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1.
Nat Protoc ; 15(2): 421-449, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932771

RESUMO

This protocol describes the design, fabrication and use of a 3D physiological and pathophysiological motor unit model consisting of motor neurons coupled to skeletal muscles interacting via the neuromuscular junction (NMJ) within a microfluidic device. This model facilitates imaging and quantitative functional assessment. The 'NMJ chip' enables real-time, live imaging of axonal outgrowth, NMJ formation and muscle maturation, as well as synchronization of motor neuron activity and muscle contraction under optogenetic control for the study of normal physiological events. The proposed protocol takes ~2-3 months to be implemented. Pathological behaviors associated with various neuromuscular diseases, such as regression of motor neuron axons, motor neuron death, and muscle degradation and atrophy can also be recapitulated in this system. Disease models can be created by the use of patient-derived induced pluripotent stem cells to generate both the motor neurons and skeletal muscle cells used. This is demonstrated by the use of cells from a patient with sporadic amyotrophic lateral sclerosis but can be applied more generally to models of neuromuscular disease, such as spinal muscular atrophy, NMJ disorder and muscular dystrophy. Models such as this hold considerable potential for applications in precision medicine, drug screening and disease risk assessment.


Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Procedimentos Analíticos em Microchip/métodos , Doenças Neuromusculares/tratamento farmacológico , Medicina de Precisão/instrumentação , Humanos , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Neuromusculares/patologia , Doenças Neuromusculares/fisiopatologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Medição de Risco
3.
Muscle Nerve ; 60(6): 687-692, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31478199

RESUMO

INTRODUCTION: Nerve imaging has a limited role in axonal and muscle fiber loss. In this study, we sought to explore the utility of standardized muscle ultrasound (US) assessment in these clinical scenarios. METHODS: We performed a prospective study from March to August 2018 of patients attending the neuromuscular clinic. All patients underwent clinical evaluation and standardized muscle thickness measurement by US in seven muscles. RESULTS: The study cohort consisted of 114 participants, including patients with polyneuropathy, motor neuron disease, and myopathy. The smallest distal muscle thickness was found in patients with polyneuropathy, while the smallest proximal muscle thickness was found in patients with myopathy. Muscle thickness was strongly correlated with muscle strength (r 2 = 0.62), electrophysiological findings (r 2 : 0.44-0.55), and disability score (r 2 = 0.53). DISCUSSION: Standardized muscle thickness measured by US shows diagnostic usefulness in a spectrum of neuromuscular disorders and correlates with clinical and electrophysiological findings.


Assuntos
Músculo Esquelético/diagnóstico por imagem , Doenças Neuromusculares/diagnóstico por imagem , Potenciais de Ação/fisiologia , Adulto , Idoso , Esclerose Amiotrófica Lateral/diagnóstico por imagem , Esclerose Amiotrófica Lateral/patologia , Esclerose Amiotrófica Lateral/fisiopatologia , Eletromiografia , Feminino , Humanos , Masculino , Sintomas Inexplicáveis , Pessoa de Meia-Idade , Força Muscular , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular Espinal/diagnóstico por imagem , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Doenças Musculares/diagnóstico por imagem , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Condução Nervosa/fisiologia , Doenças Neuromusculares/patologia , Doenças Neuromusculares/fisiopatologia , Tamanho do Órgão , Polineuropatias/diagnóstico por imagem , Polineuropatias/patologia , Polineuropatias/fisiopatologia , Estudos Prospectivos , Radiculopatia/diagnóstico por imagem , Radiculopatia/patologia , Radiculopatia/fisiopatologia , Ultrassonografia
4.
Elife ; 82019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31364990

RESUMO

Cytoplasmic dynein plays critical roles within the developing and mature nervous systems, including effecting nuclear migration, and retrograde transport of various cargos. Unsurprisingly, mutations in dynein are causative of various developmental neuropathies and motor neuron diseases. These 'dyneinopathies' define a broad spectrum of diseases with no known correlation between mutation identity and disease state. To circumvent complications associated with dynein studies in human cells, we employed budding yeast as a screening platform to characterize the motility properties of seventeen disease-correlated dynein mutants. Using this system, we determined the molecular basis for several classes of etiologically related diseases. Moreover, by engineering compensatory mutations, we alleviated the mutant phenotypes in two of these cases, one of which we confirmed with recombinant human dynein. In addition to revealing molecular insight into dynein regulation, our data provide additional evidence that the type of disease may in fact be dictated by the degree of dynein dysfunction.


