Caregiver Burden with Duchenne and Becker Muscular Dystrophy in Japan: A Clinical Observation Study.

Objective Skeletal muscle weakness and cardiomyopathy can be seen in carriers of dystrophinopathy. Therefore, the health management of caregivers of Duchenne/Becker muscular dystrophy (DMD/BMD) patients who are themselves carriers is an important issue. However, few studies have focused on caregivers who have dystrophin mutations. Methods In this cross-sectional study conducted at five hospitals, the daily living, situation medical treatment status, genetic testing, physical assessment, care burden, and quality of life of caregivers of DMD/BMD patients were surveyed. Results The subjects were 36 main caregivers (mean age 55.7±8.4 years old), of whom 52.8% were diagnosed as carriers, 8.3% were noncarriers, and 38.9% were not confirmed. In addition, half of the caregivers were not examined regularly at medical institutions. Of all caregivers, 54.3% had muscle or cardiac symptoms, and 75% had elevated serum creatine kinase levels. The mean Zarit Caregiver Burden Interview (ZBI) total score of current caregivers was 20.9±13.1. The frequency of a ZBI total score ≥25 was significantly higher in caregivers diagnosed as carriers than in caregivers unexamined as carriers (p=0.04). The health-related quality of life score (Short Form 36; SF-36) in caregivers was slightly lower than the Japanese standard scores in the sections of physical functioning, role limitations-physical, bodily pain, and social functioning. Conclusion Some caregivers of DMD/BMD patients can themselves have muscular or cardiac symptoms and a heavy care burden. It is therefore necessary for carrier caregivers, especially women, to undergo regular health checkups and receive appropriate health management.

Specificities of the DMD Gene Mutation Spectrum in Russian Patients.

Duchenne/Becker muscular dystrophy (DMD/BMD) is the most common form of muscular dystrophy, accounting for over 50% of all cases. In this regard, in Russia we carry out a program of selective screening for DMD/BMD, which mainly involves male patients. The main inclusion criteria are an increase in the level of creatine phosphokinase (>2000 U/L) or an established clinical diagnosis. At the first stage of screening, patients are scanned for extended deletions and duplications in the DMD gene using multiplex ligase-dependent probe amplification (MLPA SALSA P034 and P035 DMD probemix, MRC-Holland). The second stage is the search for small mutations using a custom NGS panel, which includes 31 genes responsible for various forms of limb-girdle muscular dystrophy. In a screening of 1025 families with a referral Duchenne/Becker diagnosis, pathogenic and likely pathogenic variants in the DMD gene were found in 788 families (in 76.9% of cases). In the current study, we analyzed the mutation spectrum of the DMD gene in Russian patients and noted certain differences between the examined cohort and the multi-ethnic cohort. The analysis of the DMD gene mutation spectrum is essential for patients with DMD/BMD because the exact mutation type determines the application of a specific therapeutic method.

Combining Protein Expression and Molecular Data Improves Mutation Characterization of Dystrophinopathies.

Duchenne and Becker muscular dystrophy are X-linked recessive inherited disorders characterized by progressive weakness due to skeletal muscle degeneration. Different mutations in the DMD gene, which encodes for dystrophin protein, are responsible for these disorders. The aim of our study was to investigate the relationship between type, size, and location of the mutation that occurs in the DMD gene and their effect on dystrophin protein expression in a cohort of 40 male dystrophinopathy patients and nine females, possible carriers. We evaluated the expression of dystrophin by immunofluorescence and immunoblotting. The mutational spectrum of the DMD gene was established by MLPA for large copy number variants, followed by HRM analysis for point mutations and sequencing of samples with an abnormal melting profile. MLPA revealed 30 deletions (75%) and three duplications (7.5%). HRM analysis accounted for seven-point mutations (17.5%). We also report four novel small mutations (c. 8507G>T, c.3021delG, c.9563_9563+1insAGCATGTTTATGATACAGCA, c.7661-60T>A) in DMD gene. Our work shows that the DNA translational open reading frame and the location of the mutation both influence the expression of dystrophin and disease severity phenotype. The proposed algorithm used in this study demonstrates its accuracy for the characterization of dystrophinopathy patients.

Genetic Profile of the Dystrophin Gene Reveals New Mutations in Colombian Patients Affected with Muscular Dystrophinopathy.

