Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.193
Filtrar
1.
Methods Mol Biol ; 2224: 203-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606217

RESUMO

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder affecting many children. The disease is caused by the lack of dystrophin production and characterized by muscle wasting. The most common causes of death are respiratory failure and heart failure. Antisense oligonucleotide-mediated exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is a promising therapeutic approach for the treatment of DMD. In preclinical studies, dystrophic mouse models are commonly used for the development of therapeutic oligos. We employ a humanized model carrying the full-length human DMD transgene along with the complete knockout of the mouse Dmd gene. In this model, the effects of human-targeting AOs can be tested without cross-reaction between mouse sequences and human sequences (note that mdx, a conventional dystrophic mouse model, carries a nonsense point mutation in exon 23 and express the full-length mouse Dmd mRNA, which is a significant complicating factor). To determine if dystrophin expression is restored, the Western blotting analysis is commonly performed; however, due to the extremely large protein size of dystrophin (427 kDa), detection and accurate quantification of full-length dystrophin can be a challenge. Here, we present methodologies to systemically inject PMOs into humanized DMD model mice and determine levels of dystrophin restoration via Western blotting. Using a tris-acetate gradient SDS gel and semi-dry transfer with three buffers, including the Concentrated Anode Buffer, Anode Buffer, and Cathode Buffer, less than 1% normal levels of dystrophin expression are easily detectable. This method is fast, easy, and sensitive enough for the detection of dystrophin from both cultured muscle cells and muscle biopsy samples.


Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/genética , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Éxons/genética , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , Transgenes/genética
2.
Nat Commun ; 12(1): 1034, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589617

RESUMO

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano , RNA Guia/genética , Pareamento de Bases , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Bases de Dados Genéticas , Doença de Fabry/genética , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Hemofilia A/genética , Hemofilia A/metabolismo , Hemofilia A/patologia , Humanos , Modelos Biológicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mutação , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
3.
Obstet Gynecol ; 137(2): 345-350, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416279

RESUMO

Advances in genetic technology have allowed for the development of multiplex panels that can test for hundreds of genetic disorders at the same time; these large panels are referred to as expanded carrier screening. This process can screen couples for far more conditions than the gene-by-gene approach used with traditional carrier screening; however, although expanded carrier screening has been promoted as an efficient means of detecting many more disorders, the complexities of genetic sequencing raise substantial challenges and concerns. In our practice, we have seen a number of complex cases in which only attention to detail on the part of thorough genetic counselors allowed identification of misclassified variants that could have resulted in significant patient harm. We raise issues that require urgent attention by professional societies, including: whether to endorse testing that uses sequencing compared with genotyping; required components of pretest and posttest counseling; reclassification of variants; whether obstetric health care professionals have a responsibility to assure that patients understand the iterative process of variant interpretation and how it relates to carrier screening results; and the question of rescreening in subsequent pregnancies. Implementation of expanded carrier screening needs to be considered thoughtfully in light of the complexity of genetic sequencing and limited knowledge of genetics of most front-line obstetric health care professionals.


Assuntos
Triagem de Portadores Genéticos , Heterozigoto , Adulto , Fibrose Cística/genética , Febre Familiar do Mediterrâneo/genética , Feminino , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , Rim Policístico Autossômico Recessivo/genética , Gravidez , Síndrome de Zellweger/genética
4.
Expert Opin Investig Drugs ; 30(2): 167-176, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33393390

RESUMO

INTRODUCTION: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin (DMD) gene. Most patients die from respiratory failure or cardiomyopathy. There are significant unmet needs for treatments for DMD as the standard of care is principally limited to symptom relief through treatments including steroids. AREAS COVERED: This review summarizes safety and efficacy in promising areas of DMD therapeutics - small molecules, stop codon readthrough, gene replacement, and exon skipping - under clinical examination from 2015-2020 as demonstrated in the NIH Clinical Trials and PubMed search engines. EXPERT OPINION: Currently, steroids persist as the most accessible medicine for DMD. Stop-codon readthrough, gene replacement, and exon-skipping therapies all aim to restore dystrophin expression. Of these strategies, gene replacement therapy has recently gained momentum while exon-skipping retains great traction. The  FDA approval of three exon-skipping antisense oligonucleotides illustrate this regulatory momentum, though the effectiveness and sequence design of eteplirsen remain controversial. Cell-penetrating peptides promise to more efficaciously treat DMD-related cardiomyopathy.The recent success of antisense therapies, however, poses major regulatory challenges. To fully realize the benefits of exon-skipping, including cocktail oligonucleotide-mediated multiple exon-skipping and oligonucleotide drugs for very rare mutations, regulatory challenges need to be addressed in coordination with scientific advances.


