RESUMO
A boy with nonambulatory Duchenne muscular dystrophy (DMD) tested positive for exon 63 duplication and exhibited intellectual disability, overweight and dyslipidaemia. The patient underwent a comprehensive multidisciplinary approach involving pharmacological and non-pharmacological interventions. Despite challenges, such as socioeconomic constraints and limited access to advanced therapies, the patient received tailored care. The management included prednisone medication, dietary modifications and psychological support. The patient's journey highlighted the complex interplay of medical and psychosocial factors affecting DMD patients in resource-limited settings. Regular monitoring and the involvement of the patient's family in a peer group were arranged to improve overall quality of life. The case underscores the need for accessible and holistic care for DMD patients, addressing both medical and psychosocial challenges.
Assuntos
Éxons , Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Masculino , Criança , Qualidade de Vida , Prednisona/uso terapêutico , Deficiência Intelectual/genéticaRESUMO
BACKGROUND: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. METHODS: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. RESULTS: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. CONCLUSIONS: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.
Assuntos
Sistemas CRISPR-Cas , Distrofina , Éxons , Edição de Genes , Terapia Genética , Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofina/genética , Edição de Genes/métodos , Terapia Genética/métodos , Linhagem Celular , Deleção de Sequência , Mioblastos/metabolismoRESUMO
BACKGROUND: Duchenne Muscular Dystrophy (DMD) is an X-linked disorder caused by mutations in the DMD gene, with large deletions being the most common type of mutation. Inversions involving the DMD gene are a less frequent cause of the disorder, largely because they often evade detection by standard diagnostic methods such as multiplex ligation probe amplification (MLPA) and whole exome sequencing (WES). CASE PRESENTATION: Our research identified two intrachromosomal inversions involving the dystrophin gene in two unrelated families through Long-read sequencing (LRS). These variants were subsequently confirmed via Sanger sequencing. The first case involved a pericentric inversion extending from DMD intron 47 to Xq27.3. The second case featured a paracentric inversion between DMD intron 42 and Xp21.1, inherited from the mother. In both cases, simple repeat sequences (SRS) were present at the breakpoints of these inversions. CONCLUSIONS: Our findings demonstrate that LRS is an effective tool for detecting atypical mutations. The identification of SRS at the breakpoints in DMD patients enhances our understanding of the mechanisms underlying structural variations, thereby facilitating the exploration of potential treatments.
Assuntos
Inversão Cromossômica , Distrofina , Distrofia Muscular de Duchenne , Humanos , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Masculino , Inversão Cromossômica/genética , Pontos de Quebra do Cromossomo , Feminino , Linhagem , Criança , Análise de Sequência de DNARESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is an incurable neuromuscular disease leading to progressive skeletal muscle weakness and fatigue. Cell transplantation in murine models has shown promise in supplementing the lack of the dystrophin protein in DMD muscles. However, the establishment of novel, long-term, relevant methods is needed to assess its efficiency on the DMD motor function. By applying newly developed methods, this study aimed to evaluate the functional and molecular effects of cell therapy-mediated dystrophin supplementation on DMD muscles. METHODS: Dystrophin was supplemented in the gastrocnemius of a 5-week-old immunodeficient DMD mouse model (Dmd-null/NSG) by intramuscular xenotransplantation of healthy human immortalized myoblasts (Hu5/KD3). A long-term time-course comparative study was conducted between wild-type, untreated DMD, and dystrophin supplemented-DMD mouse muscle functions and histology. A novel GO-ATeam2 transgenic DMD mouse model was also generated to assess in vivo real-time ATP levels in gastrocnemius muscles during repeated contractions. RESULTS: We found that 10.6% dystrophin supplementation in DMD muscles was sufficient to prevent low values of gastrocnemius maximal isometric contraction torque (MCT) at rest, while muscle fatigue tolerance, assessed by MCT decline after treadmill running, was fully ameliorated in 21-week-old transplanted mice. None of the dystrophin-supplemented fibers were positive for muscle damage markers after treadmill running, with 85.4% demonstrating the utilization of oxidative metabolism. Furthermore, ATP levels in response to repeated muscle contractions tended to improve, and mitochondrial activity was significantly enhanced in dystrophin supplemented-fibers. CONCLUSIONS: Cell therapy-mediated dystrophin supplementation efficiently improved DMD muscle functions, as evaluated using newly developed evaluation methods. The enhanced muscle fatigue tolerance in 21-week-old mice was associated with the preferential regeneration of damage-resistant and oxidative fibers, highlighting increased mitochondrial activity, after cell transplantation. These findings significantly contribute to a more in-depth understanding of DMD pathogenesis.