BACKGROUND: Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common human dystrophinopathies with recessive X-linked inheritance. Dystrophin gene deletions and duplications are the most common mutations, followed by point mutations. The aim of this study is to characterize the mutational profile of the dystrophin gene in Colombian patients with DMD/BMD. MATERIAL AND METHODS: Mutational profiling was determined in 69 affected patients using Sanger sequencing, next-generation sequencing (NGS) and/or multiplex ligation dependent-probes amplification (MLPA). Genetic variants were classified according to molecular consequence and new variants were determined through database and literature analysis. RESULTS: Mutational profile in affected patients revealed that large deletions/duplications analyzed by MLPA accounted for 72.5% of all genetic variations. By using Sanger sequencing or NGS, we identified point mutations in 15.9% and small deletions in 11.6% of the patients. New mutations were found, most of them were point mutations or small deletions (10.1%). CONCLUSION: Our results described the genetic profile of the dystrophin gene in Colombian patients with DMD and contribute to efforts to identify molecular variants in Latin American populations. For our population, 18.8% of cases could be treated with FDA or MDA approved molecular therapies based on specific mutations. These data contribute to the establishment of appropriate genetic counseling and potential treatment.

Comprehensive Molecular Analysis of DMD Gene Increases the Diagnostic Value of Dystrophinopathies: A Pilot Study in a Southern Italy Cohort of Patients.

Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked neuromuscular disease due to pathogenic sequence variations in the dystrophin (DMD) gene, one of the largest human genes. More than 70% of DMD gene defects result from genomic rearrangements principally leading to large deletions, while the remaining are small nucleotide variants, including nonsense and missense variants, small insertions/deletions or splicing alterations. Considering the large size of the gene and the wide mutational spectrum, the comprehensive molecular diagnosis of DMD/BMD is complex and may require several laboratory methods, thus increasing the time and costs of the analysis. In an attempt to simplify DMD/BMD molecular diagnosis workflow, we tested an NGS method suitable for the detection of all the different types of genomic variations that may affect the DMD gene. Forty previously analyzed patients were enrolled in this study and re-analyzed using the next generation sequencing (NGS)-based single-step procedure. The NGS results were compared with those from multiplex ligation-dependent probe amplification (MLPA)/multiplex PCR and/or Sanger sequencing. Most of the previously identified deleted/duplicated exons and point mutations were confirmed by NGS and 1 more pathogenic point mutation (a nonsense variant) was identified. Our results show that this NGS-based strategy overcomes limitations of traditionally used methods and is easily transferable to routine diagnostic procedures, thereby increasing the diagnostic power of DMD molecular analysis.

Antisense Oligonucleotide Treatment in a Humanized Mouse Model of Duchenne Muscular Dystrophy and Highly Sensitive Detection of Dystrophin Using Western Blotting.

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder affecting many children. The disease is caused by the lack of dystrophin production and characterized by muscle wasting. The most common causes of death are respiratory failure and heart failure. Antisense oligonucleotide-mediated exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is a promising therapeutic approach for the treatment of DMD. In preclinical studies, dystrophic mouse models are commonly used for the development of therapeutic oligos. We employ a humanized model carrying the full-length human DMD transgene along with the complete knockout of the mouse Dmd gene. In this model, the effects of human-targeting AOs can be tested without cross-reaction between mouse sequences and human sequences (note that mdx, a conventional dystrophic mouse model, carries a nonsense point mutation in exon 23 and express the full-length mouse Dmd mRNA, which is a significant complicating factor). To determine if dystrophin expression is restored, the Western blotting analysis is commonly performed; however, due to the extremely large protein size of dystrophin (427 kDa), detection and accurate quantification of full-length dystrophin can be a challenge. Here, we present methodologies to systemically inject PMOs into humanized DMD model mice and determine levels of dystrophin restoration via Western blotting. Using a tris-acetate gradient SDS gel and semi-dry transfer with three buffers, including the Concentrated Anode Buffer, Anode Buffer, and Cathode Buffer, less than 1% normal levels of dystrophin expression are easily detectable. This method is fast, easy, and sensitive enough for the detection of dystrophin from both cultured muscle cells and muscle biopsy samples.