Assuntos
Dineínas/genética , Dineínas/metabolismo , Regulação da Expressão Gênica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Doenças Neuromusculares/patologia , Doenças Neuromusculares/fisiopatologia , Humanos , Modelos Teóricos , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supressão Genética
5.
Nutrients ; 11(8)2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31412596

RESUMO

Autophagy is the major intracellular machinery for degrading proteins, lipids, polysaccharides, and organelles. This cellular process is essential for the maintenance of the correct cellular balance in both physiological and stress conditions. Because of its role in maintaining cellular homeostasis, dysregulation of autophagy leads to various disease manifestations, such as inflammation, metabolic alterations, aging, and neurodegeneration. A common feature of many neurologic and neuromuscular diseases is the alteration of the autophagy-lysosomal pathways. For this reason, autophagy is considered a target for the prevention and/or cure of these diseases. Dietary intake of polyphenols has been demonstrated to prevent/ameliorate several of these diseases. Thus, natural products that can modulate the autophagy machinery are considered a promising therapeutic strategy. In particular, curcumin, a phenolic compound widely used as a dietary supplement, exerts an important effect in modulating autophagy. Herein, we report on the current knowledge concerning the role of curcumin in modulating the autophagy machinery in various neurological and neuromuscular diseases as well as its role in restoring the autophagy molecular mechanism in several cell types that have different effects on the progression of neurological and neuromuscular disorders.


Assuntos
Autofagia/efeitos dos fármacos , Curcumina/uso terapêutico , Suplementos Nutricionais , Músculo Esquelético/efeitos dos fármacos , Doenças do Sistema Nervoso/tratamento farmacológico , Sistema Nervoso/efeitos dos fármacos , Doenças Neuromusculares/tratamento farmacológico , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Curcumina/efeitos adversos , Suplementos Nutricionais/efeitos adversos , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Sistema Nervoso/metabolismo , Sistema Nervoso/patologia , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/patologia , Transdução de Sinais
6.
J Surg Res ; 243: 27-32, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31151034

RESUMO

BACKGROUND: An operative biopsy is an important component in the diagnosis and treatment of neuromuscular disorders (NMDs). However, recent advances in molecular genetics suggest less invasive genetic testing should be the initial approach. The purpose of our study was to demonstrate the diagnostic value of muscle or nerve biopsy within the pediatric population at a pediatric academic center and offer recommendations for genetic testing in relation to biopsy to achieve the highest diagnostic yield. METHODS: Following institutional review board approval, we retrospectively reviewed the electronic medical record of 221 pediatric patients who underwent muscle and/or nerve biopsy for suspicion of NMD from January 2007 to March 2018. Demographics, family history, clinical presentations, genetic testing results, pathology results, anesthesia complications, clinical diagnoses, and clinic follow-up data were collected. Chi-square analysis was done for statistical significance. RESULTS: A total of 220 underwent muscle biopsy, and 15 underwent nerve biopsy. Not all patients received genetic testing. The average age at biopsy was 7.7 y. Biopsy revealed significant histologic abnormalities in 62.9% (139), directly leading to a specific clinical diagnosis in 33.9% (75). When genetic testing was done before biopsy, definite pathogenic variants were found in 7.6% (9). When genetic testing was done after biopsy, definite pathogenic variants were found in 45.0% (27). Genetic testing yield for pathogenic variants was higher when done after biopsy (P value < 0.00001). CONCLUSIONS: Muscle and nerve biopsies may provide significant diagnostic value. Biopsy helped to rule in or out NMD and guide genetic testing. Our data suggest NMD genetic testing yield was higher when done after biopsy.