Assuntos
Modelos Animais de Doenças , Distrofina , Fadiga Muscular , Músculo Esquelético , Distrofia Muscular de Duchenne , Animais , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofina/genética , Distrofina/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Humanos , Mioblastos/metabolismo , Camundongos Endogâmicos mdx , Masculino , Contração Muscular , Transplante de Células/métodosAssuntos
Ensaios Clínicos Fase III como Assunto , Hemofilia A , Distrofia Muscular de Duchenne , Humanos , Hemofilia A/terapia , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Fator VIII/uso terapêutico , Fator VIII/metabolismo , Fator VIII/genética , Indústria Farmacêutica , Terapia Genética/métodosRESUMO
Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder with progressive proximal weakness as the principal sign. Glucocorticoids and physical therapy are the mainstay of treatment. Exercise intolerance is the hallmark of metabolic myopathies, which require a combination of laboratory testing, electrodiagnostic testing, and muscle biopsy for diagnosis. Joint hypermobility may be an isolated finding or be associated with hypermobility Ehlers-Danlos syndrome (EDS), other variants of EDS, or marfanoid syndromes. The latter conditions are associated with aortic and cardiac valvular abnormalities. Osteogenesis imperfecta encompasses a group of disorders characterized by bone fragility presenting with a low-impact fracture as a result of minimal trauma. Management includes multidiscipline specialists. Down syndrome (DS), or trisomy 21, is the most common chromosome abnormality identified in live births. Routine evaluation of atlantoaxial instability with x-ray is no longer recommended for children with DS without symptoms of atlantoaxial instability; however, clinical evaluation of symptoms is required for sports preparticipation. Achondroplasia is the most common skeletal dysplasia. Clinical signs are macrocephaly, short limb, short stature with disproportionately shorter humerus and femur, along with characteristic findings in pelvis and lumbar spine x-rays. Caregivers should be educated on proper positioning and handling to avoid complications, including car seat-related deaths.
Assuntos
Acondroplasia , Síndrome de Ehlers-Danlos , Osteogênese Imperfeita , Humanos , Criança , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/terapia , Adolescente , Acondroplasia/diagnóstico , Acondroplasia/genética , Acondroplasia/terapia , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/terapia , Instabilidade Articular/diagnóstico , Instabilidade Articular/terapia , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Síndrome de Down/complicações , Síndrome de Down/diagnóstico , Doenças Musculoesqueléticas/diagnóstico , Doenças Musculoesqueléticas/terapia , Síndrome de Marfan/diagnóstico , Síndrome de Marfan/terapia , Síndrome de Marfan/complicações , Síndrome de Marfan/genética , Glucocorticoides/uso terapêutico , Modalidades de FisioterapiaRESUMO
BACKGROUND: Deletion or duplication in the DMD gene is one of the most common causes of Duchenne and Becker muscular dystrophy (DMD/BMD). However, the pathogenicity of complex rearrangements involving DMD, especially segmental duplications with unknown breakpoints, is not well understood. This study aimed to evaluate the structure, pattern, and potential impact of rearrangements involving DMD duplication. METHODS: Two families with DMD segmental duplications exhibiting phenotypical differences were recruited. Optical genome mapping (OGM) was used to explore the cryptic pattern of the rearrangements. Breakpoints were validated using long-range polymerase chain reaction combined with next-generation sequencing and Sanger sequencing. RESULTS: A multi-copy duplication involving exons 64-79 of DMD was identified in Family A without obvious clinical symptoms. Family B exhibited typical DMD neuromuscular manifestations and presented a duplication involving exons 10-13 of DMD. The rearrangement in Family A involved complex in-cis tandem repeats shown by OGM but retained a complete copy (reading frame) of DMD inferred from breakpoint validation. A reversed insertion with a segmental repeat was identified in Family B by OGM, which was predicted to disrupt the normal structure and reading frame of DMD after confirming the breakpoints. CONCLUSIONS: Validating breakpoint and rearrangement pattern is crucial for the functional annotation and pathogenic classification of genomic structural variations. OGM provides valuable insights into etiological analysis of DMD/BMD and enhances our understanding for cryptic effects of complex rearrangements.
