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

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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Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy

Human Genetics ◽

10.1007/s00439-019-02107-4 ◽

2020 ◽

Vol 139 (2) ◽

pp. 247-255 ◽

Cited By ~ 2

Author(s):

Mariko Okubo ◽

Satoru Noguchi ◽

Shinichiro Hayashi ◽

Harumasa Nakamura ◽

Hirofumi Komaki ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Nonsense Mutation ◽

Frameshift Mutation ◽

Exon Skipping ◽

Becker Muscular Dystrophy ◽

Intronic Sequence ◽

Frameshift Mutations ◽

Dmd Gene ◽

Accurate Quantification

AbstractDuchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/frameshift mutations in DMD and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in DMD in the Japanese Registry of Muscular Dystrophy. For each DMD mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in DMD gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in DMD and for interpreting the phenotypes of the BMD patients.

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Comparison of Long-term Ambulatory Function in Patients with Duchenne Muscular Dystrophy Treated with Eteplirsen and Matched Natural History Controls

Journal of Neuromuscular Diseases ◽

10.3233/jnd-200548 ◽

2021 ◽

pp. 1-11

Author(s):

Jerry R. Mendell ◽

Navid Khan ◽

Nanshi Sha ◽

Helen Eliopoulos ◽

Craig M. McDonald ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Gene Mutations ◽

Exon Skipping ◽

Term Treatment ◽

Long Term Treatment ◽

The Difference ◽

Dmd Gene ◽

6 Minute Walk Test

Background: Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by DMD gene mutations. A relationship between exon skipping and dystrophin production in exon 51-amenable patients treated with eteplirsen (EXONDYS 51 ®) is established. Once-weekly eteplirsen significantly increased dystrophin, with slower decline in ambulatory function compared to baseline. Long-term treatment with eteplirsen leads to accumulation of dystrophin over time and observed functional benefits in patients with DMD. Objective: Compare long-term ambulatory function in eteplirsen-treated patients versus controls. Methods: Study 201/202 included 12 eteplirsen-treated patients assessed twice/year for ambulatory function over 4 years. Ambulatory evaluations (6-minute walk test [6MWT], loss of ambulation, and North Star Ambulatory Assessment [NSAA]) were compared with matched controls from Italian Telethon and Leuven registries. Results: At Years 3 and 4, eteplirsen-treated patients demonstrated markedly greater mean 6MWT than controls (difference in change from baseline of 132 m [95%CI (29, 235), p = 0.015] at Year 3 and 159 m [95%CI (66, 253), p = 0.002] at Year 4). At Year 4, a significantly greater proportion of eteplirsen-treated patients were still ambulant versus controls (10/12 vs 3/11; p = 0.020). At Year 3, eteplirsen-treated patients had milder NSAA decline than controls (difference in change from baseline of 2.6, 95%CI [-6, 11]), however, the difference was not statistically significant; Year 4 control NSAA data were not available. Conclusion: In this retrospective matched control study, eteplirsen treatment resulted in attenuation of ambulatory decline over a 4-year observation period, supporting long-term benefit in patients with DMD.

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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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New Approach for Antisense Oligonucleotide-Mediated Exon Skipping in Duchenne Muscular Dystrophy

Journal of Advanced Computational Intelligence and Intelligent Informatics ◽

10.20965/jaciii.2012.p0521 ◽

2012 ◽

Vol 16 (4) ◽

pp. 521-526

Author(s):

Yoshitsugu Aoki ◽

◽

Tetsuya Nagata ◽

Shin’ichi Takeda

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Antisense Oligonucleotide ◽

Antisense Oligonucleotides ◽

Exon Skipping ◽

New Approach ◽

Reading Frame ◽

Promising Therapeutic Approach ◽

Dmd Gene ◽

Dystrophin Protein

Duchenne Muscular Dystrophy (DMD) is a lethalmuscle disorder characterized by mutations in the DMD gene. These mutations primarily disrupt the reading frame, resulting in the absence of functional dystrophin protein. Exon skipping, which involves the use of antisense oligonucleotides is a promising therapeutic approach for DMD, and clinical trials on exon skipping are currently underway in DMD patients. Recently, stable and less-toxic antisense oligonucleotides with higher efficacy have been developed in mouse and dog models of DMD. This review highlights a new approach for antisense oligonucleotide-based therapeutics for DMD, particularly for exon skipping-based methods.

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Unexpected DMD Gene Mutations Detected by CMA and CNV-seq in amniotic Fluid and Aborted Fetus Samples

10.21203/rs.3.rs-376139/v1 ◽

2021 ◽

Author(s):

Qiuhua Wu ◽

Lihui Yang ◽

Qiujie Jin ◽

Rui Wang ◽

Wen Zhai ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Muscular Dystrophy ◽

Amniotic Fluid ◽

Spontaneous Abortion ◽

Gene Mutations ◽

Dystrophin Gene ◽

Becker Muscular Dystrophy ◽

Hereditary Diseases ◽

Muscle Dysfunction ◽

Dmd Gene

Abstract Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X chromosome-linked recessive hereditary diseases. The mechanism is that the exon mutations of anti-myatrophy protein gene (Dystrophin gene) and lead to muscle dysfunction. Prenatal diagnosis can prevent the birth of children with defects and have good clinical significance. Methods: CMA and CNV-seq were used to detect the amniotic fluid after amniocentesis,. CNV-seq was used to detect spontaneous abortion tissue. The DMD gene mutations were found in 6 amniotic fluid samples and one spontaneous abortion sample. DMD gene mutations were confirmed by MLPA and new DMD mutations were found.Results: CMA found DMD mutations :1.Xp21.1, 75.5kb del (E52-53); 2.Xp21.2, 334.92kb dup (E61-79); 3.Xp21.2, 292.25kb dup (E58-74); 4.Xp21.1, 374.20 kb dup (E45-51). CNV-seq found DMD mutations: 5.X p21.2, E64-79 dup; 6.X p21.1, E1-7dup; 7.Xp21.1, E 44-52 del. Conclusions: 4 fetuses harboring DMD gene mutations were found by CMA, 2 fetuses and 1 induced abortion carrying DMD gene mutations was detected by CNV-seq. CMA/CNV-seq jointed with MLPA test can provide more comprehensive evidence for prenatal diagnosis.

