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1.
Hum Genet ; 139(2): 247-255, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31919629

RESUMO

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


Assuntos
Códon sem Sentido , Distrofina/genética , Éxons/genética , Mutação da Fase de Leitura , Distrofia Muscular de Duchenne/genética , Adolescente , Adulto , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
2.
PLoS Genet ; 15(10): e1008408, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626629

RESUMO

Satellite cells (SCs) are muscle stem cells that remain quiescent during homeostasis and are activated in response to acute muscle damage or in chronic degenerative conditions such as Duchenne Muscular Dystrophy. The activity of SCs is supported by specialized cells which either reside in the muscle or are recruited in regenerating skeletal muscles, such as for instance macrophages (MΦs). By using a dystrophic mouse model of transient MΦ depletion, we describe a shift in identity of muscle stem cells dependent on the crosstalk between MΦs and SCs. Indeed MΦ depletion determines adipogenic conversion of SCs and exhaustion of the SC pool leading to an exacerbated dystrophic phenotype. The reported data could also provide new insights into therapeutic approaches targeting inflammation in dystrophic muscles.


Assuntos
Diferenciação Celular/genética , Macrófagos/metabolismo , Distrofia Muscular de Duchenne/genética , Regeneração/genética , Animais , Linhagem da Célula/genética , Modelos Animais de Doenças , Distrofina/genética , Humanos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia
3.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(10): 1015-1018, 2019 Oct 10.
Artigo em Chinês | MEDLINE | ID: mdl-31598949

RESUMO

OBJECTIVE: To carry out genetic testing and prenatal diagnosis for a family affected with Duchenne muscular dystrophy (DMD). METHODS: Multiplex ligation dependent probe amplification (MLPA) was used to detect potential deletion and duplication of the Dystrophin gene. Haplotype analysis was performed using five short tandem repeat polymorphism loci (3'-STR, 5'-STR, 45-STR, 49-STR, 50-STR of the DMD gene. RESULTS: A same deletional mutation (exons 51-55) of the DMD gene was detected in two brothers but not in their mother. The patients and fetus have inherited different haplotypes of the Dystrophin gene from their mother, suggesting that the fetus was unaffected. CONCLUSION: The mother was very likely to harbor germline mosaicism for the Dystrophin gene variant. Genetic testing of peripheral blood samples cannot rule out germline mosaicism in the mother. Prenatal diagnosis should be provided for subsequent pregnancies in this family.


Assuntos
Distrofina/genética , Deleção de Genes , Mutação em Linhagem Germinativa , Mosaicismo , Distrofia Muscular de Duchenne/genética , Éxons , Feminino , Humanos , Masculino , Gravidez , Diagnóstico Pré-Natal
4.
Medicina (B Aires) ; 79 Suppl 3: 77-81, 2019.
Artigo em Espanhol | MEDLINE | ID: mdl-31603849

RESUMO

Duchenne muscular dystrophy is a genetically determined disease, linked to the X chromosome, c haracterized clinically by producing progressive muscle weakness, with an incidence of 1 per 3500-6000 males born. It is caused by the mutation of the DMD gene, which encodes dystrophin, a sub-sarcolemmal protein essential for structural muscle stability. The genetic defects in the DMD gene are divided into: deletions (65%) duplications (5.10%) and point mutations (10-15%). At present there is no curative treatment, the only drug that has been shown to modify the natural history of the disease (independently of the genetic mutation) are corticosteroids, currently indicated in early stages of the disease. In relation to clinical trials, in the last ten years, has experienced great advances in the field of therapeutic options, divided into two major therapeutic targets: 1) the area of gene therapies and 2) trying to reverse or block the pathophysiological processes of the disease, such as inflammation, fibrosis, muscle regeneration, etc. It is likely that an effective treatment for Duchenne muscular dystrophy requires combinations of therapies that address both the primary defect and its secondary pathophysiological consequences.


Assuntos
Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Animais , Sistemas CRISPR-Cas , Distrofina/genética , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Fenótipo
5.
Nat Med ; 25(10): 1505-1511, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31591596

RESUMO

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.


