RESUMO
Duchenne muscular dystrophy (DMD), caused by mutations at the dystrophin gene, is the most common form of muscular dystrophy. There is no cure for DMD and current therapeutic approaches to restore dystrophin expression are only partially effective. The absence of dystrophin in muscle results in dysregulation of signaling pathways, which could be targets for disease therapy and drug discovery. Previously, we identified two exceptional Golden Retriever muscular dystrophy (GRMD) dogs that are mildly affected, have functional muscle, and normal lifespan despite the complete absence of dystrophin. Now, our data on linkage, whole-genome sequencing, and transcriptome analyses of these dogs compared to severely affected GRMD and control animals reveals that increased expression of Jagged1 gene, a known regulator of the Notch signaling pathway, is a hallmark of the mild phenotype. Functional analyses demonstrate that Jagged1 overexpression ameliorates the dystrophic phenotype, suggesting that Jagged1 may represent a target for DMD therapy in a dystrophin-independent manner. PAPERCLIP.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Distrofia Muscular de Duchenne/genética , Animais , Proliferação de Células , Doenças do Cão/genética , Cães , Distrofina/deficiência , Distrofina/genética , Feminino , Estudo de Associação Genômica Ampla , Proteína Jagged-1 , Masculino , Camundongos , Distrofia Muscular Animal/genética , Linhagem , Penetrância , Proteínas Serrate-Jagged , Transcriptoma , Peixe-Zebra , Proteínas de Peixe-ZebraRESUMO
INTRODUCTION/AIMS: Heterogeneous nuclear ribonucleoprotein A1 is involved in nucleic acid homeostatic functions. The encoding gene HNRNPA1 has been associated with several neuromuscular disorders including an amyotrophic lateral sclerosis-like phenotype, distal hereditary motor neuropathy, multisystem proteinopathy, and various myopathies. We report two unrelated individuals with monoallelic stop loss variants affecting the same codon of HNRNPA1. METHODS: Two individuals with unsolved juvenile-onset myopathy were enrolled under approved institutional protocols. Phenotype data were collected and genetic analyses were performed, including whole-exome sequencing (WES). RESULTS: The two probands (MNOT002-01 and K1440-01) showed a similar onset of slowly progressive extremity and facial weakness in early adolescence. K1440-01 presented with facial weakness, winged scapula, elevated serum creatine kinase (CK) levels, and mild neck weakness. MNOT002-01 also exhibited elevated CK levels along with facial weakness, cardiomyopathy, respiratory dysfunction, pectus excavatum, a mildly rigid spine, and loss of ambulation. On quadriceps muscle biopsy, K1440-01 displayed rounded myofibers, mild variation in fiber diameter, and type 2 fiber hypertrophy, while MNOT002-01 displayed rimmed vacuoles. Monoallelic stop-loss variants in HNRNPA1 were identified for both probands: c.1119A>C p.*373Tyrext*6 (K1440-01) and c.1118A>C p.*373Serext*6 (MNOT002-01) affect the same codon and are both predicted to lead to the addition of six amino acids before termination at an alternative stop codon. DISCUSSION: Both stop-loss variants in our probands are likely pathogenic. Our findings contribute to the disease characterization of pathogenic variants in HNRNPA1. This gene should be screened in clinical diagnostic testing of unsolved cases of sporadic or dominant juvenile-onset myopathy.
RESUMO
DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.