Assuntos
Distrofina , Éxons , Distrofia Muscular de Duchenne , Linhagem , Fenótipo , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Distrofina/genética , Masculino , Éxons/genética , Feminino , Mapeamento Cromossômico , Rearranjo Gênico/genética , Criança , Duplicações Segmentares Genômicas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Duplicação Gênica/genética , AdolescenteRESUMO
Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.
Assuntos
Sistemas CRISPR-Cas , Distrofina , Éxons , Duplicação Gênica , Edição de Genes , Distrofia Muscular de Duchenne , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Humanos , Éxons/genética , Distrofina/genética , Edição de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas/genética , Terapia Genética/métodos , Fibras Musculares Esqueléticas/metabolismoRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is a rare disease that causes progressive muscle degeneration resulting in life-threatening cardiac complications. The objective of this systematic literature review was to describe and grade the published evidence of predictors of cardiac disease in DMD. METHODS: The review encompassed searches of Embase, MEDLINE ALL, and the Cochrane Database of Systematic Reviews from January 1, 2000, to December 31, 2022, for predictors of cardiac disease in DMD. The certainty of evidence (i.e., very low to high) was assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) framework. RESULTS: We included 33 publications encompassing 9,232 patients with DMD. We found moderate- to high-quality evidence that cardiac medication (i.e., ACE inhibitors [enalapril and perindopril], ß-blockers [carvedilol], and mineralocorticoid receptor antagonists [eplerenone]) are significantly associated with preserved left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), and left ventricular circumferential strain (LVCS). DMD mutations in exons 51 and 52 were found to be significantly associated with lower risk of cardiomyopathy; deletions treatable by exon 53 skipping and mutations in the Dp116 coding region with improved LVEF and prolonged cardiac dysfunction-free survival; and exons 45-50 and 52 with early left ventricular systolic dysfunction (low/very low-quality evidence). We found high-quality evidence that glucocorticoids (deflazacort) are significantly associated with preserved LVEF and improved fractional shortening (FS), and low-quality evidence that glucocorticoids (deflazacort, prednisone, and/or prednisolone) are associated with improved ejection fraction (EF) and lower risk of cardiomyopathy, ventricular dysfunction, and heart failure-related mortality. Full-time mechanical ventilation was found to be significantly correlated with LVEF (low-quality evidence), muscle strength with FS (low-quality evidence), and genetic modifiers (i.e., LTBP4 rs10880 and ACTN3) with LVEF, lower risk of cardiomyopathy and left ventricular dilation (low-quality evidence). CONCLUSION: Several sources of cardiac disease heterogeneity are well-studied in patients with DMD. Yet, the certainty of evidence is generally low, and little is known of the contribution of non-pharmacological interventions, as well as the impact of different criteria for initiation of specific treatments. Our findings help raise awareness of prevailing unmet needs, shape expectations of treatment outcomes, and inform the design of future research.