Assuntos
Terapia Genética , Distrofias Musculares/terapia , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Utrofina/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Cães , Distrofina/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Contração Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Transgenes/genética , Utrofina/uso terapêutico
6.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(8): 773-776, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31400125

RESUMO

OBJECTIVE: To discuss the value of chromosomal microarray analysis (CMA) for the identification of DMD gene deletions during prenatal diagnosis. METHODS: G-banded karyotyping and CMA were performed on fetuses with ultrasonographic soft markers but no family history for Duchenne/Becker muscular dystrophy (DMD/BMD). Denaturing high-performance liquid chromatograghy (DHPLC) was used to detect DMD gene mutations in umbilical cord blood and peripheral blood samples from the mothers. RESULTS: For fetus 1, analysis of amniocytes showed a normal karyotype, while CMA detected a 119 kb deletion at Xp21.1 (32 565 489 - 32 681 461), which encompassed exons 10 to 16 of the DMD gene. The result was confirmed by DHPLC analysis. The mother was found to have loss of heterozygosity in the same region. For fetus 2, karyotyping of amniocytes also showed a normal male karyotype, while CMA detected a 254 kb deletion at Xp21.1 (32 104 604 - 32 358 874), which encompassed exons 41 to 44 of the DMD gene. The same deletion was not detected in the mother. DHPLC analysis confirmed the presence of both deletions. CONCLUSION: Two fetuses harboring DMD gene deletions but without a family history were discovered. CMA can improve the efficiency for detecting single gene diseases caused by deletions.


Assuntos
Distrofina/genética , Deleção de Genes , Achados Incidentais , Análise em Microsséries , Distrofia Muscular de Duchenne/genética , Éxons , Feminino , Feto , Humanos , Masculino , Gravidez
7.
Int J Mol Sci ; 20(17)2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31443395

RESUMO

Duchenne muscular dystrophy (DMD) is a devastating disease featuring skeletal muscle wasting, respiratory insufficiency, and cardiomyopathy. Historically, respiratory failure has been the leading cause of mortality in DMD, but recent improvements in symptomatic respiratory management have extended the life expectancy of DMD patients. With increased longevity, the clinical relevance of heart disease in DMD is growing, as virtually all DMD patients over 18 year of age display signs of cardiomyopathy. This review will focus on the pathophysiological basis of DMD in the heart and discuss the therapeutic approaches currently in use and those in development to treat dystrophic cardiomyopathy. The first section will describe the aspects of the DMD that result in the loss of cardiac tissue and accumulation of fibrosis. The second section will discuss cardiac small molecule therapies currently used to treat heart disease in DMD, with a focus on the evidence supporting the use of each drug in dystrophic patients. The final section will outline the strengths and limitations of approaches directed at correcting the genetic defect through dystrophin gene replacement, modification, or repair. There are several new and promising therapeutic approaches that may protect the dystrophic heart, but their limitations suggest that future management of dystrophic cardiomyopathy may benefit from combining gene-targeted therapies with small molecule therapies. Understanding the mechanistic basis of dystrophic heart disease and the effects of current and emerging therapies will be critical for their success in the treatment of patients with DMD.


Assuntos
Cardiomiopatias/etiologia , Distrofia Muscular de Duchenne/complicações , Animais , Cálcio/metabolismo , Cardiomiopatias/diagnóstico , Cardiomiopatias/terapia , Gerenciamento Clínico , Suscetibilidade a Doenças , Distrofina/genética , Éxons , Fibrose , Terapia Genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo
8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(7): 666-671, 2019 Jul 10.
Artigo em Chinês | MEDLINE | ID: mdl-31302907

RESUMO

OBJECTIVE: To study the correlation of splicing mutations at the 5' end of the DMD gene with their phenotypes. METHODS: DMD gene mutations were analyzed using Multiplex Ligation Probe Amplification (MLPA) and Sanger sequencing. Co-segregation analysis was performed for the pedigrees of the probands. Influence of mutations on protein function was predicted by bioinformatic analysis. RESULTS: Three novel splicing mutations were identified in three patients with different phenotypes. Patient 1 carried a c.31+3insT mutation and presented primarily with dilated cardiomyopathy (XLDC). There was no clinical signs of skeletal myopathy. Bioinformatic analysis predicted that the mutation may inactivate the splicing donor of intron 1 and lead to premature termination of protein translation. Patient 2 carried a c.264_264+4delTGTAA mutation, which led to loss of splicing donor site for intron 4, and manifested Becker muscular dystrophy (BMD). The mutation was predicted to result in skipping of exon 4. The defective protein may still retain most of its function. Patient 3 had Duchenne muscular dystrophy (DMD) and carried a c.832-3C>T mutation which was predicted to decrease the activity of splicing acceptor of intron 8, resulting in usage of alternative acceptor site or retain of intron 8. All related transcripts may cause premature termination of protein translation and complete loss of protein function. The three mutations were all inherited from the mothers of the patients. CONCLUSION: Three novel splicing mutations were discovered at the 5' end of DMD gene in three patients with different disease phenotypes. Our study may facilitate understanding of the influence of splicing mutations at the 5' end of the DMD gene on dystrophin function and the correlation between genotypes and phenotypes.


Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Mutação , Processamento de RNA , Humanos , Fenótipo
9.
Life Sci ; 232: 116654, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31306657

RESUMO

AIMS: Immuno-inflammation contributes to the pathogenesis of Duchenne muscular dystrophy (DMD), characterized by progressive muscle degeneration and weakness. The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is crucial for initiating innate immunity. Ghrelin is a circulating hormone that exerts anti-inflammatory activity in several inflammatory diseases. However, the role of ghrelin in DMD and underlying mechanism are still unstated. Therefore, we investigated the effect and potential mechanism of ghrelin on muscle morphology and muscular function of mdx mice, a mouse model of DMD. MAIN METHODS: 4-Week-old male mdx mice were injected intraperitoneally with ghrelin (100 µg/kg of body weight/day) or saline for 4 weeks. Then, muscle performance was evaluated by behavioral tests. Skeletal muscles samples were collected and relevant parameters were measured by using histopathological analysis and molecular biology techniques both in mdx muscles and primary myoblasts. KEY FINDINGS: Ghrelin significantly improved motor performance, alleviated muscle pathology and decreased inflammatory cell infiltration in mdx mice. Importantly, ghrelin dramatically inhibited NLRP3 inflammasome activation and reduced the production of mature IL-1ß both in dystrophic muscles and in lipopolysaccharide (LPS)-primed primary myoblasts induced by the NLRP3 inflammasome activator benzylated ATP (BzATP). Furthermore, the inhibition of NLRP3 inflammasome by ghrelin was partly mediated by the suppression of JAK2-STAT3 and p38 MAPK signaling pathway. SIGNIFICANCE: Our findings reveal that ghrelin suppresses muscle inflammation and ameliorates disease phenotype through inhibition of NLRP3 inflammasome activation and the production of IL-1ß in mdx mice, which suggests new therapeutic potential of ghrelin in DMD.


Assuntos
Distrofina/fisiologia , Grelina/fisiologia , Músculo Esquelético/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Animais , Distrofina/genética , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx
10.
Medicine (Baltimore) ; 98(26): e15858, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31261494

RESUMO

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.


Assuntos
Morfolinos/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Criança , Progressão da Doença , Doenças em Gêmeos , Método Duplo-Cego , Distrofina/genética , Distrofina/metabolismo , Humanos , Masculino , Morfolinos/efeitos adversos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Índice de Gravidade de Doença , Resultado do Tratamento , Teste de Caminhada , Caminhada
11.
Hum Genet ; 138(7): 771-785, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31168774

RESUMO

Modulation of dystrophin pre-mRNA splicing is an attractive strategy to ameliorate the severe phenotype of Duchenne muscular dystrophy (DMD), although this requires a better understanding of the mechanism of splicing regulation. Aberrant splicing caused by gene mutations provides a good model to study splicing regulatory cis-elements and binding proteins. In this study, we identified skipping of in-frame exon 25 induced by a nonsense mutation (NM_004006.2:c.3340A > T;p.Lys1114*) in the DMD gene. Site-directed mutagenesis study in minigenes suggested that c.3340A > T converts an exonic splicing enhancer sequence (ESE) to a silencer element (ESS). Indeed, RNA pull-down and functional study provided evidence that c.3340A > T abolishes the binding of the splicing enhancer protein Tra2ß and promotes interactions with the repressor proteins hnRNP A1, hnRNP A2, and hnRNP H. By carefully analyzing the sequence motif encompassing the mutation site, we concluded that the skipping of exon 25 was due to disruption of a Tra2ß-dependent ESE and the creation of a new ESS associated with hnRNP A1 and hnRNP A2, which in turn increased the recruitment of hnRNP H to a nearby binding site. Finally, we demonstrated that c.3340A > T impairs the splicing of upstream intron 24 in a splicing minigene assay. In addition, we showed that the correct splicing of exon 25 is finely regulated by multiple splicing regulators that function in opposite directions by binding to closely located ESE and ESS. Our results clarify the detailed molecular mechanism of exon skipping induced by the nonsense mutation c.3340A > T and also provide information on exon 25 splicing.