Assuntos
Transtorno do Espectro Autista , Distrofias Musculares , Neuropeptídeos , Camundongos , Humanos , Animais , Criança , Distrofina/genética , Distrofina/metabolismo , Transtorno do Espectro Autista/metabolismo , Distrofias Musculares/metabolismo , Distroglicanas/metabolismo , Processamento Alternativo , Músculo Esquelético/patologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Associadas à Distrofina/genética , Proteínas Associadas à Distrofina/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene. Absence of dystrophin protein leads to progressive degradation of skeletal and cardiac function and leads to premature death. Over the years, zebrafish have been increasingly used for studying DMD and are a powerful tool for drug discovery and therapeutic development. In our study, a birefringence screening assay led to identification of phosphodiesterase 10A (PDE10A) inhibitors that reduced the manifestation of dystrophic muscle phenotype in dystrophin-deficient sapje-like zebrafish larvae. PDE10A has been validated as a therapeutic target by pde10a morpholino-mediated reduction in muscle pathology and improvement in locomotion, muscle, and vascular function as well as long-term survival in sapje-like larvae. PDE10A inhibition in zebrafish and DMD patient-derived myoblasts were also associated with reduction of PITPNA expression that has been previously identified as a protective genetic modifier in two exceptional dystrophin-deficient golden retriever muscular dystrophy (GRMD) dogs that escaped the dystrophic phenotype. The combination of a phenotypic assay and relevant functional assessments in the sapje-like zebrafish enhances the potential for the prospective discovery of DMD therapeutics. Indeed, our results suggest a new application for a PDE10A inhibitor as a potential DMD therapeutic to be investigated in a mouse model of DMD.
Assuntos
Distrofina/metabolismo , Distrofia Muscular Animal/prevenção & controle , Distrofia Muscular de Duchenne/prevenção & controle , Mioblastos/efeitos dos fármacos , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Diester Fosfórico Hidrolases/química , Pirazóis/farmacologia , Quinolinas/farmacologia , Animais , Cães , Distrofina/genética , Humanos , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Peixe-ZebraRESUMO
INTRODUCTION: RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. METHODS: Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. RESULTS: Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. DISCUSSION: Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Distrofia Miotônica/genética , Adulto , Animais , Linhagem Celular , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mioblastos/patologia , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Peixe-ZebraRESUMO
Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disease that is caused by the loss of functional dystrophin protein in cardiac and skeletal muscles. DMD patient muscles become weakened, leading to eventual myofiber breakdown and replacement with fibrotic and adipose tissues. Inflammation drives the pathogenic processes through releasing inflammatory cytokines and other factors that promote skeletal muscle degeneration and contributing to the loss of motor function. Selective inhibitors of nuclear export (SINEs) are a class of compounds that function by inhibiting the nuclear export protein exportin 1 (XPO1). The XPO1 protein is an important regulator of key inflammatory and neurological factors that drive inflammation and neurotoxicity in various neurological and neuromuscular diseases. Here, we demonstrate that SINE compound KPT-350 can ameliorate dystrophic-associated pathologies in the muscles of DMD models of zebrafish and mice. Thus, SINE compounds are a promising novel strategy for blocking dystrophic symptoms and could be used in combinatorial treatments for DMD.
Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Carioferinas/antagonistas & inibidores , Distrofia Muscular de Duchenne/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Peixe-Zebra/genética , Administração Oral , Animais , Biomarcadores/sangue , Citocinas/antagonistas & inibidores , Citocinas/sangue , Modelos Animais de Doenças , Locomoção/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos DBA , Camundongos Endogâmicos mdx , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação , Proteínas de Peixe-Zebra/genética , Proteína Exportina 1RESUMO
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-α (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.
Assuntos
Distrofia Muscular de Duchenne/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/fisiologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Cães , Distrofina/genética , Distrofina/metabolismo , Humanos , Células Musculares/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/fisiopatologia , Mutação , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Peixe-Zebra/metabolismoRESUMO
Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency.
Assuntos
Distrofia Muscular do Cíngulo dos Membros/genética , Mutação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Adulto , Animais , Animais Geneticamente Modificados , Respiração Celular/genética , Células Cultivadas , Drosophila , Proteínas de Drosophila/genética , Feminino , Humanos , Masculino , Camundongos , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia , Mioblastos/patologia , Linhagem , Arábia Saudita , SudãoRESUMO
Current molecular genomic approaches to human genetic disorders have led to an explosion in the identification of the genes and their encoded proteins responsible for these disorders. The identification of the gene altered by mutations in Duchenne and Becker muscular dystrophy was one of the earliest examples of this paradigm. The nearly 30 years of research partly outlined here exemplifies the road that similar current gene discovery protocols will be expected to travel, albeit much more rapidly owing to improved diagnosis of genetic disorders and an understanding of the spectrum of mutations thought to cause them. The identification of the protein dystrophin has led to a new understanding of the muscle cell membrane and the proteins involved in membrane stability, as well as new candidate genes for additional forms of muscular dystrophy. Animal models identified with naturally occurring mutations and developed by genetic manipulation have furthered the understanding of disease progression and underlying pathology. The biochemistry and molecular analysis of patient samples have led to the different dystrophin-dependent and -independent therapies that are currently close to or in human clinical trials. The lessons learned from decades of research on dystrophin have benefited the field of human genetics.
