients with mutations in exons 63-79 appear to have greater declines in ALMI after age 10 y than DMD patients with the other 3 genotypes. Our data on ALMI trajectories in pediatric patients with dystrophinopathies can provide insight for clinical care management needs as well as inform clinical trial design for therapeutic trials in DMD and BMD.

DMD/BMD – OUTCOME MEASURES: EP.129 Value of global longitudinal strain for identification and monitoring of left ventricular dysfunction in Becker muscular dystrophy

Cardiac involvement is the main cause of death in Becker muscular dystrophy (BMD). Identification of left ventricular (LV) function is crucial, but standard echocardiographic measures such as LV ejection fraction (EF) might not be sensitive enough to detect early myocardial dysfunction. We explored the value of LV global longitudinal strain (GLS) as a more accurate echocardiographic parameter to detect and monitor LV dysfunction in BMD. Furthermore, we studied possible factors associated with LV dysfunction and progression. 40 BMD patients (age 39.0±13.2 years) and 21 matched controls were included. Clinical variables, pulmonary tests, serum biomarkers, and echocardiograms were collected at baseline and after two years. LV systolic function was assessed by LVEF and LV GLS; a significant progression in LV dysfunction was defined as an absolute LV GLS decline ≥15%. Responsiveness to cardiac disease progression was determined using standardized response means (SRM). BMD patients showed impaired LVEF and LV GLS as compared to controls (p<0.001). Of interest, 31 patients (77.5%) showed impaired LV GLS (defined as>-18%) while only 24 patients (60%) had reduced LVEF. LV GLS and LVEF correlated with troponin-I and NT-proBNP (0.52<rho≤0.68), but not with skeletal muscle or pulmonary function. At follow-up (2.0±0.5 years, n=29), LV GLS worsened significantly (-1.3±0.8%, p=0.002, SRM=0.70, annually 0.60%) while LVEF remained stable. No risk factors for LV dysfunction progression were identified. In BMD, LV GLS is frequently impaired and shows deterioration over time as compared to LVEF. LV GLS could be used as a more sensitive parameter to identify and monitor LV dysfunction.

Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects

BackgroundExercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy.MethodsHere, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography–mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof’s effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models.ResultsWe demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity.ConclusionsOur study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.

Validation of Chemokine Biomarkers in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease involving complex skeletal muscle pathogenesis. The pathogenesis is triggered by sarcolemma instability due to the lack of dystrophin protein expression, leading to Ca2+ influx, muscle fiber apoptosis, inflammation, muscle necrosis, and fibrosis. Our lab recently used two high-throughput multiplexing techniques (e.g., SomaScan® aptamer assay and tandem mass tag-(TMT) approach) and identified a series of serum protein biomarkers tied to different pathobiochemical pathways. In this study, we focused on validating the circulating levels of three proinflammatory chemokines (CCL2, CXCL10, and CCL18) that are believed to be involved in an early stage of muscle pathogenesis. We used highly specific and reproducible MSD ELISA assays and examined the association of these chemokines with DMD pathogenesis, age, disease severity, and response to glucocorticoid treatment. As expected, we confirmed that these three chemokines were significantly elevated in serum and muscle samples of DMD patients relative to age-matched healthy controls (p-value < 0.05, CCL18 was not significantly altered in muscle samples). These three chemokines were not significantly elevated in Becker muscular dystrophy (BMD) patients, a milder form of dystrophinopathy, when compared in a one-way ANOVA to a control group but remained significantly elevated in the age-matched DMD group (p < 0.05). CCL2 and CCL18 but not CXCL10 declined with age in DMD patients, whereas all three chemokines remained unchanged with age in BMD and controls. Only CCL2 showed significant association with time to climb four steps in the DMD group (r = 0.48, p = 0.038) and neared significant association with patients’ reported outcome in the BMD group (r = 0.39, p = 0.058). Furthermore, CCL2 was found to be elevated in a serum of the mdx mouse model of DMD, relative to wild-type mouse model. This study suggests that CCL2 might be a suitable candidate biomarker for follow-up studies to demonstrate its physiological significance and clinical utility in DMD.

Intrafamilial phenotypic heterogeneity related to a new DMD splice site variant

Dystrophinopathies are a group of X-linked neuromuscular disorders that result from pathogenic variants in the DMD gene. Their pathophysiological substrate is the defective expression of dystrophin in many tissues. While patients from the same pedigree usually present similar dystrophin expression and clinical course, the extent of cardiac and skeletal muscle involvement may not correlate in the same individual. We identified a new splice site variant c.2803+5G>C (NM_004006) ClinVar VCV000803902, located in intron 22 of DMD in a Brazilian family that present a broad phenotypic and histological heterogeneity. One of the subjects had a typical Duchenne muscular dystrophy (DMD) phenotype, whereas the others had Becker muscular dystrophy (BMD). Cardiac involvement was remarkable in some of the BMD patients, but not in the DMD patient. Western blot analysis of skeletal muscle revealed much lower levels of calsequestrin in the most severely affected patient compared to his brother, whose phenotype is BMD, highlighting the potential role of proteins involved in skeletal muscle calcium homeostasis in differential degrees of dystrophinopathies.