Assuntos
Distrofina/genética , Elementos Facilitadores Genéticos , Éxons , Distrofia Muscular de Duchenne/genética , Mutação de Sentido Incorreto , Processamento de RNA , Elementos Silenciadores Transcricionais , Adolescente , Regulação da Expressão Gênica , Humanos , Masculino , Distrofia Muscular de Duchenne/patologia
12.
Genet Test Mol Biomarkers ; 23(7): 468-472, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31157985

RESUMO

Introduction: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are inherited X-linked recessive genetic disorders caused by defects in the dystrophin gene. Abnormality in the dystrophin protein causes progressive muscle damage and weakness leading to long-term disability. Objective: To investigate the spectrum of dystrophin gene variants (deletions and duplications) in Pakistani patients suspected of having DMD/BMD or of being DMD/BMD carriers. Methods: A single center (Aga Khan University Hospital, Karachi, Pakistan) retrospective review of 46 cases was conducted to characterize dystrophin gene variants (deletion/duplication) in DMB/BMD patients using the multiplex ligation-dependent probe amplification-based method to provide coverage for all 79 exons. Results: Dystrophin gene deletions were identified in 40 of 46 cases, whereas duplications were present in 6 of 46 cases. The majority of the deletions were present between exons 45 and 52 followed by the region between exons 8 and 18. The most frequently deleted was exon 46 (8%) followed by exon 49 (7%). Dystrophin gene duplications were clustered between exons 3 and 7. The average deletion or duplication size was five exons for both kinds of variants. Conclusions: The applicability of exon skipping drugs depends on the specific mutational frequencies within populations. Our data suggest that for the Pakistani patients, multiple exon skipping between exons 46 and 49 could potentially be a target for exon skipping therapy. However, a larger nationwide study is required to further identify the predominant deletion/duplication dystrophin gene variants present in the population.


Assuntos
Distrofina/genética , Deleção de Genes , Duplicação Gênica , Distrofia Muscular de Duchenne/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Paquistão , Estudos Retrospectivos , Adulto Jovem
13.
Nat Rev Neurol ; 15(7): 373-386, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31147635

RESUMO

Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein. Improvements in patient care and disease management have slowed down disease progression, but current treatments cannot stop the relentless loss of muscle tissue and function, which leads to premature death. Research is ongoing to develop effective therapies for DMD. Gene-addition, exon-skipping, stop codon readthrough and genome-editing therapies can restore the expression of partially functional dystrophin protein, whereas other therapeutic approaches aim to improve muscle function and quality by targeting pathways involved in the pathogenesis of DMD. This Review outlines important developments in these research areas and specifically focuses on new therapies that are in the clinical trial phase or have already been approved.


Assuntos
Terapia Genética , Distrofia Muscular de Duchenne/terapia , Anti-Inflamatórios/uso terapêutico , Distrofina/genética , Éxons , Edição de Genes , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética
14.
Muscle Nerve ; 60(3): 328-335, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31228273

RESUMO

INTRODUCTION: No etiologic therapy is available for Duchenne muscular dystrophy (DMD), but mesenchymal stem cells were shown to be effective in preclinical models of DMD. The objective of this study is to investigate the effect of microfragmented fat extracted on a murine model of DMD. METHODS: Fat tissue was extracted from healthy human participants and injected IM into DMD mice. Histological analysis, cytokines, and force measurement were performed up to 4 weeks after injection. RESULTS: Duchenne muscular dystrophy mice injected with microfragmented fat exhibited an improved muscle phenotype (decreased necrosis and fibrosis), a decrease of inflammatory cytokines, and increased strength. DISCUSSION: Administration of microfragmented fat in key muscles may improve muscular phenotype in patients with DMD. Muscle Nerve, 2019.