Assuntos
Distrofia Muscular de Duchenne , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Humanos , CardiopatiasRESUMO
Duchenne muscular dystrophy is secondarily accompanied by Ca2+ excess in muscle fibers. Part of the Ca2+ accumulates in the mitochondria, contributing to the development of mitochondrial dysfunction and degeneration of muscles. In this work, we assessed the effect of intraperitoneal administration of rhodacyanine MKT077 (5 mg/kg/day), which is able to suppress glucose-regulated protein 75 (GRP75)-mediated Ca2+ transfer from the sarcoplasmic reticulum (SR) to mitochondria, on the Ca2+ overload of skeletal muscle mitochondria in dystrophin-deficient mdx mice and the concomitant mitochondrial dysfunction contributing to muscle pathology. MKT077 prevented Ca2+ overload of quadriceps mitochondria in mdx mice, reduced the intensity of oxidative stress, and improved mitochondrial ultrastructure, but had no effect on impaired oxidative phosphorylation. MKT077 eliminated quadriceps calcification and reduced the intensity of muscle fiber degeneration, fibrosis level, and normalized grip strength in mdx mice. However, we noted a negative effect of MKT077 on wild-type mice, expressed as a decrease in the efficiency of mitochondrial oxidative phosphorylation, SR stress development, ultrastructural disturbances in the quadriceps, and a reduction in animal endurance in the wire-hanging test. This paper discusses the impact of MKT077 modulation of mitochondrial dysfunction on the development of skeletal muscle pathology in mdx mice.
Assuntos
Cálcio , Distrofina , Camundongos Endogâmicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne , Animais , Camundongos , Cálcio/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofina/metabolismo , Distrofina/deficiência , Distrofina/genética , Estresse Oxidativo/efeitos dos fármacos , Masculino , Fosforilação Oxidativa/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/patologia , Mitocôndrias Musculares/ultraestruturaRESUMO
The approval of splice-switching oligonucleotides with phosphorodiamidate morpholino oligomers (PMOs) for treating Duchenne muscular dystrophy (DMD) has advanced the field of oligonucleotide therapy. Despite this progress, PMOs encounter challenges such as poor tissue uptake, particularly in the heart, diaphragm, and central nervous system (CNS), thereby affecting patient's prognosis and quality of life. To address these limitations, we have developed a PMOs-based heteroduplex oligonucleotide (HDO) technology. This innovation involves a lipid-ligand-conjugated complementary strand hybridized with PMOs, significantly enhancing delivery to key tissues in mdx mice, normalizing motor functions, muscle pathology, and serum creatine kinase by restoring internal deleted dystrophin expression. Additionally, PMOs-based HDOs normalized cardiac and CNS abnormalities without adverse effects. Our technology increases serum albumin binding to PMOs and improves blood retention and cellular uptake. Here we show that PMOs-based HDOs address the limitations in oligonucleotide therapy for DMD and offer a promising approach for diseases amenable to exon-skipping therapy.
Assuntos
Modelos Animais de Doenças , Distrofina , Camundongos Endogâmicos mdx , Morfolinos , Distrofia Muscular de Duchenne , Animais , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Morfolinos/administração & dosagem , Morfolinos/genética , Camundongos , Distrofina/genética , Distrofina/metabolismo , Splicing de RNA , Humanos , Éxons/genética , Masculino , Músculo Esquelético/metabolismo , Terapia Genética/métodos , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/farmacocinéticaRESUMO
BACKGROUND: The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. RESULTS: To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. CONCLUSIONS: Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.
Assuntos
Sistemas CRISPR-Cas , Modelos Animais de Doenças , Camundongos Transgênicos , Distrofia Muscular de Duchenne , Transgenes , Animais , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Camundongos , Humanos , Edição de Genes/métodos , Distrofina/genética , Duplicação Gênica , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications.