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Distrofina/metabolismo , Distrofias Musculares/fisiopatologia , Distrofias Musculares/terapia , Animais , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Distrofina/genética , Terapia Genética/métodos , Humanos , Terapia de Alvo Molecular/métodos , Mutação , Diester Fosfórico Hidrolases/metabolismo , Esteroides/uso terapêutico , Utrofina/genética , Utrofina/metabolismoRESUMO
The current study characterizes a cohort of limb-girdle muscular dystrophy (LGMD) in the United States using whole-exome sequencing. Fifty-five families affected by LGMD were recruited using an institutionally approved protocol. Exome sequencing was performed on probands and selected parental samples. Pathogenic mutations and cosegregation patterns were confirmed by Sanger sequencing. Twenty-two families (40%) had novel and previously reported pathogenic mutations, primarily in LGMD genes, and also in genes for Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital myopathy, myofibrillar myopathy, inclusion body myopathy and Pompe disease. One family was diagnosed via clinical testing. Dominant mutations were identified in COL6A1, COL6A3, FLNC, LMNA, RYR1, SMCHD1 and VCP, recessive mutations in ANO5, CAPN3, GAA, LAMA2, SGCA and SGCG, and X-linked mutations in DMD. A previously reported variant in DMD was confirmed to be benign. Exome sequencing is a powerful diagnostic tool for LGMD. Despite careful phenotypic screening, pathogenic mutations were found in other muscle disease genes, largely accounting for the increased sensitivity of exome sequencing. Our experience suggests that broad sequencing panels are useful for these analyses because of the phenotypic overlap of many neuromuscular conditions. The confirmation of a benign DMD variant illustrates the potential of exome sequencing to help determine pathogenicity.
Assuntos
Exoma/genética , Testes Genéticos/métodos , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Distrofia Muscular do Cíngulo dos Membros/genética , Sequência de Bases , Miopatias Distais/diagnóstico , Miopatias Distais/genética , Feminino , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Doença de Depósito de Glicogênio Tipo II/genética , Humanos , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular Facioescapuloumeral/diagnóstico , Distrofia Muscular Facioescapuloumeral/genética , Mutação/genética , Miopatias Congênitas Estruturais/diagnóstico , Miopatias Congênitas Estruturais/genética , Análise de Sequência de DNA/métodos , Estados UnidosRESUMO
Sapje zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. However, the extent that sapje mimic the muscle dysfunction of higher vertebrate models of DMD is unclear. We used an optical birefringence assay to differentiate affected dystrophic sapje larvae from their unaffected siblings and then studied trunk muscle contractility at 4-7 days postfertilization. Preparation cross-sectional area (CSA) was similar for affected and unaffected larvae, yet tetanic forces of affected preparations were only 30-60% of normal. ANCOVA indicated that the linear relationship observed between tetanic force and CSA for unaffected preparations was absent in the affected population. Consequently, the average force/CSA of affected larvae was depressed 30-70%. Disproportionate reductions in twitch vs. tetanic force, and a slowing of twitch tension development and relaxation, indicated that the myofibrillar disorganization evident in the birefringence assay could not explain the entire force loss. Single eccentric contractions, in which activated preparations were lengthened 5-10%, resulted in tetanic force deficits in both groups of larvae. However, deficits of affected preparations were three- to fivefold greater at all strains and ages, even after accounting for any recovery. Based on these functional assessments, we conclude that the sapje mutant zebrafish is a phenotypically severe model of DMD. The severe contractile deficits of sapje larvae represent novel physiological endpoints for therapeutic drug screening.
