scholarly journals Repurposing Pathogenic Variants of DMD Gene and its Isoforms for DMD Exon Skipping Intervention

2020 ◽  
Vol 20 (7) ◽  
pp. 519-530 ◽  
Author(s):  
Rahul Tyagi ◽  
Sumit Kumar ◽  
Ashwin Dalal ◽  
Faruq Mohammed ◽  
Manju Mohanty ◽  
...  
Keyword(s):  
Exon Skipping ◽  
Population Based ◽  
Neuromuscular Disorder ◽  
Copy Number Variations ◽  
Reading Frame ◽  
The North ◽  
Pathogenic Variants ◽  
Mutation Database ◽  
Dmd Gene ◽  
Dependent Probe

Background: Duchenne Muscular Dystrophy (DMD) is a progressive, fatal neuromuscular disorder caused by mutations in the DMD gene. Emerging antisense oligomer based exon skipping therapy provides hope for the restoration of the reading frame. Objectives: Population-based DMD mutation database may enable exon skipping to be used for the benefit of patients. Hence, we planned this study to identify DMD gene variants in North Indian DMD cases. Methods: A total of 100 DMD cases were recruited and Multiplex ligation-dependent probe amplification (MLPA) analysis was performed to obtain the deletion and duplication profile. Results: Copy number variations (deletion/duplication) were found in 80.85% of unrelated DMD cases. Sixty-eight percent of cases were found to have variations in the distal hotspot region (Exon 45- 55) of the DMD gene. Exon 44/45 variations were found to be the most prominent among single exon variations, whereas exon 49/50 was found to be the most frequently mutated locations in single/ multiple exon variations. As per Leiden databases, 86.84% cases harboured out-of-frame mutations. Domain wise investigation revealed that 68% of mutations were localized in the region of spectrin repeats. Dp140 isoform was predicted to be absent in 62/76 (81.57%) cases. A total of 45/80 (56.25 %) and 23/80 (28.70%) DMD subjects were predicted to be amenable to exon 51 and exon 45 skipping trials, respectively. Conclusion: A major proportion of DMD subjects (80%) could be diagnosed by the MLPA technique. The data generated from our study may be beneficial for strengthening of mutation database in the North Indian population.

scholarly journals Identification of Exonic Copy Number Variations in Dystrophin Gene Using Mlpa / Identificarea Variaţiilor Numărului de Copii în Gena Distrofinei Folosind Metoda Mlpa

2014 ◽  
Vol 22 (4) ◽  
Author(s):  
Cristina Rusu ◽  
Adriana Sireteanu ◽  
Lăcrămioara Butnariu ◽  
Monica Pânzaru ◽  
Elena Braha ◽  
...  
Keyword(s):  
Copy Number ◽  
Mutation Screening ◽  
Gene Mutations ◽  
Dystrophin Gene ◽  
Copy Number Variations ◽  
Sequencing Analysis ◽  
Reading Frame ◽  
Muscle Disorders ◽  
Dmd Gene ◽  
Dependent Probe

AbstractDuchenne and Becker muscular dystrophies (DMD/BMD) are X-linked progressive muscle disorders determined by mutations of the dystrophin (DMD) gene. Multiplex Ligation - Dependent Probe Amplification (MLPA) is a simple, inexpensive and reliable test for molecular diagnosis of DMD gene mutations. It identifies exonic copy number variations in the DMD gene, but the test should be completed with sequencing analysis in case of single exon deletions/duplications. The aim of this study was to evaluate the efficiency of MLPA as a DMD mutation screening tool in affected males and carrier females, as well as to appreciate the frequency of different types of mutations and to check the validity of the “reading frame rule”. We have used MLPA for the detection of deletions/ duplications in DMD gene in 53 individuals (30 affected males and 23 asymptomatic female relatives) referred for evaluation and genetic counseling due to the clinical suspicion of DMD/BMD. In the affected males (21 DMD and 9 BMD) MLPA had a detection rate of 63.5% (53.5% deletions and 10% duplications). The most frequently deleted exon was exon 45 and the most frequent duplication involved exons 3-5, confirming the presence of the two hotspot mutation regions reported in the literature. Mutations detected in our study have a slightly different location compared to literature data. Reading frame rule was valid in 84% of our cases.