Assuntos
Peptídeos Penetradores de Células/uso terapêutico , Distrofina/genética , Terapia Genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos/uso terapêutico , Animais , Peptídeos Penetradores de Células/efeitos adversos , Códon de Terminação , Desenvolvimento de Medicamentos , Drogas em Investigação/efeitos adversos , Drogas em Investigação/uso terapêutico , Éxons , Regulação da Expressão Gênica , Predisposição Genética para Doença , Terapia Genética/efeitos adversos , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Mutação , Oligonucleotídeos/efeitos adversos , Esteroides/efeitos adversos , Esteroides/uso terapêutico , Resultado do Tratamento
6.
Cells ; 9(12)2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33322031

RESUMO

Myostatin inhibition therapy has held much promise for the treatment of muscle wasting disorders. This is particularly true for the fatal myopathy, Duchenne Muscular Dystrophy (DMD). Following on from promising pre-clinical data in dystrophin-deficient mice and dogs, several clinical trials were initiated in DMD patients using different modality myostatin inhibition therapies. All failed to show modification of disease course as dictated by the primary and secondary outcome measures selected: the myostatin inhibition story, thus far, is a failed clinical story. These trials have recently been extensively reviewed and reasons why pre-clinical data collected in animal models have failed to translate into clinical benefit to patients have been purported. However, the biological mechanisms underlying translational failure need to be examined to ensure future myostatin inhibitor development endeavors do not meet with the same fate. Here, we explore the biology which could explain the failed translation of myostatin inhibitors in the treatment of DMD.


Assuntos
Anticorpos/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Miostatina/antagonistas & inibidores , Animais , Anticorpos/farmacologia , Ensaios Clínicos como Assunto , Humanos , Camundongos , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/genética , Falha de Tratamento
7.
PLoS One ; 15(12): e0244215, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362201

RESUMO

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.


Assuntos
Modelos Animais de Doenças , Distrofina/genética , Deleção de Genes , Distrofia Muscular de Duchenne/genética , Fenótipo , Transgenes , Animais , Fenômenos Biomecânicos , Distrofina/metabolismo , Éxons , Marcha , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/patologia
8.
PLoS One ; 15(9): e0239468, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970732

RESUMO

Most Duchenne muscular dystrophy (DMD) cases are caused by deletions or duplications of one or more exons that disrupt the reading frame of DMD mRNA. Restoring the reading frame allows the production of partially functional dystrophin proteins, and result in less severe symptoms. Antisense oligonucleotide mediated exon skipping has been approved for DMD, but this strategy needs repeated treatment. CRISPR/Cas9 can also restore dystrophin reading frame. Although recent in vivo studies showed the efficacy of the single-cut reframing/exon skipping strategy, methods to find the most efficient single-cut sgRNAs for a specific mutation are lacking. Here we show that the insertion/deletion (INDEL) generating efficiency and the INDEL profiles both contribute to the reading frame restoring efficiency of a single-cut sgRNA, thus assays only examining INDEL frequency are not able to find the best sgRNAs. We therefore developed a GFP-reporter assay to evaluate single-cut reframing efficiency, reporting the combined effects of both aspects. We show that the GFP-reporter assay can reliably predict the performance of sgRNAs in myoblasts. This GFP-reporter assay makes it possible to efficiently and reliably find the most efficient single-cut sgRNA for restoring dystrophin expression.