Duchenne and Becker Muscular Dystrophies' Prevalence in MD STARnet Surveillance Sites: An Examination of Racial and Ethnic Differences.

INTRODUCTION: Previous studies indicated variability in the prevalence of Duchenne and Becker muscular dystrophies (DBMD) by racial/ethnic groups. The Centers for Disease Control and Prevention's (CDC) Muscular Dystrophy Surveillance, Tracking, and Research network (MD STARnet) conducts muscular dystrophy surveillance in multiple geographic areas of the USA and continues to enroll new cases. This provides an opportunity to continue investigating differences in DBMD prevalence by race and ethnicity and to compare the impact of using varying approaches for estimating prevalence. OBJECTIVE: To estimate overall and race/ethnicity-specific prevalence of DBMD among males aged 5-9 years and compare the performance of three prevalence estimation methods. METHODS: The overall and race/ethnicity-specific 5-year period prevalence rates were estimated with MD STARnet data using three methods. Method 1 used the median of 5-year prevalence, and methods 2 and 3 calculated prevalence directly with different birth cohorts. To compare prevalence between racial/ethnic groups, Poisson modeling was used to estimate prevalence ratios (PRs) with non-Hispanic (NH) whites as the referent group. Comparison between methods was also conducted. RESULTS: In the final population-based sample of 1,164 DBMD males, the overall 5-year prevalence for DBMD among 5-9 years of age ranged from 1.92 to 2.48 per 10,000 males, 0.74-1.26 for NH blacks, 1.78-2.26 for NH whites, 2.24-4.02 for Hispanics, and 0.61-1.83 for NH American Indian or Alaska Native and Asian or Native Hawaiian or Pacific Islander (AIAN/API). The PRs for NH blacks/NH whites, Hispanics/NH whites, and NH AIAN/API/NH whites were 0.46 (95% CI: 0.36-0.59), 1.37 (1.17-1.61), and 0.61 (0.40-0.93), respectively. CONCLUSIONS: In males aged 5-9 years, compared to the prevalence of DBMD in NH whites, prevalence in NH blacks and NH AIAN/API was lower and higher in Hispanics. All methods produced similar prevalence estimates; however, method 1 produced narrower confidence intervals and method 2 produced fewer zero prevalence estimates than the other two methods.

Cardiac magnetic resonance in patients with muscular dystrophies.

Muscular dystrophies are inherited disorders sharing similar clinical features and dystrophic changes on muscle biopsy. Duchenne muscular dystrophy is the most common inherited muscle disease of childhood, and Becker muscular dystrophy is a milder allelic variant with a slightly lower prevalence. Myotonic dystrophy is the most frequent form in adults. Cardiac magnetic resonance is the gold standard technique for the quantification of cardiac chamber volumes and function, and also enables a characterisation of myocardial tissue. Most cardiac magnetic resonance studies in the setting of muscular dystrophy were carried out at single centres, evaluated small numbers of patients and used widely heterogeneous protocols. Even more importantly, those studies analysed more or less extensively the patterns of cardiac involvement, but usually did not try to establish the added value of cardiac magnetic resonance to standard echocardiography, the evolution of cardiac disease over time and the prognostic significance of cardiac magnetic resonance findings. As a result, the large and heterogeneous amount of information on cardiac involvement in muscular dystrophies cannot easily be translated into recommendations on the optimal use of cardiac magnetic resonance. In this review, whose targets are cardiologists and neurologists who manage patients with muscular dystrophy, we try to summarise cardiac magnetic resonance findings in patients with muscular dystrophy, and the results of studies evaluating the role of cardiac magnetic resonance as a tool for diagnosis, risk stratification and follow-up. Finally, we provide some practical recommendations about the need and timing of cardiac magnetic resonance examination for the management of patients with muscular dystrophy.

Iranian Registry of Duchenne and Becker Muscular Dystrophies: Characterization and Preliminary Data.