Assuntos
Testes Genéticos , Músculo Esquelético/patologia , Doenças Neuromusculares/diagnóstico , Nervos Periféricos/patologia , Biópsia , Criança , Feminino , Seguimentos , Humanos , Masculino , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Estudos Retrospectivos
8.
Am J Hum Genet ; 104(5): 847-860, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31051113

RESUMO

Collagen type IV alpha 1 and alpha 2 chains form heterotrimers ([α1(IV)]2α2(IV)) that represent a fundamental basement membrane constituent. Dominant COL4A1 and COL4A2 mutations cause a multisystem disorder that is marked by clinical heterogeneity and variable expressivity and that is generally characterized by the presence of cerebrovascular disease with ocular, renal, and muscular involvement. Despite the fact that muscle pathology is reported in up to one-third of individuals with COL4A1 and COL4A2 mutations and in animal models with mutations in COL4A1 and COL4A2 orthologs, the pathophysiological mechanisms underlying COL4A1-related myopathy are unknown. In general, mutations are thought to impair [α1(IV)]2α2(IV) secretion. Whether pathogenesis results from intracellular retention, extracellular deficiency, or the presence of mutant proteins in basement membranes represents an important gap in knowledge and a major obstacle for developing targeted interventions. We report that Col4a1 mutant mice develop progressive neuromuscular pathology that models human disease. We demonstrate that independent muscular, neural, and vascular insults contribute to neuromyopathy and that there is mechanistic heterogeneity among tissues. Importantly, we provide evidence of a COL4A1 functional subdomain with disproportionate significance for tissue-specific pathology and demonstrate that a potential therapeutic strategy aimed at promoting [α1(IV)]2α2(IV) secretion can ameliorate or exacerbate myopathy in a mutation-dependent manner. These data have important translational implications for prediction of clinical outcomes based on genotype, development of mechanism-based interventions, and genetic stratification for clinical trials. Collectively, our data underscore the importance of the [α1(IV)]2α2(IV) network as a multifunctional signaling platform and show that allelic and tissue-specific mechanistic heterogeneities contribute to the variable expressivity of COL4A1 and COL4A2 mutations.


Assuntos
Colágeno Tipo IV/genética , Doenças Musculares/etiologia , Mutação , Doenças Neuromusculares/etiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Musculares/patologia , Doenças Neuromusculares/patologia , Especificidade de Órgãos , Fenótipo
9.
J Hum Genet ; 64(6): 551-559, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30867548

RESUMO

Genetic diagnoses are becoming a routine in the medical practice of neuromuscular diseases. Many diagnoses, however, can have an influence on relatives and family members and thus must be handled carefully by genetic counseling (GC). Here, we aimed to assess the purpose of undergoing GC to verify the utility of collaborations between clinical and genetic divisions. We investigated consecutive GC cases of neuromuscular disease and examined the role of GC. Our study included 102 cases who underwent GC in our hospital from July 2005 to March 2018: 86.3% were women and 45.1% were in their 30's. Disease explanation was the most common reason for attending GC (29.4%), followed by prenatal diagnosis (25.5%), pre-symptomatic diagnosis (17.6%), and carrier diagnosis (14.7%). Clients typically visited the hospital for GC when some kind of life event occurred, such as marriage, had a desire to bear a child, or a change in the condition of the proband. Clinicians should be conscious of such life events from the perspective of both the client and their relatives, and guide the GC at an appropriate time. Overall, the degree of recognition of genetic risk by clients differed; thus, it is important for GC to determine the status of each unique situation and respond individually.


