Aminoglycoside-induced mutation suppression (stop codon readthrough) as a therapeutic strategy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by progressive muscle weakness, is caused by the absence or a decreased amount of the muscle cytoskeletal protein dystrophin. Currently, several therapeutic approaches to cure DMD are being investigated, which can be categorized into two groups: therapies that aim to restore dystrophin expression, and those that aim to compensate for the lack of dystrophin. Therapies that restore dystrophin expression include read-through therapy, exon skipping, vector-mediated gene therapy, and cell therapy. Of these approaches, the most advanced are the read-through and exon skipping therapies. In 2014, ataluren, a drug that can promote ribosomal read-through of mRNA containing a premature stop codon, was conditionally approved in Europe. In 2016, eteplirsen, a morpholino-based chemical capable of skipping exon 51 in premature mRNA, received conditional approval in the USA. Clinical trials on vector-mediated gene therapy carrying micro- and mini- dystrophin are underway. More innovative therapeutic approaches include CRISPR/Cas9-based genome editing and stem cell-based cell therapies. Here we review the current status of therapeutic approaches for DMD, focusing on therapeutic approaches that can restore dystrophin.

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Multiple regulatory events controlling the expression and localization of utrophin in skeletal muscle fibers: insights into a therapeutic strategy for Duchenne muscular dystrophy

Journal of Physiology-Paris ◽

10.1016/s0928-4257(01)00078-x ◽

2002 ◽

Vol 96 (1-2) ◽

pp. 31-42 ◽

Cited By ~ 20

Author(s):

Bernard J Jasmin ◽

Lindsay M Angus ◽

Guy Bélanger ◽

Joe V Chakkalakal ◽

Anthony O Gramolini ◽

...

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Therapeutic Strategy ◽

Muscle Fibers ◽

Skeletal Muscle Fibers

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Hybrid minigene splicing assay verified the pathogenicity of a novel splice site variant in the dystrophin gene of a Chinese patient with typical Duchenne muscular dystrophy phenotype

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2015-1042 ◽

2016 ◽

Vol 54 (9) ◽

pp. 1435-1440 ◽

Cited By ~ 4

Author(s):

Zhihong Wang ◽

Yanhong Lin ◽

Liping Qiu ◽

Dezhu Zheng ◽

Aizhen Yan ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Splice Site ◽

Sanger Sequencing ◽

Stop Codon ◽

Premature Stop Codon ◽

Splice Site Mutation ◽

Dystrophin Gene ◽

Site Mutation ◽

Aberrant Splicing

AbstractBackground:Duchenne muscular dystrophy (DMD) is typically caused by disrupting the reading frame of the dystrophin gene: approximately 70%–80% of mutational events are represented by deletions or duplications of one or more exons in the dystrophin gene, and the remaining cases by subtle mutations, including point mutations, small indels, small inversions, and complex small rearrangements. The dystrophin gene is the largest known gene with one of the highest known rates of new mutations.Methods:Deletions and duplications were detected in theDMDgene of the proband by using multiple ligation-dependent probe amplification (MLPA). Targeted next-generation sequencing (NGS) was used in the subtle mutation detection, followed by Sanger sequencing confirmation. The effect of the mutation on the splicing of theDMDgene was assessed by bioinformatics prediction and hybrid minigene splicing assay (HMSA).Results:Neither duplication nor deletion was found in theDMDgene of the proband. While a novel splice site mutation c.6762+1G>C was identified in the proband by NGS and Sanger sequencing, and his mother was heterozygous at the same site. Bioinformatics predicted that the 5′ donor splice site of intron 46 disappeared because of the mutation, which would lead to aberrant splicing and introduce premature stop codon. The HMSA results were in agreement with the prediction.Conclusions:The novel splice site mutation caused DMD in the proband by aberrant splicing. We suggested that combined applications of MLPA, NGS, HMSA and bioinformatics are comprehensive and effective methods for diagnosis and aberrant splicing study of DMD.

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Theragnosis for Duchenne Muscular Dystrophy

Frontiers in Pharmacology ◽

10.3389/fphar.2021.648390 ◽

2021 ◽

Vol 12 ◽

Author(s):

Leonela Luce ◽

Micaela Carcione ◽

Chiara Mazzanti ◽

Paula I. Buonfiglio ◽

Viviana Dalamón ◽

...

Keyword(s):

Differential Diagnosis ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Latin American ◽

Stop Codon ◽

Meta Analysis ◽

Premature Stop Codon ◽

Exon Skipping ◽

Corticosteroid Treatment ◽

Severe Form

Dystrophinopathies cover a spectrum of rare progressive X-linked muscle diseases, arising from DMD mutations. They are among the most common pediatric muscular dystrophies, being Duchenne muscular dystrophy (DMD) the most severe form. Despite the fact that there is still no cure for these serious diseases, unprecedented advances are being made for the development of therapies for DMD. Some of which are already conditionally approved: exon skipping and premature stop codon read-through. The present work aimed to characterize the mutational spectrum of DMD in an Argentinian cohort, to identify candidates for available pharmacogenetic treatments and finally, to conduct a comparative analysis of the Latin American (LA) frequencies of mutations amenable for available DMD therapies. We studied 400 patients with clinical diagnosis of dystrophinopathy, implementing a diagnostic molecular algorithm including: MLPA/PCR/Sanger/Exome and bioinformatics. We also performed a meta-analysis of LA’s metrics for DMD available therapies. The employed algorithm resulted effective for the achievement of differential diagnosis, reaching a detection rate of 97%. Because of this, corticosteroid treatment was correctly indicated and validated in 371 patients with genetic confirmation of dystrophinopathy. Also, 20 were eligible for exon skipping of exon 51, 21 for exon 53, 12 for exon 45 and another 70 for premature stop codon read-through therapy. We determined that 87.5% of DMD patients will restore the reading frame with the skipping of only one exon. Regarding nonsense variants, UGA turned out to be the most frequent premature stop codon observed (47%). According to the meta-analysis, only four LA countries (Argentina, Brazil, Colombia and Mexico) provide the complete molecular algorithm for dystrophinopathies. We observed different relations among the available targets for exon skipping in the analyzed populations, but a more even proportion of nonsense variants (∼40%). In conclusion, this manuscript describes the theragnosis carried out in Argentinian dystrophinopathy patients. The implemented molecular algorithm proved to be efficient for the achievement of differential diagnosis, which plays a crucial role in patient management, determination of the standard of care and genetic counseling. Finally, this work contributes with the international efforts to characterize the frequencies and variants in LA, pillars of drug development and theragnosis.