Assuntos
Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Peixe-Zebra/fisiologia , Animais , Modelos Animais de Doenças , Cinética , Contração Muscular , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Lenta/patologia , Análise de Regressão , Sarcômeros/metabolismo , Tetania/fisiopatologiaRESUMO
Development of novel therapeutics requires good animal models of disease. Disorders for which good animal models do not exist have very few drugs in development or clinical trial. Even where there are accepted, albeit imperfect models, the leap from promising preclinical drug results to positive clinical trials commonly fails, including in disorders of skeletal muscle. The main alternative model for early drug development, tissue culture, lacks both the architecture and, usually, the metabolic fidelity of the normal tissue in vivo. Herein, we demonstrate the feasibility and validity of human to mouse xenografts as a preclinical model of myopathy. Human skeletal muscle biopsies transplanted into the anterior tibial compartment of the hindlimbs of NOD-Rag1(null) IL2rγ(null) immunodeficient host mice regenerate new vascularized and innervated myofibers from human myogenic precursor cells. The grafts exhibit contractile and calcium release behavior, characteristic of functional muscle tissue. The validity of the human graft as a model of facioscapulohumeral muscular dystrophy is demonstrated in disease biomarker studies, showing that gene expression profiles of xenografts mirror those of the fresh donor biopsies. These findings illustrate the value of a new experimental model of muscle disease, the human muscle xenograft in mice, as a feasible and valid preclinical tool to better investigate the pathogenesis of human genetic myopathies and to more accurately predict their response to novel therapeutics.
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Marcadores Genéticos , Xenoenxertos/fisiologia , Músculo Esquelético/transplante , Distrofia Muscular Facioescapuloumeral/cirurgia , Animais , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/patologiaRESUMO
Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx(5cv) mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Distrofina/genética , Heme Oxigenase-1/biossíntese , Distrofia Muscular de Duchenne/tratamento farmacológico , Animais , GMP Cíclico/biossíntese , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Distrofina/deficiência , Heme Oxigenase-1/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , RNA Mensageiro/genética , Transdução de Sinais/genética , Citrato de Sildenafila , Sulfonas/farmacologia , Regulação para Cima , Peixe-Zebra/genéticaRESUMO
Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetically determined muscle disorders with a primary or predominant involvement of the pelvic or shoulder girdle musculature. More than 20 genes with autosomal recessive (LGMD2A to LGMD2Q) and autosomal dominant inheritance (LGMD1A to LGMD1H) have been mapped/identified to date. Mutations are known for six among the eight mapped autosomal dominant forms: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin-3), LGMD1D (desmin), LGMD1E (DNAJB6), and more recently for LGMD1F (transportin-3). Our group previously mapped the LGMD1G gene at 4q21 in a Caucasian-Brazilian family. We now mapped a Uruguayan family with patients displaying a similar LGMD1G phenotype at the same locus. Whole genome sequencing identified, in both families, mutations in the HNRPDL gene. HNRPDL is a heterogeneous ribonucleoprotein family member, which participates in mRNA biogenesis and metabolism. Functional studies performed in S. cerevisiae showed that the loss of HRP1 (yeast orthologue) had pronounced effects on both protein levels and cell localizations, and yeast proteome revealed dramatic reorganization of proteins involved in RNA-processing pathways. In vivo analysis showed that hnrpdl is important for muscle development in zebrafish, causing a myopathic phenotype when knocked down. The present study presents a novel association between a muscular disorder and a RNA-related gene and reinforces the importance of RNA binding/processing proteins in muscle development and muscle disease. Understanding the role of these proteins in muscle might open new therapeutic approaches for muscular dystrophies.