Duchenne (DMD) and Becker muscular dystrophies (BMD) are rare neuromuscular disorders caused by mutations in the dystrophin gene and failure in its protein production. The absence or the reduced expression of dystrophin render muscles prone to damage, including the cardiac and respiratory muscles with reduced life expectancy. Careful planning for clinical trials will require a sufficient number of confirmed cases to meet the inclusion criteria. National registries for rare disorders serve as an essential tool for personalized medicines or mutation-specific trials to facilitate patient recruitment. The Iranian Registry of DMD and BMD (IRDAB) collects detailed molecular data of Iranian DMD/BMD patients and carriers according to the TREAT-NMD Global Neuromuscular Network guidelines. As of March 2020, five hundred and twenty-two cases are registered. The registry incorporates multi-level web and database technologies, where registrants can access their data and compare it to the cumulative data. The registry's objectives are to recruit eligible patients for clinical trials and provide sufficient data for the national program of disease surveillance and social planning. Furthermore, the registry provides accurate epidemiological data, phenotype/genotype correlation, and evaluate the standards of care in Iran.

Corrigendum: Serum MyomiRs as Biomarkers for Female Carriers of Duchenne/Becker Muscular Dystrophy.

[This corrects the article DOI: 10.3389/fneur.2020.563609.].

Serum MyomiRs as Biomarkers for Female Carriers of Duchenne/Becker Muscular Dystrophy.

Background: Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked recessive lethal neuromuscular disease. MicroRNAs expressed in striated muscle, myomiRs, have been proposed as its potential biomarkers. Serum creatine kinase (CK) is commonly used as a biomarker in clinical practice, but it is not reliable. The aim of this study was to assess whether serum levels of myomiRs has diagnostic value for detection of female DMD/BMD carriers with normal or elevated CK. Methods: Thirty four female carriers and 33 age-matched healthy female controls were enrolled. Peripheral blood samples were collected and serum miRNAs were extracted for measurement of miR-1, miR-133a, miR-133b, miR-206, miR-208a, miR-208b, and miR-499 by quantitative real-time polymerase chain reaction. Results: MiR-1, miR-133a, miR-133b, miR-206, miR-208a, miR-208b, and miR-499 were upregulated in all female carriers in comparison to healthy controls. MiR-1 (Spearman's rho = +0.406, p = 0.017) was correlated with CK in the female carrier group. Receiver operating characteristic curve analysis of all seven myomiRs showed that the area under the curve (AUC) for miR-499, miR-133b, miR-1, miR-208b, and miR-133a exceeded 70.0%, and for miR-206 and miR-208a exceeded 60.0%. MiR-133b and miR-499 were significantly increased in all female carriers, even those with normal CK. AUC for the combination of all seven miRNAs was 87.2%. CK (OR 0.406, 95% CI 0.000-0.001, p < 0.0001) and miR-499 (OR 0.323, 95% CI 0.023-0.106, p = 0.003) were considered to be independent predictors for female carriers presence in the multivariable regression analysis model. Conclusions: MiR-133b and miR-499 are potentially useful biomarkers for female carriers with DMD/BMD (including those with normal CK). The combination of all seven serum miRNAs and their respective combinations with CK have better diagnostic value for female carriers than either CK or any separate miRNA.

Molecular Diagnosis and Novel Therapies for Neuromuscular Diseases.

With the development of novel targeted therapies, including exon skipping/inclusion and gene replacement therapy, the field of neuromuscular diseases has drastically changed in the last several years. Until 2016, there had been no FDA-approved drugs to treat Duchenne muscular dystrophy (DMD), the most common muscular dystrophy. However, several new personalized therapies, including antisense oligonucleotides eteplirsen for DMD exon 51 skipping and golodirsen and viltolarsen for DMD exon 53 skipping, have been approved in the last 4 years. We are witnessing the start of a therapeutic revolution in neuromuscular diseases. However, the studies also made clear that these therapies are still far from a cure. Personalized genetic medicine for neuromuscular diseases faces several key challenges, including the difficulty of obtaining appropriate cell and animal models and limited its applicability. This Special Issue "Molecular Diagnosis and Novel Therapies for Neuromuscular/Musculoskeletal Diseases" highlights key areas of research progress that improve our understanding and the therapeutic outcomes of neuromuscular diseases in the personalized medicine era.

The analysis of DMD gene deletions by multiplex PCR in Indonesian DMD/BMD patients: the era of personalized medicine.