Assuntos
Distrofina/genética , Células-Tronco Mesenquimais/citologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Tecido Adiposo/patologia , Animais , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Força Muscular/fisiologia
15.
Genes Chromosomes Cancer ; 58(10): 731-736, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31066955

RESUMO

Conventional osteosarcoma is the most common primary malignancy of bone. This group of neoplasms is subclassified according to specific histological features, but hitherto there has been no correlation between subtype, treatment, and prognosis. By in-depth genetic analyses of a chondroblastoma-like osteosarcoma, we detect a genetic profile that is distinct from those previously reported in benign and malignant bone tumors. The overall genomic copy number profile was less complex than that typically associated with conventional osteosarcoma, and there was no activating point mutation in any of H3F3A, H3F3B, IDH1, IDH2, BRAF, or GNAS. Instead, we found a homozygous CDKN2A deletion, a DMD microdeletion and an FN1-FGFR1 gene fusion. The latter alteration has been described in phosphaturic mesenchymal tumor. This tumor type shares some morphological features with chondroblastoma-like osteosarcoma and we cannot rule out that the present case actually represents an FN1-FGFR1 positive malignant phosphaturic mesenchymal tumor of bone without osteomalacia.


Assuntos
Neoplasias Ósseas/genética , Condroblastoma/genética , Deleção de Genes , Mesenquimoma/genética , Fusão Oncogênica , Osteossarcoma/genética , Neoplasias Ósseas/patologia , Condroblastoma/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Distrofina/genética , Fibronectinas/genética , Homozigoto , Humanos , Masculino , Mesenquimoma/metabolismo , Pessoa de Meia-Idade , Osteossarcoma/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
16.
Nat Biomed Eng ; 3(6): 427-437, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31097816

RESUMO

Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR-Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR-Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR-Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR-Chip expands the applications of CRISPR-Cas9 technology to the on-chip electrical detection of nucleic acids.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Grafite/química , Proteínas Imobilizadas/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Transistores Eletrônicos , DNA/genética , Distrofina/genética , Éxons/genética , Genoma , Células HEK293 , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , Mutação/genética , RNA Guia/metabolismo
17.
Zhonghua Fu Chan Ke Za Zhi ; 54(4): 226-231, 2019 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-31006187

RESUMO

Objective: To explore the mutation characteristics of DMD gene in patients with Duchenne or Becker muscular dystrophy and female carriers, to provide effective prenatal diagnosis. Methods: Samples were collected from 94 male patients clinically diagnosed with Duchenne or Becker muscular dystrophy and 121 corresponding female relatives from Qingdao Women and Children's Hospital from June 2011 to October 2018. Multiplex ligation-dependent probe amplification (MLPA) was used to detect their DMD gene, and 23 high risk pregnants were performed prenatal diagnosis. Any candidate of DMD gene single-exon deletion was validated by further PCR amplification. The sample with whole DMD gene deletion was confirmed by chromosomal microarray analysis (CMA) to detect copy number variations and break site. Results: Among 94 clinical Duchenne or Becker muscular dystrophy patients, 66(70.2%, 66/94) were detected gene mutation; 56 cases were exon deletion mutation and 10 cases were duplication mutation. In 121 female relatives, 48 cases (39.7%, 48/121) were diagnosed as carriers. The mutation carrying rate, was 64.5% (40/62) identified in 62 mothers of Duchenne or Becker muscular dystrophy patients. Five Duchenne or Becker muscular dystrophy fetuses and 5 carrier fetuses were prenatally diagnosed in 23 high risk pregnants. Two children with the entire DMD gene deletion were identified more deletions at Xp21, with deletions of 6.66 Mb and 10.64 Mb respectively. Conclusions: MLPA may be an important method to detect DMD gene mutation of deletion and duplication. Therefore, the diagnosis of probands, female carriers and making an effective prenatal diagnosis are essential to reduce the birth of children with Duchenne or Becker muscular dystrophy.