Assuntos
Família , Aconselhamento Genético , Doenças Neuromusculares/diagnóstico , Adulto , Feminino , Triagem de Portadores Genéticos , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Linhagem , Fatores de Risco , Adulto Jovem
10.
Theranostics ; 9(5): 1232-1246, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30867827

RESUMO

The study of human neuromuscular diseases has traditionally been performed in animal models, due to the difficulty of performing studies in human subjects. Despite the unquestioned value of animal models, inter-species differences hamper the translation of these findings to clinical trials. Tissue-engineered models of the neuromuscular junction (NMJ) allow for the recapitulation of the human physiology in tightly controlled in vitro settings. Methods: Here we report the first human patient-specific tissue-engineered model of the neuromuscular junction (NMJ) that combines stem cell technology with tissue engineering, optogenetics, microfabrication and image processing. The combination of custom-made hardware and software allows for repeated, quantitative measurements of NMJ function in a user-independent manner. Results: We demonstrate the utility of this model for basic and translational research by characterizing in real time the functional changes during physiological and pathological processes. Principal Conclusions: This system holds great potential for the study of neuromuscular diseases and drug screening, allowing for the extraction of quantitative functional data from a human, patient-specific system.


Assuntos
Modelos Teóricos , Doenças Neuromusculares/patologia , Doenças Neuromusculares/fisiopatologia , Optogenética/métodos , Engenharia Tecidual/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiologia , Junção Neuromuscular/fisiopatologia
12.
PLoS One ; 14(2): e0212198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794581

RESUMO

INTRODUCTION: The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (cas) is a new technology that allows easier manipulation of the genome. Its potential to edit genes opened a new door in treatment development for incurable neurological monogenic diseases (NMGDs). The aim of this systematic review was to summarise the findings on the current development of CRISPR-cas for therapeutic purposes in the most frequent NMGDs and provide critical assessment. METHODS AND DATA ACQUISITION: We searched the MEDLINE and EMBASE databases, looking for original studies on the use of CRISPR-cas to edit pathogenic variants in models of the most frequent NMGDs, until end of 2017. We included all the studies that met the following criteria: 1. Peer-reviewed study report with explicitly described experimental designs; 2. In vitro, ex vivo, or in vivo study using human or other animal biological systems (including cells, tissues, organs, organisms); 3. focusing on CRISPR as the gene-editing method of choice; and 5. featured at least one NMGD. RESULTS: We obtained 404 papers from MEDLINE and 513 from EMBASE. After removing the duplicates, we screened 490 papers by title and abstract and assessed them for eligibility. After reading 50 full-text papers, we finally selected 42 for the review. DISCUSSION: Here we give a systematic summary on the preclinical development of CRISPR-cas for therapeutic purposes in NMGDs. Furthermore, we address the clinical interpretability of the findings, giving a comprehensive overview of the current state of the art. Duchenne's muscular dystrophy (DMD) paves the way forward, with 26 out of 42 studies reporting different strategies on DMD gene editing in different models of the disease. Most of the strategies aimed for permanent exon skipping by deletion with CRISPR-cas. Successful silencing of the mHTT gene with CRISPR-cas led to successful reversal of the neurotoxic effects in the striatum of mouse models of Huntington's disease. Many other strategies have been explored, including epigenetic regulation of gene expression, in cellular and animal models of: myotonic dystrophy, Fraxile X syndrome, ataxias, and other less frequent dystrophies. Still, before even considering the clinical application of CRISPR-cas, three major bottlenecks need to be addressed: efficacy, safety, and delivery of the systems. This requires a collaborative approach in the research community, while having ethical considerations in mind.


Assuntos
Sistemas CRISPR-Cas , Expansão das Repetições de DNA , Edição de Genes/métodos , Técnicas de Transferência de Genes , Doenças Genéticas Inatas , Doenças Neuromusculares , Animais , Modelos Animais de Doenças , Edição de Genes/tendências , Inativação Gênica , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Doenças Genéticas Inatas/terapia , Humanos , MEDLINE , Camundongos , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Doenças Neuromusculares/terapia
13.
Sci Rep ; 9(1): 23, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631115

RESUMO

We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.