Assuntos
Éxons/genética , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Fases de Leitura/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Distrofina/genética , Distrofina/metabolismo , Genes Reporter/genética , Humanos , Mutação INDEL/genética , Distrofia Muscular de Duchenne/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/genética
10.
Georgian Med News ; (303): 79-85, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32841186

RESUMO

The purpose of the study is to examine in depth and analyze the clinical and some paraclinical characteristics for a family history of Duchenne muscular dystrophy. We analyzed the follow up clinical and laboratory data of Duchenne muscular dystrophy in two brothers-german, aged 16 and 14, respectively. The patients underwent a standardized examination, involving studying the medical case history, general clinical data, determining Sheldon's somatotype and the constitutional type, the detailed neurological status examination, testing a personality type, laboratory and instrumental examinations. Through the laboratory examination we determined the general blood test indicators, total serum protein levels, total cholesterol, the ALAT, ASAT, CPK levels, the indicators of the immunogram, myositis profile and the genetic markers of the disease. The instrumental examination included the ultrasound of the abdominal organs, muscles, as well as echo-cardiography, electroneuromyography. A complete examination fragment of 42 patients with myodystrophies is presented. The paper presents the neurological examination results, the genetic study data and the CPK level indicators in the representatives of Duchenne muscular dystrophy family history. The given family history of Duchenne muscular dystrophy showed two brothers-german to have differences both in the defective dystrophin gene exons at Xp21 and in the disease clinical picture. Thus, patient A., who is an elder brother was detected to have exon 47, 48, 50 and 52 deletion, and patient B., who is a younger brother, was found to have exon 45-43 deletion. The presented family history of Duchenne muscular dystrophy acknowledges the fact that the clinical, genetic, biochemical and other characteristics in patients with dystrophinopathies warrant further comprehensive investigations in order to update diagnostic and prognostic techniques, considering the great medical and social significance of this disabling pathology. However, the onset age of the disease, the clinical course, and the changes in the CPK level were different. Due to the muscle ultrasound both patients were detected to have degenerative changes in the proximal upper and lower limbs.


Assuntos
Distrofia Muscular de Duchenne/genética , Miosite , Idade de Início , Idoso , Distrofina/genética , Éxons , Humanos , Masculino
11.
Pediatrics ; 146(3)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32826339

RESUMO

Disorders of central and peripheral nervous system should be considered in floppy infants with ventilator dependence. Workup for neuromuscular disorders should be undertaken in infants with hypotonia, weakness, contractures, feeding difficulties, or failed attempts at extubation. We present the case of a preterm infant with hypotonia and ventilator dependence where despite a positive result, further investigations were undertaken because of lack of clinical correlation. The infant had a rare combination of 2 neuromuscular conditions: X-linked myotubular myopathy and Duchenne muscular dystrophy. One was the reason for immediate clinical manifestation and the other influenced the prognosis and decision-making in determining reorientation of care. This case demonstrates the value of interpretation of a positive result that did not explain the clinical picture and warranted consideration of further diagnosis. This case also emphasizes the importance of discussions with family about the prognosis of 2 conditions that influenced decision making.


Assuntos
Recém-Nascido Prematuro , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/diagnóstico , Miopatias Congênitas Estruturais/complicações , Miopatias Congênitas Estruturais/diagnóstico , Evolução Fatal , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro/fisiologia , Masculino , Distrofia Muscular de Duchenne/genética , Miopatias Congênitas Estruturais/genética
12.
PLoS One ; 15(8): e0236689, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785240

RESUMO

OBJECTIVE: To compare the effects of photobiomodulation therapy (PBMT) and pharmacological therapy (glucocorticoids and non-steroidal anti-inflammatory drugs) applied alone and in different combinations in mdx mice. METHODS: The animals were randomized and divided into seven experimental groups treated with placebo, PBMT, prednisone, non-steroidal anti-inflammatory drug (NSAIDs), PBMT plus prednisone and PBMT plus NSAID. Wild type animals were used as control. All treatments were performed during 14 consecutive weeks. Muscular morphology, protein expression of dystrophin and functional performance were assessed at the end of the last treatment. RESULTS: Both treatments with prednisone and PBMT applied alone or combined, were effective in preserving muscular morphology. In addition, the treatments with PBMT (p = 0.0005), PBMT plus prednisone (p = 0.0048) and PBMT plus NSAID (p = 0.0021) increased dystrophin gene expression compared to placebo-control group. However, in the functional performance the PBMT presented better results compared to glucocorticoids (p<0.0001). In contrast, the use of NSAIDs did not appear to add benefits to skeletal muscle tissue in mdx mice. CONCLUSION: We believe that the promising and optimistic results about the PBMT in skeletal muscle of mdx mice may in the future contribute to this therapy to be considered a safe alternative for patients with Duchenne Muscular Dystrophy (DMD) in a washout period (between treatment periods with glucocorticoids), allowing them to remain receiving effective and safe treatment in this period, avoiding at this way periods without administration of any treatment.