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Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação , Ribonucleoproteínas/genética , Adulto , Animais , Mapeamento Cromossômico , Feminino , Expressão Gênica , Loci Gênicos , Humanos , Masculino , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Linhagem , Fenótipo , Processamento Pós-Transcricional do RNA , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Peixe-Zebra/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
INTRODUCTION: The genetic causes of limb-girdle muscular dystrophy (LGMD) have been studied in numerous countries, but such investigations have been limited in Egypt. METHODS: A cohort of 30 families with suspected LGMD from Assiut, Egypt, was studied using immunohistochemistry, homozygosity mapping, Sanger sequencing, and whole exome sequencing. RESULTS: Six families were confirmed to have pathogenic mutations, 4 in SGCA and 2 in DMD. Of these, 3 families harbored a single nonsense mutation in SGCA, suggesting that this may be a common mutation in Assiut, Egypt, originating from a founder effect. CONCLUSIONS: The Assiut region in Egypt appears to share at least several of the common LGMD genes found in other parts of the world. It is notable that 4 of the 6 mutations were ascertained by means of whole exome sequencing, even though it was the last approach adopted. This illustrates the power of this technique for identifying causative mutations for muscular dystrophies. Muscle Nerve 54: 690-695, 2016.
Assuntos
Códon sem Sentido/genética , Homozigoto , Distrofia Muscular do Cíngulo dos Membros/epidemiologia , Distrofia Muscular do Cíngulo dos Membros/genética , Sarcoglicanas/genética , Egito/epidemiologia , Feminino , Humanos , Masculino , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , LinhagemRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy characterized by an asymmetric progressive weakness and wasting of the facial, shoulder and upper arm muscles, frequently accompanied by hearing loss and retinal vasculopathy. FSHD is an autosomal dominant disease linked to chromosome 4q35, but the causative gene remains controversial. DUX4 is a leading candidate gene as causative of FSHD. However, DUX4 expression is extremely low in FSHD muscle, and there is no DUX4 animal model that mirrors the pathology in human FSHD. Here, we show that the misexpression of very low levels of human DUX4 in zebrafish development recapitulates the phenotypes seen in human FSHD patients. Microinjection of small amounts of human full-length DUX4 (DUX4-fl) mRNA into fertilized zebrafish eggs caused asymmetric abnormalities such as less pigmentation of the eyes, altered morphology of ears, developmental abnormality of fin muscle, disorganization of facial musculature and/or degeneration of trunk muscle later in development. Moreover, DUX4-fl expression caused aberrant localization of myogenic cells marked with α-actin promoter-driven enhanced green fluorescent protein outside somite boundary, especially in head region. These abnormalities were rescued by coinjection of the short form of DUX4 (DUX4-s). Our results suggest that the misexpression of DUX4-fl, even at extremely low level, can recapitulate the phenotype observed in FSHD patients in a vertebrate model. These results strongly support the current hypothesis for a role of DUX4 in FSHD pathogenesis. We also propose that DUX4 expression during development is important for the pathogenesis of FSHD.
Assuntos
Proteínas de Homeodomínio/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Peixe-Zebra/genética , Actinas/genética , Actinas/metabolismo , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Microscopia Eletrônica de Transmissão , Músculo Esquelético/anormalidades , Distrofia Muscular Facioescapuloumeral/patologia , Óvulo/crescimento & desenvolvimento , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ombro/anormalidadesRESUMO
Skeletal muscle possesses a strong ability to regenerate following injury, a fact that has been largely attributed to satellite cells. Satellite cells are skeletal muscle stem cells located beneath the basal lamina of the myofiber, and are the principal cellular source of growth and regeneration in skeletal muscle. MicroRNAs (miRNAs) play key roles in modulating several cellular processes by targeting multiple mRNAs that comprise a single or multiple signaling pathway. Several miRNAs have been shown to regulate satellite cell activity, such as miRNA-489, which functions to maintain satellite cells in a quiescent state. Although muscle-specific miRNAs have been identified, many of the molecular mechanisms that regulate myogenesis that are regulated by miRNAs still remain unknown. In this study, we have shown that miR-128a is highly expressed in brain and skeletal muscle, and increases during myoblast differentiation. MiR-128a was found to regulate the target genes involved in insulin signaling, which include Insr (insulin receptor), Irs1 (insulin receptor substrate 1) and Pik3r1 (phosphatidylinositol 3-kinases regulatory 1) at both the mRNA and protein level. Overexpression of miR-128a in myoblasts inhibited cell proliferation by targeting IRS1. By contrast, inhibition of miR-128a induced myotube maturation and myofiber hypertrophy in vitro and in vivo. Moreover, our results demonstrate that miR-128a expression levels are negatively controlled by tumor necrosis factor α (TNF-α). TNF-α promoted myoblast proliferation and myotube hypertrophy by facilitating IRS1/Akt signaling via a direct decrease of miR-128a expression in both myoblasts and myotubes. In summary, we demonstrate that miR-128a regulates myoblast proliferation and myotube hypertrophy, and provides a novel mechanism through which IRS1-dependent insulin signaling is regulated in skeletal muscle.
