Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

ObjectiveTo report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping.MethodsPart 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry.ResultsTwelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%–4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry.ConclusionGolodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization.Clinicaltrials.gov identifierNCT02310906.Classification of evidenceThis study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.

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The Duchenne muscular dystrophy gene and cancer

Cellular Oncology ◽

10.1007/s13402-020-00572-y ◽

2020 ◽

Author(s):

Leanne Jones ◽

Michael Naidoo ◽

Lee R. Machado ◽

Karen Anthony

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Protein ◽

Neuromuscular Disorders ◽

Becker Muscular Dystrophy ◽

Gene Products ◽

Large Gene ◽

Cancer Types ◽

And Function ◽

Dystrophin Protein

Abstract Background Mutation of the Duchenne muscular dystrophy (DMD) gene causes Duchenne and Becker muscular dystrophy, degenerative neuromuscular disorders that primarily affect voluntary muscles. However, increasing evidence implicates DMD in the development of all major cancer types. DMD is a large gene with 79 exons that codes for the essential muscle protein dystrophin. Alternative promotor usage drives the production of several additional dystrophin protein products with roles that extend beyond skeletal muscle. The importance and function(s) of these gene products outside of muscle are not well understood. Conclusions We highlight a clear role for DMD in the pathogenesis of several cancers, including sarcomas, leukaemia’s, lymphomas, nervous system tumours, melanomas and various carcinomas. We note that the normal balance of DMD gene products is often disrupted in cancer. The short dystrophin protein Dp71 is, for example, typically maintained in cancer whilst the full-length Dp427 gene product, a likely tumour suppressor, is frequently inactivated in cancer due to a recurrent loss of 5’ exons. Therefore, the ratio of short and long gene products may be important in tumorigenesis. In this review, we summarise the tumours in which DMD is implicated and provide a hypothesis for possible mechanisms of tumorigenesis, although the question of cause or effect may remain. We hope to stimulate further study into the potential role of DMD gene products in cancer and the development of novel therapeutics that target DMD.

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P.145Optimisation of a high–throughput digital script for multiplexed immunofluorescent analysis of sarcolemmal dystrophin - associated protein complex (DPC) and myofibre regeneration in entire transverse sections of muscle biopsies in Duchenne muscular dystrophy

Neuromuscular Disorders ◽

10.1016/j.nmd.2019.06.201 ◽

2019 ◽

Vol 29 ◽

pp. S90

Author(s):

D. Scaglioni ◽

M. Ellis ◽

F. Catapano ◽

S. Torelli ◽

D. Chambers ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

High Throughput ◽

Protein Complex ◽

Immunofluorescent Analysis ◽

Muscle Biopsies

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Duchenne and Becker muscular dystrophy: a molecular and immunohistochemical approach

Arquivos de Neuro-Psiquiatria ◽

10.1590/s0004-282x2007000100016 ◽

2007 ◽

Vol 65 (1) ◽

pp. 73-76 ◽

Cited By ~ 13

Author(s):

Aline Andrade Freund ◽

Rosana Herminia Scola ◽

Raquel Cristina Arndt ◽

Paulo José Lorenzoni ◽

Claudia Kamoy Kay ◽

...

Keyword(s):

Muscular Dystrophy ◽

Muscular Atrophy ◽

Hot Spot ◽

Dystrophin Gene ◽

Becker Muscular Dystrophy ◽

Congenital Myopathy ◽

Specific Method ◽

Dystrophin Deficiency ◽

Muscle Biopsies ◽

Female Carriers

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the dystrophin gene. We studied 106 patients with a diagnosis of probable DMD/BMD by analyzing 20 exons of the dystrophin gene in their blood and, in some of the cases, by immunohistochemical assays for dystrophin in muscle biopsies. In 71.7% of the patients, deletions were found in at least one of the exons; 68% of these deletions were in the hot-spot 3' region. Deletions were found in 81.5% of the DMD cases and in all the BMD cases. The cases without deletions, which included the only woman in the study with DMD, had dystrophin deficiency. The symptomatic female carriers had no deletions but had abnormal dystrophin distribution in the sarcolemma (discontinuous immunostains). The following diagnoses were made for the remaining cases without deletions with the aid of a muscle biopsy: spinal muscular atrophy, congenital myopathy; sarcoglycan deficiency and unclassified limb-girdle muscular dystrophy. Dystrophin analysis by immunohistochemistry continues to be the most specific method for diagnosis of DMD/BMD and should be used when no exon deletions are found in the dystrophin gene in the blood.