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Prediction of Premature Termination Codon Suppressing Compounds for Treatment of Duchenne Muscular Dystrophy Using Machine Learning

Molecules ◽

10.3390/molecules25173886 ◽

2020 ◽

Vol 25 (17) ◽

pp. 3886

Author(s):

Kate Wang ◽

Eden L. Romm ◽

Valentina L. Kouznetsova ◽

Igor F. Tsigelny

Keyword(s):

Machine Learning ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Premature Termination ◽

Stop Codon ◽

Pharmacophore Model ◽

Dystrophin Gene ◽

Protein Translation ◽

Premature Termination Codon ◽

Termination Codon

A significant percentage of Duchenne muscular dystrophy (DMD) cases are caused by premature termination codon (PTC) mutations in the dystrophin gene, leading to the production of a truncated, non-functional dystrophin polypeptide. PTC-suppressing compounds (PTCSC) have been developed in order to restore protein translation by allowing the incorporation of an amino acid in place of a stop codon. However, limitations exist in terms of efficacy and toxicity. To identify new compounds that have PTC-suppressing ability, we selected and clustered existing PTCSC, allowing for the construction of a common pharmacophore model. Machine learning (ML) and deep learning (DL) models were developed for prediction of new PTCSC based on known compounds. We conducted a search of the NCI compounds database using the pharmacophore-based model and a search of the DrugBank database using pharmacophore-based, ML and DL models. Sixteen drug compounds were selected as a consensus of pharmacophore-based, ML, and DL searches. Our results suggest notable correspondence of the pharmacophore-based, ML, and DL models in prediction of new PTC-suppressing compounds.

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250. A Novel Approach To Identify Patients with Duchenne Muscular Dystrophy Caused by Stop Codon Mutations Using Aminoglycoside Antibiotics

Molecular Therapy ◽

10.1016/s1525-0016(16)40692-1 ◽

2003 ◽

Vol 7 (5) ◽

pp. S99

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Stop Codon ◽

Aminoglycoside Antibiotics ◽

Novel Approach

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Clinical and Genetic Characteristics in Young, Glucocorticoid-Naive Boys With Duchenne Muscular Dystrophy

Neurology ◽

10.1212/wnl.0000000000013122 ◽

2021 ◽

pp. 10.1212/WNL.0000000000013122

Author(s):

Marianela Schiava ◽

Rachel Amos ◽

Henriette VanRuiten ◽

Michael P McDermott ◽

Williams B Martens ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Stop Codon ◽

Incidental Finding ◽

Diagnostic Delay ◽

Genetic Diagnosis ◽

Neuromuscular Disorder ◽

Double Blind ◽

Genetic Characteristics ◽

The Mean

Background and ObjectivesDuchenne muscular dystrophy (DMD) is a paediatric neuromuscular disorder caused by mutations in the dystrophin gene. Geneotype-phenotype associations have been examined in glucocorticoid treated boys, but there are few data on the young glucocorticoid-naïve DMD population. A sample of young glucocorticoid-naïve DMD boys is described and genotype-phenotype associations are investigated.MethodsScreening and baseline data were collected for all the participants in the Finding the Optimum Corticosteroid Regime for Duchenne Muscular Dystrophy (FOR-DMD)study, an international, multi-centre, randomized, double-blind, clinical trial comparing three glucocorticoid regimens in glucocorticoid-naïve, genetically confirmed boys with DMD between 4 and <8 years of age.ResultsOne hundred and ninety-six boys were recruited. The meanage at randomization (+ standard deviation) was 5.8+ 1.0 years. The predominant mutation type was out of frame deletions 67.4%, (130/193) of which 68.5% (89/130) were amenable to exon skipping. The most frequent mutations were deletions amenable to exon 51 skipping 13.0% (25/193). Stop codon mutations accounted for 10.4% (20/193).The mean age at first parental concerns was 29.8 + 18.7 months, the mean age at genetic diagnosis was 53.9 + 21.9 months and the mean diagnostic delay was 25.9 + 18.2 months. The mean diagnostic delay for boys diagnosed following an incidental finding of isolated hyperCKemia (n=19) was 6.4 + 7.4 months. The mean ages at independent walking and talking in sentences were 17.1 + 4.2 and 29.0 + 10.7 months, respectively. Median height percentiles were below the 25th percentile regardless of age group. No genotype-phenotype associations were identified expect for boys with an exon 8 skippable deletions who had better performance on time to walk/run 10 meters (p=0.02)compared to boys with deletions not amenable to skipping.DiscussionThis study describes clinical and genetic characteristics of a sample of young glucocorticoid-naïve boys with DMD. A low threshold for CK testing can lead to an earlier diagnosis. Motor and speech delay were common presenting symptoms.The effects of low, pre-treatment height on growth and adults height requires further study. These findings may promote earlier recognition of DMD and inform study design for future clinical trials.

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