Assuntos
Adenosina , Citosina , Edição de Genes , Distrofia Muscular de Duchenne , Citosina/metabolismo , Animais , Edição de Genes/métodos , Adenosina/metabolismo , Camundongos , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Engenharia de Proteínas , Distrofina/genética , Distrofina/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Modelos Animais de Doenças , Éxons/genética , Células HEK293 , Sistemas CRISPR-Cas , Proteínas de Escherichia coliRESUMO
Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive degenerative disease of skeletal muscle, characterized by intramuscular inflammation, muscle regeneration disorder and replacement of muscle with fibroadipose tissue. DMD is caused by the absence of normal dystrophy. Impaired self-renew ability and limited differentiation capacity of satellite cells are proved as main reasons for muscle regeneration failure. The deficiency of estrogen impedes the process of muscle regeneration. However, the role of estrogen receptor ß (ERß) in muscle regeneration is still unclear. This study aims to investigate the role and the pharmacological effect of ERß activation on muscle regeneration in mdx mice. This study showed that mRNA levels of ERß and myogenic-related genes both witnessed increasing trends in dystrophic context. Our results revealed that treatment with selective ERß agonist (DPN, diarylpropionitrile) significantly increased myogenic differentiation 1 (MyoD-1) level and promoted muscle regeneration in mdx mice. Similarly, in mdx mice with muscle-specific estrogen receptor α (ERα) ablation, DPN treatment still promoted muscle regeneration. Moreover, we demonstrated that myoblasts differentiation was accompanied by raised nuclear accumulation of ERß. DPN treatment augmented the nuclear accumulation of ERß and, thus, contributed to myotubes formation. One important finding was that forkhead box O3A (FOXO3A), as a pivotal transcription factor in Myod-1 transcription, participated in the ERß-promoted muscle regeneration. Overall, we offered an interesting explanation about the crucial role of ERß during myogenesis.
Assuntos
Receptor beta de Estrogênio , Proteína Forkhead Box O3 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne , Proteína MyoD , Nitrilas , Propionatos , Regeneração , Animais , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Proteína MyoD/genética , Proteína MyoD/metabolismo , Regeneração/efeitos dos fármacos , Proteína Forkhead Box O3/metabolismo , Proteína Forkhead Box O3/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Nitrilas/farmacologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Camundongos , Propionatos/farmacologia , Masculino , Desenvolvimento Muscular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Diferenciação Celular/efeitos dos fármacosAssuntos
Distrofina , Terapia Genética , Vetores Genéticos , Distrofia Muscular de Duchenne , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Distrofina/genética , Terapia Genética/métodos , Humanos , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Animais , Modelos Animais de DoençasRESUMO
Dystrophinopathies are a group of neuromuscular disorders, inherited in an X-linked recessive manner, caused by pathogenic variants in the DMD gene. Copy number variation detection and next generation sequencing allow the detection of around 99 % of the pathogenic variants. However, some patients require mRNA studies from muscle biopsies to identify deep intronic pathogenic variants. Here, we report a child suspected of having Duchenne muscular dystrophy, with a muscle biopsy showing dystrophin deficiency, and negative molecular testing for deletions, duplications, and small variants. mRNA analysis from muscle biopsy revealed a pseudoexon activation that introduce a premature stop codon into the reading frame. gDNA sequencing allowed to identified a novel variant, c.832-186 T>G, which creates a cryptic donor splice site, recognizing the underlying mechanism causing the pseudoexon insertion. This case highlights the usefulness of the mRNA analysis from muscle biopsy when routine genetic testing is negative and clinical suspicion of dystrophinopathies remains the main clinical diagnosis suspicion.