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A different spectrum of DMD gene mutations in local Chinese patients with Duchenne/Becker muscular dystrophy

Chinese Medical Journal ◽

10.1097/00029330-200607010-00004 ◽

2006 ◽

Vol 119 (13) ◽

pp. 1079-1087 ◽

Cited By ~ 14

Author(s):

Lo Fai-man Ivan ◽

Lai Keung-san Kent ◽

Ming-forTong Tony ◽

Lam Tak-sum Stephen

Keyword(s):

Muscular Dystrophy ◽

Gene Mutations ◽

Becker Muscular Dystrophy ◽

Chinese Patients ◽

Dmd Gene

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Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots: Prospects and Challenges

Journal of Personalized Medicine ◽

10.3390/jpm8040041 ◽

2018 ◽

Vol 8 (4) ◽

pp. 41 ◽

Cited By ~ 20

Author(s):

Yusuke Echigoya ◽

Kenji Rowel Q. Lim ◽

Akinori Nakamura ◽

Toshifumi Yokota

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Exon Skipping ◽

Becker Muscular Dystrophy ◽

Therapeutic Modality ◽

Therapeutic Concept ◽

Patient Registration ◽

Potential Therapeutic Application ◽

Dmd Gene ◽

Retrospective Database

Duchenne muscular dystrophy (DMD), a fatal X-linked recessive disorder, is caused mostly by frame-disrupting, out-of-frame deletions in the dystrophin (DMD) gene. Antisense oligonucleotide-mediated exon skipping is a promising therapy for DMD. Exon skipping aims to convert out-of-frame mRNA to in-frame mRNA and induce the production of internally-deleted dystrophin as seen in the less severe Becker muscular dystrophy. Currently, multiple exon skipping has gained special interest as a new therapeutic modality for this approach. Previous retrospective database studies represented a potential therapeutic application of multiple exon skipping. Since then, public DMD databases have become more useful with an increase in patient registration and advances in molecular diagnosis. Here, we provide an update on DMD genotype-phenotype associations using a global DMD database and further provide the rationale for multiple exon skipping development, particularly for exons 45–55 skipping and an emerging therapeutic concept, exons 3–9 skipping. Importantly, this review highlights the potential of multiple exon skipping for enabling the production of functionally-corrected dystrophin and for treating symptomatic patients not only with out-of-frame deletions but also those with in-frame deletions. We will also discuss prospects and challenges in multiple exon skipping therapy, referring to recent progress in antisense chemistry and design, as well as disease models.

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

Journal of Personalized Medicine ◽

10.3390/jpm9010016 ◽

2019 ◽

Vol 9 (1) ◽

pp. 16 ◽

Cited By ~ 9

Author(s):

Akinori Nakamura

Keyword(s):

Muscular Dystrophy ◽

Single Gene ◽

Genetic Diagnosis ◽

Exon Skipping ◽

Becker Muscular Dystrophy ◽

Therapeutic Strategies ◽

Severe Phenotype ◽

Reading Frame ◽

Dystrophin Expression ◽

Dmd Gene

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.

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

BMC Research Notes ◽

10.1186/s13104-019-4730-1 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 3

Author(s):

Kristy Iskandar ◽

Ery Kus Dwianingsih ◽

Linda Pratiwi ◽

Alvin Santoso Kalim ◽

Hasna Mardhiah ◽

...

Keyword(s):

Multiplex Pcr ◽

Gene Deletion ◽

Hot Spot ◽

Exon Skipping ◽

Becker Muscular Dystrophy ◽

High Serum ◽

Gene Deletions ◽

Male Patients ◽

Dmd Gene ◽

Dystrophin Protein

Abstract 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.

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Duchenne Muscular Dystrophy (DMD) Treatment: Past and Present Perspectives

Muscular Dystrophy - Research Updates and Therapeutic Strategies ◽

10.5772/intechopen.92765 ◽

2020 ◽

Author(s):

Nahla O. Mousa ◽

Ahmed Osman ◽

Nagia Fahmy ◽

Ahmed Abdellatif ◽

Waheed K. Zahra

Keyword(s):

Stem Cell ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Genetic Defect ◽

Exon Skipping ◽

Disease Treatment ◽

Pulmonary Functions ◽

Progressive Muscle Weakness ◽

Dmd Gene ◽

Dystrophin Protein

Duchenne muscular dystrophy (DMD) is one of the fatal X-linked disorders that are characterized by progressive muscle weakness and occur due to mutation in the largest human gene known as the DMD gene which encodes dystrophin protein that is mandatory for keeping the muscles structurally and functionally intact. The disease always affects boys (1 from every ~5000), and in some cases the female carriers are symptomatic. The disease usually leads to impairment in cardiac and pulmonary functions leading to the death of the patients in very young ages. Understanding DMD through precise molecular diagnosis will aid in determining the suitable therapeutic approach for the cases like designing exon-skipping antisense oligonucleotides (AOs) or stem cell-based therapies in conjunction with gene editing techniques (CRISPR/Cas9). Such therapies can correct the genetic defect in the DMD gene and ameliorate the symptoms. In this chapter, we will illustrate the past and current strategies for DMD disease treatment.

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