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Evidence of Insulin Resistance and Other Metabolic Alterations in Boys with Duchenne or Becker Muscular Dystrophy

International Journal of Endocrinology ◽

10.1155/2015/867273 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 27

Author(s):

Maricela Rodríguez-Cruz ◽

Raúl Sanchez ◽

Rosa E. Escobar ◽

Oriana del Rocío Cruz-Guzmán ◽

Mardia López-Alarcón ◽

...

Keyword(s):

Insulin Resistance ◽

Muscular Dystrophy ◽

Glucose Metabolism ◽

Molecular Mechanisms ◽

Becker Muscular Dystrophy ◽

Body Fat Mass ◽

Metabolic Alterations ◽

Underweight Patients ◽

Dmd Gene ◽

Muscle Biopsies

Aim. Our aim was (1) to determine the frequency of insulin resistance (IR) in patients with Duchenne/Becker muscular dystrophy (DMD/BMD), (2) to identify deleted exons of DMD gene associated with obesity and IR, and (3) to explore some likely molecular mechanisms leading to IR.Materials and Methods. In 66 patients with DMD/BMD without corticosteroids treatment, IR, obesity, and body fat mass were evaluated. Molecules involved in glucose metabolism were analyzed in muscle biopsies. Results show that 18.3%, 22.7%, and 68% were underweight, overweight, or obese, and with high adiposity, respectively; 48.5% and 36.4% presented hyperinsulinemia and IR, respectively. Underweight patients (27.3%) exhibited hyperinsulinemia and IR. Carriers of deletions in exons 45 (OR = 9.32; 95% CI = 1.16–74.69) and 50 (OR = 8.73; 95% CI = 1.17–65.10) from DMD gene presented higher risk for IR than noncarriers. We observed a greater staining of cytoplasmic aggregates for GLUT4 in muscle biopsies than healthy muscle tissue.Conclusion. Obesity, hyperinsulinemia, and IR were observed in DMD/BMD patients and are independent of corticosteroids treatment. Carriers of deletion in exons 45 or 50 from DMD gene are at risk for developing IR. It is suggested that alteration in GLUT4 in muscle fibers from DMD patients could be involved in IR.

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The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD

Biomedicines ◽

10.3390/biomedicines9020219 ◽

2021 ◽

Vol 9 (2) ◽

pp. 219

Author(s):

Elena Gargaun ◽

Sestina Falcone ◽

Guilhem Solé ◽

Julien Durigneux ◽

Andoni Urtizberea ◽

...

Keyword(s):

Muscular Dystrophy ◽

Gene Mutations ◽

Exon Skipping ◽

Becker Muscular Dystrophy ◽

Protein Stabilization ◽

Progressive Muscular Dystrophy ◽

Dilatative Cardiomyopathy ◽

Proliferation And Differentiation ◽

Dmd Gene ◽

Dystrophin Protein

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

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Advances in Genetic Characterization and Genotype–Phenotype Correlation of Duchenne and Becker Muscular Dystrophy in the Personalized Medicine Era

Journal of Personalized Medicine ◽

10.3390/jpm10030111 ◽

2020 ◽

Vol 10 (3) ◽

pp. 111 ◽

Cited By ~ 1

Author(s):

Omar Sheikh ◽

Toshifumi Yokota

Keyword(s):

Muscular Dystrophy ◽

Personalized Medicine ◽

Genetic Diagnosis ◽

Exon Skipping ◽

Dystrophin Gene ◽

Becker Muscular Dystrophy ◽

Phenotype Correlation ◽

Related Condition ◽

Genotype Phenotype Correlation ◽

Dystrophin Protein

Currently, Duchenne muscular dystrophy (DMD) and the related condition Becker muscular dystrophy (BMD) can be usually diagnosed using physical examination and genetic testing. While BMD features partially functional dystrophin protein due to in-frame mutations, DMD largely features no dystrophin production because of out-of-frame mutations. However, BMD can feature a range of phenotypes from mild to borderline DMD, indicating a complex genotype–phenotype relationship. Despite two mutational hot spots in dystrophin, mutations can arise across the gene. The use of multiplex ligation amplification (MLPA) can easily assess the copy number of all exons, while next-generation sequencing (NGS) can uncover novel or confirm hard-to-detect mutations. Exon-skipping therapy, which targets specific regions of the dystrophin gene based on a patient’s mutation, is an especially prominent example of personalized medicine for DMD. To maximize the benefit of exon-skipping therapies, accurate genetic diagnosis and characterization including genotype–phenotype correlation studies are becoming increasingly important. In this article, we present the recent progress in the collection of mutational data and optimization of exon-skipping therapy for DMD/BMD.