Assuntos
Testes Diagnósticos de Rotina/métodos , Doenças do Sistema Nervoso/diagnóstico , Doenças Neuromusculares/diagnóstico , Expansão das Repetições de Trinucleotídeos , Humanos , Dispositivos Lab-On-A-Chip , Doenças do Sistema Nervoso/patologia , Doenças Neuromusculares/patologia , Sensibilidade e Especificidade
14.
Eur J Paediatr Neurol ; 23(2): 254-261, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30626539

RESUMO

AIMS: To define the neurological and neuropathological alterations caused by SYNE1 mutations. METHODS: We describe 5 patients (3 males, 2 females; age 3-24 years) from 3 families. The diagnostic work-up included three muscle biopsies and two nerve biopsies in three of the cases. RESULTS: Three different phenotypes were discerned. Two patients showed progressive ataxia, mental retardation, neuropathy and radially deviated thumbs (spinocerebellar ataxia, SCAR, type 8 phenotype). Two patients had mild congenital myopathy with restrictive lung disease, clubfeet and thumb anomalies (myopathic arthrogryposis). One patient had congenital myopathy with dilated cardiomyopathy and adducted thumbs (Emery-Dreifuss Muscular Dystrophy, EDMD, type 4). Light microscopy of the three muscle biopsies revealed chronic non-necrotizing myopathy without rimmed vacuoles in all cases combined with neurogenic atrophy in one case. The two nerve biopsies showed predominantly axonal neuropathy with demyelinating features. Nuclear alterations, most notably lobulation and focal widening of the space between inner and outer leaflet of the nuclear envelope, were a prominent consistent feature of myonuclei and Schwann cell nuclei in each of the three muscle specimens and one nerve specimen that could be examined by electron microscopy. CONCLUSION: Thumb abnormalities and nuclear envelope alterations are characteristic for SYNE 1 mutations. Schwann cell nuclei are affected, indicating that such nuclear envelope changes in glial cells contribute to the neurodegenerative phenotype in human nesprinopathies.


Assuntos
Proteínas do Tecido Nervoso/genética , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/genética , Polegar/anormalidades , Adolescente , Criança , Pré-Escolar , Proteínas do Citoesqueleto , Feminino , Humanos , Masculino , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Mutação , Membrana Nuclear/patologia , Fenótipo , Células de Schwann/patologia , Células de Schwann/ultraestrutura , Adulto Jovem
15.
J Lipid Res ; 60(2): 312-317, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30287524

RESUMO

Phosphoinositide-specific phospholipases C (PI-PLCs) are involved in signaling pathways related to critical cellular functions, such as cell cycle regulation, cell differentiation, and gene expression. Nuclear PI-PLCs have been studied as key enzymes, molecular targets, and clinical prognostic/diagnostic factors in many physiopathologic processes. Here, we summarize the main studies about nuclear PI-PLCs, specifically, the imbalance of isozymes such as PI-PLCß1 and PI-PLCζ, in cerebral, hematologic, neuromuscular, and fertility disorders. PI-PLCß1 and PI-PLCÉ£1 affect epilepsy, depression, and bipolar disorder. In the brain, PI-PLCß1 is involved in endocannabinoid neuronal excitability and is a potentially novel signature gene for subtypes of high-grade glioma. An altered quality or quantity of PI-PLCζ contributes to sperm defects that result in infertility, and PI-PLCß1 aberrant inositide signaling contributes to both hematologic and degenerative muscle diseases. Understanding the mechanisms behind PI-PLC involvement in human pathologies may help identify new strategies for personalized therapies of these conditions.