OBJECTIVE: Duchenne/Becker muscular dystrophy (DMD/BMD) is the most common genetic neuromuscular disease in children, resulting from a defect in the DMD gene located on Xp21.2. The new emerging treatment using exon skipping strategy is tailored to specific mutations, thus molecular diagnostics are particularly important. This study aimed to detect the DMD gene deletion in Indonesian DMD/BMD patients and analyze the potential amenability by exon skipping therapy. RESULTS: Thirty-four male patients were enrolled in this study, 23 of them (67.6%) underwent muscle biopsy and showed the absence or partially expressed dystrophin protein in immunohistochemistry staining. All patients had very high serum CK levels (10.529 ± 9.97 IU/L). Multiplex PCR revealed the DMD gene deletions in 15 (44.1%) cases. Seventy-eight percent of deletions were clustered in the hot-spot region of exon 43 to 52. Furthermore, seven (20.5%) patients were potentially amenable to exon skipping treatment. Therefore, multiplex PCR is one feasible method to detect DMD gene deletion in Indonesian DMD/BMD patients that can further determine the potential amenability of exon skipping therapy. In addition, this study is the first report of DMD gene deletion analysis in Indonesia.

Mutation-Based Therapeutic Strategies for Duchenne Muscular Dystrophy: From Genetic Diagnosis to Therapy.

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscle disorders caused by mutations of the DMD gene, which encodes the subsarcolemmal protein dystrophin. In DMD, dystrophin is not expressed due to a disruption in the reading frame of the DMD gene, resulting in a severe phenotype. Becker muscular dystrophy exhibits a milder phenotype, having mutations that maintain the reading frame and allow for the production of truncated dystrophin. To date, various therapeutic approaches for DMD have been extensively developed. However, the pathomechanism is quite complex despite it being a single gene disorder, and dystrophin is expressed not only in a large amount of skeletal muscle but also in cardiac, vascular, intestinal smooth muscle, and nervous system tissue. Thus, the most appropriate therapy would be complementation or restoration of dystrophin expression, such as gene therapy using viral vectors, readthrough therapy, or exon skipping therapy. Among them, exon skipping therapy with antisense oligonucleotides can restore the reading frame and yield the conversion of a severe phenotype to one that is mild. In this paper, I present the significance of molecular diagnosis and the development of mutation-based therapeutic strategies to complement or restore dystrophin expression.

Tips to Design Effective Splice-Switching Antisense Oligonucleotides for Exon Skipping and Exon Inclusion.

Antisense-mediated exon skipping and exon inclusion have proven to be powerful tools for treating neuromuscular diseases. The approval of Exondys 51 (eteplirsen) and Spinraza (nusinersen) for the treatment of patients with Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) was the most noteworthy accomplishment in 2016. Exon skipping uses short DNA-like molecules called antisense oligonucleotides (AONs) to correct the disrupted reading frame, allowing the production of functional quasi-dystrophin proteins, and ameliorate the progression of the disease. Exon inclusion for SMA employs an AON targeting an intronic splice silencer site to include an exon which is otherwise spliced out. Recently, these strategies have also been explored in many other genetic disorders, including dysferlin-deficient muscular dystrophy (e.g., Miyoshi myopathy; MM, limb-girdle muscular dystrophy type 2B; LGMD2B, and distal myopathy with anterior tibial onset; DMAT), laminin α2 chain (merosin)-deficient congenital muscular dystrophy (MDC1A), sarcoglycanopathy (e.g., limb-girdle muscular dystrophy type 2C; LGMD2C), and Fukuyama congenital muscular dystrophy (FCMD). A major challenge in exon skipping and exon inclusion is the difficulty in designing effective AONs. The mechanism of mRNA splicing is highly complex, and the efficacy of AONs is often unpredictable. We will discuss the design of effective AONs for exon skipping and exon inclusion in this chapter.

Quantitative Evaluation of Exon Skipping in Immortalized Muscle Cells In Vitro.