scholarly journals CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 342 ◽  
Author(s):  
Kenji Rowel Q. Lim ◽  
Quynh Nguyen ◽  
Kasia Dzierlega ◽  
Yiqing Huang ◽  
Toshifumi Yokota
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Animal Models ◽  
Mechanical Stress ◽  
Muscle Cell ◽  
Neuromuscular Disorder ◽  
Reading Frame ◽  
Advantages And Disadvantages ◽  
Short Palindromic Repeat ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.

scholarly journals Genotype–Phenotype Correlations in Duchenne and Becker Muscular Dystrophy Patients from the Canadian Neuromuscular Disease Registry

2020 ◽  
Vol 10 (4) ◽  
pp. 241
Author(s):  
Kenji Rowel Q. Lim ◽  
Quynh Nguyen ◽  
Toshifumi Yokota
Keyword(s):  
Muscular Dystrophy ◽  
Neuromuscular Disease ◽  
Becker Muscular Dystrophy ◽  
Neuromuscular Disorder ◽  
Test Results ◽  
Disease Registry ◽  
Reading Frame ◽  
Negative Results ◽  
Genetic Test Results ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder generally caused by out-of-frame mutations in the DMD gene. In contrast, in-frame mutations usually give rise to the milder Becker muscular dystrophy (BMD). However, this reading frame rule does not always hold true. Therefore, an understanding of the relationships between genotype and phenotype is important for informing diagnosis and disease management, as well as the development of genetic therapies. Here, we evaluated genotype–phenotype correlations in DMD and BMD patients enrolled in the Canadian Neuromuscular Disease Registry from 2012 to 2019. Data from 342 DMD and 60 BMD patients with genetic test results were analyzed. The majority of patients had deletions (71%), followed by small mutations (17%) and duplications (10%); 2% had negative results. Two deletion hotspots were identified, exons 3–20 and exons 45–55, harboring 86% of deletions. Exceptions to the reading frame rule were found in 13% of patients with deletions. Surprisingly, C-terminal domain mutations were associated with decreased wheelchair use and increased forced vital capacity. Dp116 and Dp71 mutations were also linked with decreased wheelchair use, while Dp140 mutations significantly predicted cardiomyopathy. Finally, we found that 12.3% and 7% of DMD patients in the registry could be treated with FDA-approved exon 51- and 53-skipping therapies, respectively.

scholarly journals X-Linked Retinitis Pigmentosa Caused by Non-Canonical Splice Site Variants in RPGR

2021 ◽  
Vol 22 (2) ◽  
pp. 850
Author(s):  
Friederike Kortüm ◽  
Sinja Kieninger ◽  
Pascale Mazzola ◽  
Susanne Kohl ◽  
Bernd Wissinger ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Splice Site ◽  
Exon Skipping ◽  
Open Reading Frame ◽  
Aberrant Splicing ◽  
Reading Frame ◽  
Pathogenic Variants ◽  
Nucleotide Addition ◽  
Intron 2

We aimed to validate the effect of non-canonical splice site variants in the RPGR gene in five patients from four families diagnosed with retinitis pigmentosa. Four variants located in intron 2 (c.154 + 3_154 + 6del), intron 3 (c.247 + 5G>A), intron 7 (c.779-5T>G), and intron 13 (c.1573-12A>G), respectively, were analyzed by means of in vitro splice assays. Splicing analysis revealed different aberrant splicing events, including exon skipping and intronic nucleotide addition, which are predicted to lead either to an in-frame deletion affecting relevant protein domains or to a frameshift of the open reading frame. Our data expand the landscape of pathogenic variants in RPGR, thereby increasing the genetic diagnostic rate in retinitis pigmentosa and allowing patients harboring the analyzed variants to be enrolled in clinical trials.

scholarly journals Detailed genetic and functional analysis of the hDMDdel52/mdx mouse model

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244215
Author(s):  
Alper Yavas ◽  
Rudie Weij ◽  
Maaike van Putten ◽  
Eleni Kourkouta ◽  
Chantal Beekman ◽  
...  
Keyword(s):  
Mouse Model ◽  
Mrna Level ◽  
Exon Skipping ◽  
Neuromuscular Disorder ◽  
Motor Deficit ◽  
Mdx Mouse ◽  
Transcription Activator ◽  
Reading Frame ◽  
New Findings ◽  
Significant Difference

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.