Assuntos
Proteínas Substratos do Receptor de Insulina/metabolismo , Insulina/metabolismo , MicroRNAs/metabolismo , Desenvolvimento Muscular/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Diferenciação Celular/genética , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Feminino , Hipertrofia/genética , Hipertrofia/metabolismo , Hipertrofia/patologia , Insulina/genética , Proteínas Substratos do Receptor de Insulina/genética , Camundongos , MicroRNAs/genética , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Mioblastos/metabolismo , Mioblastos/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Previously, we identified family with sequence similarity 65, member B (Fam65b), as a protein transiently up-regulated during differentiation and fusion of human myogenic cells. Silencing of Fam65b expression results in severe reduction of myogenin expression and consequent lack of myoblast fusion. The molecular function of Fam65b and whether misregulation of its expression could be causative of muscle diseases are unknown. Protein pulldowns were used to identify Fam65b-interacting proteins in differentiating human muscle cells and regenerating muscle tissue. In vitro, human muscle cells were treated with histone-deacetylase (HDAC) inhibitors, and expression of Fam65b and interacting proteins was studied. Nontreated cells were used as controls. In vivo, expression of Fam65b was down-regulated in developing zebrafish to determine the effects on muscle development. Fam65b binds to HDAC6 and dysferlin, the protein mutated in limb girdle muscular dystrophy 2B. The tricomplex Fam65b-HDAC6-dysferlin is transient, and Fam65b expression is necessary for the complex to form. Treatment of myogenic cells with pan-HDAC or HDAC6-specific inhibitors alters Fam65b expression, while dysferlin expression does not change. Inhibition of Fam65b expression in developing zebrafish results in abnormal muscle, with low birefringence, tears at the myosepta, and increased embryo lethality. Fam65b is an essential component of the HDAC6-dysferlin complex. Down-regulation of Fam65b in developing muscle causes changes consistent with muscle disease.-Balasubramanian, A., Kawahara, G., Gupta, V. A., Rozkalne, A., Beauvais, A., Kunkel, L. M., Gussoni, E. Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.
Assuntos
Diferenciação Celular/genética , Histona Desacetilases/metabolismo , Proteínas de Membrana/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteínas Musculares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular , Células Cultivadas , Regulação para Baixo/genética , Disferlina , Desacetilase 6 de Histona , Histona Desacetilases/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Ligação Proteica/genética , Alinhamento de Sequência , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Peixe-ZebraRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder caused by contractions of repetitive elements within the macrosatellite D4Z4 on chromosome 4q35. The pathophysiology of FSHD is unknown and, as a result, there is currently no effective treatment available for this disease. To better understand the pathophysiology of FSHD and develop mRNA-based biomarkers of affected muscles, we compared global analysis of gene expression in two distinct muscles obtained from a large number of FSHD subjects and their unaffected first-degree relatives. Gene expression in two muscle types was analyzed using GeneChip Gene 1.0 ST arrays: biceps, which typically shows an early and severe disease involvement; and deltoid, which is relatively uninvolved. For both muscle types, the expression differences were mild: using relaxed cutoffs for differential expression (fold change ≥1.2; nominal P value <0.01), we identified 191 and 110 genes differentially expressed between affected and control samples of biceps and deltoid muscle tissues, respectively, with 29 genes in common. Controlling for a false-discovery rate of <0.25 reduced the number of differentially expressed genes in biceps to 188 and in deltoid to 7. Expression levels of 15 genes altered in this study were used as a "molecular signature" in a validation study of an additional 26 subjects and predicted them as FSHD or control with 90% accuracy based on biceps and 80% accuracy based on deltoids.