Assuntos
Encefalopatias/enzimologia , Núcleo Celular/enzimologia , Doenças Hematológicas/enzimologia , Infertilidade/enzimologia , Doenças Neuromusculares/enzimologia , Fosfolipases Tipo C/metabolismo , Animais , Encefalopatias/patologia , Doenças Hematológicas/patologia , Humanos , Infertilidade/patologia , Isoenzimas/metabolismo , Doenças Neuromusculares/patologia
16.
Clin Dysmorphol ; 28(1): 17-21, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30303820

RESUMO

Glycogen storage disease IV (GSD IV), caused by a defect in GBE1, is a clinically heterogeneous disorder. A classical hepatic form and a neuromuscular form have been described. The severe neuromuscular form presents as a fetal akinesia deformation sequence or a congenital subtype. We ascertained three unrelated families with fetuses/neonates who presented with fetal akinesia deformation sequence to our clinic for genetic counseling. We performed a detailed clinical evaluation, exome sequencing, and histopathology examination of two fetuses and two neonates from three unrelated families presenting with these perinatally lethal neuromuscular forms of GSD IV. Exome sequencing in the affected fetuses/neonates identified four novel pathogenic variants (c.1459G>T, c.144-1G>A, c.1680C>G, and c.1843G>C) in GBE1 (NM_000158). Histopathology examination of tissues from the affected fetuses/neonate was consistent with the diagnosis. Here, we add three more families with the severe perinatally lethal neuromuscular forms of GSD IV to the GBE1 mutation spectrum.


Assuntos
Artrogripose/enzimologia , Artrogripose/genética , Sistema da Enzima Desramificadora do Glicogênio/genética , Doença de Depósito de Glicogênio Tipo IV/enzimologia , Doença de Depósito de Glicogênio Tipo IV/genética , Mutação/genética , Doenças Neuromusculares/enzimologia , Doenças Neuromusculares/genética , Artrogripose/patologia , Sequência de Bases , Feminino , Feto/patologia , Doença de Depósito de Glicogênio Tipo IV/patologia , Humanos , Recém-Nascido , Masculino , Doenças Neuromusculares/patologia , Linhagem
17.
J Vis Exp ; (138)2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-30148479

RESUMO

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.


Assuntos
Regulação da Expressão Gênica/genética , Terapia Genética/métodos , MicroRNAs/genética , Doenças Neuromusculares/genética , Doenças Neuromusculares/terapia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Doenças Neuromusculares/patologia , Roedores , Sorogrupo
18.
Curr Opin Gastroenterol ; 34(5): 295-300, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30067562

RESUMO

PURPOSE OF REVIEW: Accurate diagnosis of gastrointestinal neuromuscular diseases requires full thickness biopsy for adequate histologic evaluation of the enteric neuromuscular and ancillary cells. Historically, this has been achieved by surgical approaches. An overview of procedure evolution and current techniques of endoscopic full thickness biopsy (EFTB) for diagnosis of gastrointestinal neuromuscular disorders will be presented. RECENT FINDINGS: Emergence and advancement of endoscopic full thickness resection techniques has offered a less invasive, nevertheless an effective modality of tissue acquirement. Recently, clip-assisted close-then-cut EFTB has been utilized in clinical practice under research protocol. Early experience has shown that this technique provides an adequate full-thickness specimen including the myenteric plexus and ganglia cells, with acceptable safety profiles. SUMMARY: EFTB is a promising means in diagnosing the nature of the disease and guiding therapy. Available animal and human studies have shown the noninferiority of endoscopic methods to surgical ones in term of adequacy of tissue samples, while potentially decreasing the risk and occurrence of complications. Further large prospective studies are needed to assess its efficacy, safety and impacts on patient's outcomes.