Assuntos
Distrofina/genética , Terapia com Luz de Baixa Intensidade , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/efeitos da radiação , Distrofia Muscular de Duchenne/terapia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Terapia Combinada , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Glucocorticoides/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Prednisona/farmacologia
13.
PLoS One ; 15(8): e0237803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813700

RESUMO

A nonsense mutation adds a premature stop signal that hinders any further translation of a protein-coding gene, usually resulting in a null allele. To investigate the possible exceptions, we used the DMD gene as an ideal model. First, because dystrophin absence causes Duchenne muscular dystrophy (DMD), while its reduction causes Becker muscular dystrophy (BMD). Second, the DMD gene is X-linked and there is no second allele that can interfere in males. Third, databases are accumulating reports on many mutations and phenotypic data. Finally, because DMD mutations may have important therapeutic implications. For our study, we analyzed large databases (LOVD, HGMD and ClinVar) and literature and revised critically all data, together with data from our internal patients. We totally collected 2593 patients. Positioning these mutations along the dystrophin transcript, we observed a nonrandom distribution of BMD-associated mutations within selected exons and concluded that the position can be predictive of the phenotype. Nonsense mutations always cause DMD when occurring at any point in fifty-one exons. In the remaining exons, we found milder BMD cases due to early 5' nonsense mutations, if reinitiation can occur, or due to late 3' nonsense when the shortened product retains functionality. In the central part of the gene, all mutations in some in-frame exons, such as in exons 25, 31, 37 and 38 cause BMD, while mutations in exons 30, 32, 34 and 36 cause DMD. This may have important implication in predicting the natural history and the efficacy of therapeutic use of drug-stimulated translational readthrough of premature termination codons, also considering the action of internal natural rescuers. More in general, our survey confirm that a nonsense mutation should be not necessarily classified as a null allele and this should be considered in genetic counselling.


Assuntos
Códon sem Sentido/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mutação/genética , Inquéritos e Questionários , Sequência de Aminoácidos , Sequência de Bases , Distrofina/química , Éxons/genética , Humanos , Fenótipo
14.
Am J Physiol Heart Circ Physiol ; 319(3): H582-H603, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32762558

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac ß-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.


Assuntos
Arginina/análogos & derivados , Cardiomiopatias/metabolismo , Circulação Coronária , Tolerância ao Exercício , Heterozigoto , Distrofia Muscular de Duchenne/metabolismo , Miocárdio/metabolismo , Músculo Quadríceps/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Arginina/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Miocárdio/patologia , Necrose , Músculo Quadríceps/patologia , Músculo Quadríceps/fisiopatologia , Função Ventricular Esquerda
15.
Am J Pathol ; 190(10): 2136-2145, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650001

RESUMO

Secondary mitochondrial damage in skeletal muscles is a common feature of different neuromuscular disorders, which fall outside the mitochondrial cytopathies. The common cause of mitochondrial dysfunction and structural changes in skeletal muscle tissue remains to be discovered. Although they are associated with different clinical, genetic, and pathologic backgrounds, the pathomechanisms underlying neuromuscular disorders might be attributed to the complex interaction and cross talk between mitochondria and the associated miRNAs. This study aimed to identify the common miRNA signatures that are associated with mitochondrial damage in different muscular dystrophies (MDs; Duchenne muscular dystrophy, megaconial congenital muscular dystrophy, Ullrich congenital muscular dystrophy, and α-dystroglycanopathy). The miRNome profiles of skeletal muscle biopsies acquired from four different MD groups and control individuals were analyzed by miRNA microarray. We identified 17 common up-regulated miRNAs in all of the tested MD groups. A specific bioinformatics approach identified 10 of these miRNAs to be specifically related to the mitochondrial pathways. Six miRNAs, miR-134-5p, miR-199a-5p, miR-382-5p, miR-409-3p, miR-497-5p, and miR-708-5p, were associated with the top four mitochondrial pathways and were thus selected as priority candidates for further validation by quantitative real-time PCR analysis. We demonstrate, for the first time, common up-regulated miRNAs that are associated with mitochondrial damage in different MD groups, therefore contributing to the pathophysiology. Our findings may open a new gate toward therapeutics.