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Confocal analysis of the dystrophin protein complex in muscular dystrophy

Muscle & Nerve ◽

10.1002/1097-4598(200102)24:2<262::aid-mus120>3.0.co;2-3 ◽

2001 ◽

Vol 24 (2) ◽

pp. 262-272 ◽

Cited By ~ 14

Author(s):

Romesh Draviam ◽

Lynn Billington ◽

Andy Senchak ◽

Eric P. Hoffman ◽

Simon C. Watkins

Keyword(s):

Muscular Dystrophy ◽

Protein Complex ◽

Dystrophin Protein

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High-Throughput Digital Image Analysis Reveals Distinct Patterns of Dystrophin Expression in Dystrophinopathy Patients

Journal of Neuropathology & Experimental Neurology ◽

10.1093/jnen/nlab088 ◽

2021 ◽

Author(s):

Silvia Torelli ◽

Domenic Scaglioni ◽

Valentina Sardone ◽

Matthew J Ellis ◽

Joana Domingos ◽

...

Keyword(s):

Image Analysis ◽

Muscular Dystrophy ◽

High Throughput ◽

Expression Patterns ◽

Becker Muscular Dystrophy ◽

Clear Understanding ◽

Incurable Disease ◽

Dystrophin Expression ◽

Dmd Gene ◽

Muscle Biopsies

Abstract Duchenne muscular dystrophy (DMD) is an incurable disease caused by out-of-frame DMD gene deletions while in frame deletions lead to the milder Becker muscular dystrophy (BMD). In the last decade several antisense oligonucleotides drugs have been developed to induce a partially functional internally deleted dystrophin, similar to that produced in BMD, and expected to ameliorate the disease course. The pattern of dystrophin expression and functionality in dystrophinopathy patients is variable due to multiple factors, such as molecular functionality of the dystrophin and its distribution. To benchmark the success of therapeutic intervention, a clear understanding of dystrophin expression patterns in dystrophinopathy patients is vital. Recently, several groups have used innovative techniques to quantify dystrophin in muscle biopsies of children but not in patients with milder BMD. This study reports on dystrophin expression using both Western blotting and an automated, high-throughput, image analysis platform in DMD, BMD, and intermediate DMD/BMD skeletal muscle biopsies. Our results found a significant correlation between Western blot and immunofluorescent quantification indicating consistency between the different methodologies. However, we identified significant inter- and intradisease heterogeneity of patterns of dystrophin expression in patients irrespective of the amount detected on blot, due to variability in both fluorescence intensity and dystrophin sarcolemmal circumference coverage. Our data highlight the heterogeneity of the pattern of dystrophin expression in BMD, which will assist the assessment of dystrophin restoration therapies.

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Challenges of Interpreting Dystrophin Content by Western Blot

US Neurology ◽

10.17925/usn.2019.15.1.40 ◽

2019 ◽

Vol 15 (1) ◽

pp. 40 ◽

Cited By ~ 3

Author(s):

Frederick J Schnell ◽

Diane Frank ◽

Sue Fletcher ◽

Russell D Johnsen ◽

Steve D Wilton ◽

...

Keyword(s):

Clinical Trials ◽

Data Analysis ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Western Blot ◽

Becker Muscular Dystrophy ◽

Human Muscle ◽

Healthy Human ◽

Dystrophin Protein ◽

Western Blot Data

The Duchenne muscular dystrophy community has recently seen the first approved therapy for the restoration of dystrophin, based on its ability to increase levels of dystrophin protein, as determined by western blot. The approval, along with the initiation of clinical trials evaluating other dystrophin-restoring therapies, highlights the importance of accurate dystrophin quantitation. Nonoptimized western blot methods can reflect inaccurate results, especially in the quantitation of low dystrophin levels. A few key changes to standards and data analysis parameters can result in a low level of dystrophin (<0.5% of a healthy biopsy) being inaccurately interpreted as >20% of the levels reported in healthy human muscle. A review of the dystrophin western blot data on Duchenne and Becker muscular dystrophy biopsies is conducted, along with a thorough investigation of methodologies to quantify dystrophin.

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