Assuntos
Biópsia/métodos , Endoscopia Gastrointestinal/métodos , Gastroenteropatias/patologia , Intestinos/patologia , Plexo Mientérico/patologia , Doenças Neuromusculares/patologia , Humanos , Intestinos/inervação
19.
J Neurol ; 265(11): 2506-2524, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30155738

RESUMO

Variants in the skeletal muscle ryanodine receptor 1 gene (RYR1) result in a spectrum of RYR1-related disorders. Presentation during infancy is typical and ranges from delayed motor milestones and proximal muscle weakness to severe respiratory impairment and ophthalmoplegia. We aimed to elucidate correlations between genotype, protein structure and clinical phenotype in this rare disease population. Genetic and clinical data from 47 affected individuals were analyzed and variants mapped to the cryo-EM RyR1 structure. Comparisons of clinical severity, motor and respiratory function and symptomatology were made according to the mode of inheritance and affected RyR1 structural domain(s). Overall, 49 RYR1 variants were identified in 47 cases (dominant/de novo, n = 35; recessive, n = 12). Three variants were previously unreported. In recessive cases, facial weakness, neonatal hypotonia, ophthalmoplegia/paresis, ptosis, and scapular winging were more frequently observed than in dominant/de novo cases (all, p < 0.05). Both dominant/de novo and recessive cases exhibited core myopathy histopathology. Clinically severe cases were typically recessive or had variants localized to the RyR1 cytosolic shell domain. Motor deficits were most apparent in the MFM-32 standing and transfers dimension, [median (IQR) 85.4 (18.8)% of maximum score] and recessive cases exhibited significantly greater overall motor function impairment compared to dominant/de novo cases [79.7 (18.8)% vs. 87.5 (17.7)% of maximum score, p = 0.03]. Variant mapping revealed patterns of clinical severity across RyR1 domains, including a structural plane of interest within the RyR1 cytosolic shell, in which 84% of variants affected the bridging solenoid. We have corroborated genotype-phenotype correlations and identified RyR1 regions that may be especially sensitive to structural modification.


Assuntos
Doenças Neuromusculares/genética , Doenças Neuromusculares/fisiopatologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Acetilcisteína/uso terapêutico , Adolescente , Adulto , Estudos Transversais , Método Duplo-Cego , Feminino , Estudos de Associação Genética , Variação Genética , Humanos , Masculino , Fármacos Neuromusculares/uso terapêutico , Doenças Neuromusculares/tratamento farmacológico , Doenças Neuromusculares/patologia , Estudos Prospectivos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Relação Estrutura-Atividade , Adulto Jovem
20.
BMC Med Genet ; 19(1): 95, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29879922

RESUMO

BACKGROUND: We investigated a South African family of admixed ancestry in which the first generation (G1) developed insidious progressive distal to proximal weakness in their twenties, while their offspring (G2) experienced severe unexpected symptoms of myalgia and cramps since adolescence. Our aim was to identify deleterious mutations that segregate with the affected individuals in this family. METHODS: Exome sequencing was performed on five cases, which included three affected G1 siblings and two pauci-symptomatic G2 offspring. As controls we included an unaffected G1 sibling and a spouse of one of the G1 affected individuals. Homozygous or potentially compound heterozygous variants that were predicted to be functional and segregated with the affected G1 siblings, were further evaluated. Additionally, we considered variants in all genes segregating exclusively with the affected (G1) and pauci-symptomatic (G2) individuals to address the possibility of a pseudo-autosomal dominant inheritance pattern in this family. RESULTS: All affected G1 individuals were homozygous for a novel truncating p.Tyr1433Ter DYSF (dysferlin) mutation, with their asymptomatic sibling and both pauci-symptomatic G2 offspring carrying only a single mutant allele. Sanger sequencing confirmed segregation of the variant. No additional potentially contributing variant was found in the DYSF or any other relevant gene in the pauci-symptomatic carriers. CONCLUSION: Our finding of a truncating dysferlin mutation confirmed dysferlinopathy in this family and we propose that the single mutant allele is the primary contributor to the neuromuscular symptoms seen in the second-generation pauci-symptomatic carriers.


Assuntos
Disferlina/genética , Exoma/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Mutação , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Adolescente , Adulto , Feminino , Seguimentos , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Prognóstico , Irmãos , Sequenciamento Completo do Exoma , Adulto Jovem
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