Assuntos
Mitocôndrias/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofia Muscular de Duchenne/genética , Esclerose/genética , Adolescente , Criança , Pré-Escolar , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Lactente , Masculino , MicroRNAs/genética
16.
Life Sci ; 257: 118069, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659370

RESUMO

AIM: Up-regulation of inflammasome proteins was reported in dystrophin-deficient muscles. However, it remains to be determined whether inflammasome activation plays a role in the pathogenesis of Duchenne muscular dystrophy. This study was therefore set out to investigate whether genetic disruption of the inflammasome pathway impacts the disease progression in mdx mice. MAIN METHODS: Mice deficient in both dystrophin and ASC (encoded by Pycard [PYD And CARD Domain Containing]) were generated. The impact of ASC deficiency on muscular dystrophy of mdx mice were assessed by measurements of serum cytokines, Western blot, real-time PCR and histopathological staining. KEY FINDINGS: The pro-inflammatory cytokines such as TNF-α, IL-6, KC/GRO and IL-10 were markedly increased in the sera of 8-week-old mdx mice compared to WT. Western blotting showed that P2X7, caspase-1, ASC and IL-18 were upregulated. Disruption of ASC and dystrophin expression in the mdx/ASC-/- mice was verified by Western blot analysis. Histopathological analysis did not find significant alterations in the muscular dystrophy phenotype in mdx/ASC-/- mice as compared to mdx mice. SIGNIFICANCE: Taken together, our results show that disruption of the central adaptor ASC of the inflammasome is insufficient to alleviate muscular dystrophy phenotype in mdx mice.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Distrofina/genética , Inflamassomos/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Animais , Citocinas/sangue , Modelos Animais de Doenças , Progressão da Doença , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Distrofia Muscular de Duchenne/genética , Regulação para Cima
17.
Brain Nerve ; 72(7): 753-766, 2020 Jul.
Artigo em Japonês | MEDLINE | ID: mdl-32641572

RESUMO

There are many ethical issues in the health care of hereditary neuromuscular diseases such as Duchenne muscular dystrophy. The problems lie in the protection of personal genetic information in genetic diagnosis and genetic discrimination. The idea that allocating expensive medical care to such patients with severe disabilities is futile should be avoided. QOL as patient's individual subjective perception has not been completely understood in the academic field of clinical ethics. From the view of a third party it might be thought that the neuromuscular disease patients' QOL is extremely low. In such cases, ventilator therapy, PEG placement, and latest antisense nucleotide therapy might be considered wasteful. However, these therapies must be necessary and appropriate from the patients' own view. These dilemma on clinical ethics can be solved by focusing on enhancing the patients' subjective QOL. This can be achieved by means of modern genetics, symptom control techniques and the use of a safety net medical care system with a multidisciplinary team.


Assuntos
Distrofia Muscular de Duchenne , Doenças Neuromusculares , Ética Clínica , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Qualidade de Vida
18.
Proc Natl Acad Sci U S A ; 117(28): 16456-16464, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32616572

RESUMO

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop in DMD expression levels in the presence of PTC. We observed that the inhibition of NMD does not normalize DMD gene expression in DMD. Additionally, in situ hybridization showed that DMD messenger RNA primarily localizes in the nuclear compartment, confirming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to explore DMD transcription dynamics revealed a lower rate of DMD transcription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 in mdx mice compared to wild-type mice, indicating a chromatin conformation less prone to transcription in mdx mice. In line with this finding, treatment with the histone deacetylase inhibitor givinostat caused a significant increase in DMD transcript expression in mdx mice. Overall, our findings show that transcription dynamics across the DMD locus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process.