Assuntos
Distrofina/genética , Deleção de Genes , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Diagnóstico Pré-Natal/métodos , Criança , Variações do Número de Cópias de DNA , Éxons , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , Gravidez
18.
Cell Mol Life Sci ; 76(20): 4155-4164, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31030226

RESUMO

Evolved xCas9(3.7) variant with broad PAM compatibility has been reported in cell lines, while its editing efficiency was site-specific. Here, we show that xCas9(3.7) can recognize a broad PAMs including NGG, NGA, and NGT, in both embryos and Founder (F0) rabbits. Furthermore, the codon-optimized xCas9-derived base editors, exBE4 and exABE, can dramatically improve the base editing efficiencies in rabbit embryos. Our results demonstrated that the optimized xCas9 with expanded PAM compatibility and enhanced base editing efficiency could be used for precise gene modifications in organisms.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Efeito Fundador , Edição de Genes/métodos , Marcação de Genes/métodos , RNA Guia/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/metabolismo , Códon , Distrofina/genética , Distrofina/metabolismo , Embrião de Mamíferos , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Microinjeções , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , RNA Guia/metabolismo , Coelhos , Repetições de Trinucleotídeos , Zigoto
19.
PLoS One ; 14(4): e0215821, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31013315

RESUMO

Previous studies have shown that proteasome inhibition can have beneficial effects in dystrophic mouse models. In this study, we have investigated the effects of a new selective proteasome inhibitor, CLi, a strong caspase-like inhibitor of the 20S proteasome, on skeletal and cardiac muscle functions of mdx mice. In the first series of experiments, five-month-old male mdx mice (n = 34) were treated with 2 different doses (20 and 100 µg/kg) of CLi and in the second series of experiments, five-month-old female mdx (n = 19) and wild-type (n = 24) mice were treated with 20 µg/kg CLi and Velcade (1 mg/kg) for 1-month. All animals were treadmill exercised twice a week to worsen the dystrophic features. In the first series of experiments, our results demonstrated that 20 µg/kg CLi did not significantly increase absolute and specific maximal forces in skeletal muscle from male mdx mice. Moreover, the higher susceptibility to contraction induced skeletal muscle injury was worsened by 100 µg/kg CLi since the force drop following lengthening contractions was increased with this high dose. Furthermore, we found no differences in the mRNA levels of the molecular markers implicated in dystrophic features. Concerning cardiac function, CLi had no effect on left ventricular function since ejection and shortening fractions were unchanged in male mdx mice. Similarly, CLi did not modify the expression of genes implicated in cardiac remodeling. In the second series of experiments, our results demonstrated an improvement in absolute and specific maximal forces by CLi, whereas Velcade only increased specific maximal force in female mdx mice. In addition, exercise tolerance was not improved by CLi. Taken together, our results show that CLi treatment can only improve maximal force production in exercised female mdx mice without affecting either exercice tolerance capacity or cardiac function. In conclusion, selective inhibition of caspase-like activity of proteasome with CLi has no compelling beneficial effect in dystrophic mdx mice.


Assuntos
Inibidores de Caspase/farmacologia , Contração Muscular/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Piridonas/farmacologia , Animais , Inibidores de Caspase/química , Caspases/genética , Modelos Animais de Doenças , Distrofina/genética , Feminino , Coração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/efeitos dos fármacos , Condicionamento Físico Animal , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/química , Piridonas/química
20.
PLoS One ; 14(4): e0215335, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31017936

RESUMO

Duchenne muscular dystrophy (DMD) is caused by a lack of dystrophin protein. Next to direct effects on the muscles, this has also metabolic consequences. The influence of nutrition on disease progression becomes more and more recognized. Protein intake by DMD patients may be insufficient to meet the increased demand of the constantly regenerating muscle fibers. This led to the hypothesis that improving protein uptake by the muscles could have therapeutic effects. The present study examined the effects of a modified diet, which composition might stimulate muscle growth, on disease pathology in the D2-mdx mouse model. D2-mdx males were fed with either a control diet or modified diet, containing high amounts of branched-chain amino acids, vitamin D3 and ursolic acid, for six weeks. Our study indicates that the modified diet could not ameliorate the muscle pathology. No effects on bodyweight or weight of individual muscles were observed. Neither did the diet affect severity of fibrosis or calcification of the muscles.


Assuntos
Distrofia Muscular de Duchenne/dietoterapia , Distrofia Muscular de Duchenne/patologia , Aminoácidos de Cadeia Ramificada/administração & dosagem , Animais , Calcinose/patologia , Colecalciferol/administração & dosagem , Proteínas na Dieta/metabolismo , Modelos Animais de Doenças , Distrofina/deficiência , Distrofina/genética , Fibrose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/metabolismo , Regeneração , Triterpenos/administração & dosagem
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