Exon skipping through the use of antisense oligonucleotides (AOs) is currently one of the most promising approaches for treating Duchenne muscular dystrophy (DMD). While we now have a number of AO drug candidates in clinical trials, we are still faced with issues of poor or controversial efficacy in some of these drugs. This is the case with eteplirsen, an exon 51-skipping AO that is the first and only FDA-approved drug for DMD to date. Effective procedures must, therefore, be set up for the in vitro screening of potential AOs for DMD treatment. Here, we describe one such procedure using immortalized DMD patient-derived muscle cells. Aside from allowing for the quantitative evaluation of candidate AOs based on their exon skipping efficiency and dystrophin protein rescue levels, these immortalized cells are stable, pure, easy to grow, and not subject to confounding by senescence-related issues. This procedure enables a more reliable screening of AOs prior to their entry in clinical trials and greatly facilitates the search for more efficacious candidate exon skipping AOs for DMD treatment.

Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping.

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. To evaluate and optimize the sequence of antisense oligonucleotides (AOs), muscle cell lines with DMD mutations are useful tools. However, there are only several immortalized muscle cell lines with DMD mutations available that can be used to test the efficacy of exon skipping in vitro. In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.

An Overview of Recent Advances and Clinical Applications of Exon Skipping and Splice Modulation for Muscular Dystrophy and Various Genetic Diseases.

Exon skipping is a therapeutic approach that is feasible for various genetic diseases and has been studied and developed for over two decades. This approach uses antisense oligonucleotides (AON) to modify the splicing of pre-mRNA to correct the mutation responsible for a disease, or to suppress a particular gene expression, as in allergic diseases. Antisense-mediated exon skipping is most extensively studied in Duchenne muscular dystrophy (DMD) and has developed from in vitro proof-of-concept studies to clinical trials targeting various single exons such as exon 45 (casimersen), exon 53 (NS-065/NCNP-01, golodirsen), and exon 51 (eteplirsen). Eteplirsen (brand name Exondys 51), is the first approved antisense therapy for DMD in the USA, and provides a treatment option for ~14% of all DMD patients, who are amenable to exon 51 skipping. Eteplirsen is granted accelerated approval and marketing authorization by the US Food and Drug Administration (FDA), on the condition that additional postapproval trials show clinical benefit. Permanent exon skipping achieved at the DNA level using clustered regularly interspaced short palindromic repeats (CRISPR) technology holds promise in current preclinical trials for DMD. In hopes of achieving clinical success parallel to DMD, exon skipping and splice modulation are also being studied in other muscular dystrophies, such as Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy including limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy (DMAT), myotonic dystrophy, and merosin-deficient congenital muscular dystrophy type 1A (MDC1A). This chapter also summarizes the development of antisense-mediated exon skipping therapy in diseases such as Usher syndrome, dystrophic epidermolysis bullosa, fibrodysplasia ossificans progressiva (FOP), and allergic diseases.

Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45-55 Skipping Accompanied by Rescue of Dystrophin Expression.

Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach for the treatment of various genetic diseases and a therapy which has gained significant traction in recent years following FDA approval of new antisense-based drugs. Exon skipping for Duchenne muscular dystrophy (DMD) works by modulating dystrophin pre-mRNA splicing, preventing incorporation of frame-disrupting exons into the final mRNA product while maintaining the open reading frame, to produce a shortened-yet-functional protein as seen in milder Becker muscular dystrophy (BMD) patients. Exons 45-55 skipping in dystrophin is potentially applicable to approximately 47% of DMD patients because many mutations occur within this "mutation hotspot." In addition, patients naturally harboring a dystrophin exons 45-55 in-frame deletion mutation have an asymptomatic or exceptionally mild phenotype compared to shorter in-frame deletion mutations in this region. As such, exons 45-55 skipping could transform the DMD phenotype into an asymptomatic or very mild BMD phenotype and rescue nearly a half of DMD patients. In addition, this strategy is potentially applicable to some BMD patients as well, who have in-frame deletion mutations in this region. As the degree of exon skipping correlates with therapeutic outcomes, reliable measurements of exon skipping efficiencies are essential to the development of novel antisense-mediated exon skipping therapeutics. In the case of DMD, researchers have often relied upon human muscle fibers obtained from muscle biopsies for testing; however, this method is highly invasive and patient myofibers can display limited proliferative ability. To overcome these challenges, researchers can generate myofibers from patient fibroblast cells by transducing the cells with a viral vector containing MyoD, a myogenic regulatory factor. Here, we describe a methodology for assessing dystrophin exons 45-55 multiple skipping efficiency using antisense oligonucleotides in human muscle cells derived from DMD patient fibroblast cells.