Assuntos
Códon sem Sentido/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , RNA Mensageiro/genética , Animais , Códon sem Sentido/metabolismo , Modelos Animais de Doenças , Distrofina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/metabolismo
19.
AJNR Am J Neuroradiol ; 41(7): 1271-1278, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32616576

RESUMO

BACKGROUND AND PURPOSE: Duchenne muscular dystrophy is an X-linked disorder characterized by progressive muscle weakness and prominent nonmotor manifestations, such as a low intelligence quotient and neuropsychiatric disturbance. We investigated WM integrity in patients with Duchenne muscular dystrophy using DTI. MATERIALS AND METHODS: Fractional anisotropy and mean, axial, and radial diffusivity (DTI measures) were used to assess WM microstructural integrity along with neuropsychological evaluation in patients with Duchenne muscular dystrophy (n = 60) and controls (n = 40). Exon deletions in the DMD gene were confirmed using multiplex ligation-dependent probe amplification. Patients were classified into proximal (DMD Dp140+) and distal (DMD Dp140-) subgroups based on the location of the exon deletion and expression of short dystrophin Dp140 isoform. WM integrity was examined using whole-brain Tract-Based Spatial Statistics and atlas-based analysis of DTI data. The Pearson correlation was performed to investigate the possible relationship between neuropsychological scores and DTI metrics. RESULTS: The mean ages of Duchenne muscular dystrophy and control participants were 8.0 ± 1.2 years and 8.2 ± 1.4 years, respectively. The mean age at disease onset was 4.1 ± 1.8 years, and mean illness duration was 40.8 ± 25.2 months. Significant differences in neuropsychological scores were observed between the proximal and distal gene-deletion subgroups, with more severe impairment in the distal-deletion subgroup (P < .05). Localized fractional anisotropy changes were seen in the corpus callosum, parietal WM, and fornices in the patient subgroup with Dp140+, while widespread changes were noted in the Dp140- subgroup. The Dp140+ subgroup showed increased axial diffusivity in multiple WM regions relative to the Dp140- subgroup. No significant correlation was observed between clinical and neuropsychological scores and diffusion metrics. CONCLUSIONS: Widespread WM differences are evident in patients with Duchenne muscular dystrophy relative to healthy controls. Distal mutations in particular are associated with extensive WM abnormalities and poor neuropsychological profiles.


Assuntos
Encéfalo/diagnóstico por imagem , Distrofia Muscular de Duchenne/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , Adolescente , Encéfalo/patologia , Criança , Imagem de Tensor de Difusão/métodos , Distrofina/genética , Feminino , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mutação , Neuroimagem/métodos , Substância Branca/patologia
20.
PLoS One ; 15(6): e0232654, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559196

RESUMO

Recently DNA sequencing analysis has played a vital role in the unambiguous diagnosis of clinically suspected patients with Duchenne Muscular Dystrophy (DMD). DMD is a monogenic, X-linked, recessive, degenerative pediatric neuromuscular disorder affecting males, invariably leading to fatal cardiopulmonary failure. Early and precise diagnosis of the disease is an essential part of an effective disease management strategy as care guidelines and prevention through counseling need to be initiated at the earliest particularly since therapies are now available for a subset of patients. In this manuscript we report the DMD gene mutational profiles of 961 clinically suspected male DMD patients, 99% of whom were unrelated. We utilized a molecular diagnostic approach which is cost-effective for most patients and follows a systematic process that sequentially involves identification of hotspot deletions using mPCR, large deletions and duplications using MLPA and small insertions/ deletions and point mutations using an NGS muscular dystrophy gene panel. Pathogenic DMD gene mutations were identified in 84% of patients. Our data compared well with the frequencies and distribution of deletions and duplications reported in the DMD gene in other published studies. We also describe a number of rare in-frame mutations, which appeared to be enriched in the 5' proximal hotspot region of the DMD gene. Furthermore, we identified a family with a rare non-contiguous deletion mutation in the DMD gene where three males were affected and two females were deemed carriers. A subset of patients with mutations in the DMD gene who are likely to benefit therapeutically from new FDA and EMA approved drugs were found in our cohort. Given that the burden of care for DMD patients invariably falls on the mothers, particularly in rural India, effective genetic counseling followed by carrier screening is crucial for prevention of this disorder. We analyzed the carrier status of consented female relatives of 463 probands to gauge the percentage of patients with familial disease. Our analysis revealed 43.7% of mothers with DMD gene mutations. Our comprehensive efforts, involving complete genetic testing coupled with compassionate genetic counseling provided to DMD patients and their families, are intended to improve the quality of life of DMD patients and to empower carrier females to make informed reproductive choices to impede the propagation of this deadly disease.


Assuntos
Distrofia Muscular de Duchenne/genética , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Família , Feminino , Aconselhamento Genético , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/prevenção & controle , Distrofia Muscular de Duchenne/terapia , Mutação , Fenótipo , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...