Assuntos
Códon sem Sentido , Distrofina , Íntrons , Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/genética , Distrofina/genética , Masculino , Éxons , Criança , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Background: Duchenne muscular dystrophy (DMD) is a progressive, life-limiting, neuromuscular disorder. Clinicians play an important role in informing families about therapy options, including approved gene therapies and clinical trials of unapproved therapies. Objective: This study aimed to understand the perspectives of clinicians about gene therapy for DMD, which has not previously been studied. Methods: We conducted interviews with specialist clinicians treating patients with DMD in the United States (nâ=â8) and United Kingdom (nâ=â8). Interviews were completed in 2022, before any approved gene therapies, to gain insight into barriers and facilitators to implementing gene therapy and educational needs of clinicians. Results: Most respondents expressed cautious optimism about gene therapy. Responses varied regarding potential benefits with most expecting delayed progression and duration of benefit (1 year to lifelong). Concern about anticipated risks also varied; types of anticipated risks included immunological reactions, liver toxicity, and cardiac or renal dysfunction. Clinicians generally, but not uniformly, understood that gene therapy for DMD would not be curative. Most reported needing demonstrable clinical benefit to justify treatment-related risks. Conclusions: Our data demonstrate variability in knowledge and attitudes about gene therapy among clinicians who follow patients with DMD. As our knowledge base about DMD gene therapy grows, clinician education is vital to ensuring that accurate information is communicated to patients and families.
Assuntos
Terapia Genética , Distrofia Muscular de Duchenne , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Humanos , Terapia Genética/métodos , Atitude do Pessoal de Saúde , Estados Unidos , Reino Unido , Masculino , FemininoRESUMO
Structural variants (SVs) of unknown significance are great challenges for prenatal risk assessment, especially when involving dose-sensitive genes such as DMD The pathogenicities of 5'-terminal DMD duplications in the database remain controversial. Four prenatal cases with Xp21.1 duplications were identified by routine prenatal genomic testing, encompassing the 5'-UTR to exons 1-2 in family 1 and family 2, and to exons 1-9 in family 3. The duplication in family 4 was non-contiguous covering the 5'-UTR to exon 1 and exons 3-7. All were traced to unaffected males in the family pedigrees. A new genome-wide approach of optical genome mapping was performed in families 1, 2, and 3 to delineate the breakpoints and orientation of the duplicated fragments. The extra copies were tandemly inserted into the upstream of DMD, preserving the integrity of ORF from the second copy. The pathogenicities were thus reclassified as likely benign. Our data highlight the importance of structural delineation by optical genome mapping in prenatal risk assessment of incidentally identified SVs involving DMD and other similar large dose-sensitive genes.
Assuntos
Distrofina , Linhagem , Humanos , Feminino , Masculino , Medição de Risco/métodos , Gravidez , Distrofina/genética , Diagnóstico Pré-Natal/métodos , Mapeamento Cromossômico/métodos , Cromossomos Humanos X/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/diagnóstico , Duplicação Cromossômica/genética , Éxons/genética , Duplicação Gênica/genética , AdultoRESUMO
Sestrin2 (Sesn2) has been previously confirmed to be a stress-response molecule. However, the influence of Sesn2 on myogenic differentiation remains elusive. This study was conducted to analyze the role of Sesn2 in the myogenic differentiation of C2C12 myoblasts and related aspects in mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Our results showed that knockdown of Sesn2 reduced the myogenic differentiation capacity of C2C12 myoblasts. Predictive analysis from two databases suggested that miR-182-5p is a potential regulator of Sesn2. Further experimental validation revealed that overexpression of miR-182-5p decreased both the protein and mRNA levels of Sesn2 and inhibited myogenesis of C2C12 myoblasts. These findings suggest that miR-182-5p negatively regulates myogenesis by repressing Sesn2 expression. Extending to an in vivo model of DMD, knockdown of Sesn2 led to decreased Myogenin (Myog) expression and increased Pax7 expression, while its overexpression upregulated Myog levels and enhanced the proportion of slow-switch myofibers. These findings indicate the crucial role of Sesn2 in promoting myogenic differentiation and skeletal muscle regeneration, providing potential therapeutic targets for muscular dystrophy.