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

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.

scholarly journals CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy

2020 ◽  
Author(s):  
Kenji Rowel Q. Lim ◽  
Quynh Nguyen ◽  
Kasia Dzierlega ◽  
Yiqing Huang ◽  
Toshifumi Yokota
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Animal Models ◽  
Mechanical Stress ◽  
Muscle Cell ◽  
Muscle Cells ◽  
Neuromuscular Disorder ◽  
Reading Frame ◽  
Advantages And Disadvantages ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose quite well, each comes with their own limitations. To help overcome this, clustered regularly interspaced short palindromic repeats (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.

Functional muscle recovery following dystrophin and myostatin exon splice modulation in aged mdx mice

2019 ◽  
Author(s):  
Ngoc Lu-Nguyen ◽  
Arnaud Ferry ◽  
Frederick J Schnell ◽  
Gunnar J Hanson ◽  
Linda Popplewell ◽  
...  
Keyword(s):  
Combined Therapy ◽  
Muscle Growth ◽  
Exon Skipping ◽  
Negative Regulator ◽  
Mdx Mice ◽  
Reading Frame ◽  
Combination Approach ◽  
Dmd Gene ◽  
The One ◽  
Dystrophin Protein

Abstract Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 3500–5000 newborn boys. It is due to mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibres and their replacement with fibro-adipogenic tissue. Out-of-frame mutations in the DMD gene can be modified by using antisense oligonucleotides (AONs) to promote skipping of specific exons such that the reading frame is restored and the resulting protein produced, though truncated, is functional. We have shown that AONs can also be used to knock down myostatin, a negative regulator of muscle growth and differentiation, through disruption of the transcript reading frame, and thereby enhance muscle strength. In young mdx mice, combined dystrophin and myostatin exon skipping therapy greatly improved DMD pathology, compared to the single dystrophin skipping approach. Here we show that in aged (>15-month-old) mdx mice, when the pathology is significantly more severe and more similar to the one observed in DMD patients, the effect of the combined therapy is slightly attenuated but still beneficial in improving the disease phenotype. These results confirm the beneficial outcome of the combination approach and support its translation into DMD clinical trials.

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

2019 ◽  
Vol 9 (1) ◽  
pp. 16 ◽  
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.

scholarly journals Novel PHEX Variants and Splicing Mutations in Patients with X-Linked Hypophosphatemia

2021 ◽  
Author(s):  
Xuesha Xing ◽  
Jinlan Gao ◽  
Hongwei Ma ◽  
Lina Zhang ◽  
Fang Li ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Genetic Disorder ◽  
Exon Skipping ◽  
Hypophosphatemic Rickets ◽  
Missense Mutations ◽  
Splice Sites ◽  
Nonsense Mutations ◽  
Reading Frame ◽  
Splicing Mutations ◽  
Mutation Database

Abstract Background: X-linked hypophosphatemia rickets (XLH) is a genetic disorder of phosphate wasting that causes the majority of inherited hypophosphatemic rickets. The disease is caused by mutations in the phosphate-regulating endopeptidase gene (PHEX). All types of mutations have been detected in the PHEX gene. There is no clear preference for the position and the type of mutation.Methods: In this study, we performed DNA sequencing and ex vivo splicing analysis to study the PHEX gene in ten sporadic patients and six core families with XLH. Results: A total of 25 patients were studied. Fifteen different mutations were detected in these patients, including five missense mutations, five splicing mutations, two nonsense mutations, two small deletions, and one small insertion. Five mutations were not previously reported. We also characterized the splicing mutations identified in our study, which consisted of exon skipping and activation of new splice sites in an exon or intron. Most of the observed changes resulted in a frameshift of the PHEX open reading frame.Conclusions: The genetic etiology in patients with XLH were successfully identified. This study expands the mutation database of the PHEX gene. We also explored the pathogenesis of XLH caused by splice site mutations. These results will contribute to the diagnosis of XLH and the pathogenicity analysis of mutations in patients.

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