Caracterização da passagem da postura de bipedestação para a de sedestação no solo, em crianças portadoras de Distrofia Muscular de Duchenne

Duchenne Muscular Dystrophy (DMD) is a genetically determinated pathology, X-linked recessive A DMD child presents irreversible and progressive muscle weakness, causing difficulties in funcional activities. The objective of this study was described the transference of posture from orthostatism to sitting on the floor in 43 children with DMD, from five to ten years of age (average age of 7,4 years). They were filmed and we observed the transference of posture by videotape and the datas was correlated with the child's age and the time expended in the posture tranference. It was found two characteristic forms to realize this maneuver with five variations to each one. It was notted relationship between the child's age and the form adopted to realize the transference, but it wasn't noted any relation with the the time expended in the posture transference.A Distrofia Muscular de Duchenne (DMD) é uma patologia geneticamente determinada, recessiva, ligada ao cromossomo X. A criança portadora de DMD apresenta fraqueza progressiva e irreversível da musculatura esquelética, prejudicando a realização de atividades motoras. O objetivo deste estudo foi descrever a mudança de postura de bipedestação para sedestação no solo, em 43 crianças com idades entre cinco e dez anos (média de 7,4 anos), portadoras de DMD. As crianças foram filmadas realizando as mudanças de decúbito e posteriormente estudou-se a transferência de postura através da observação dos filmes, evitando assim, a repetição desnecessária das manobras pelas crianças. Correlacionou-se os achados sobre o desempenho nesta tarefa com a idade e o tempo utilizado para a mudança de postura. Posteriormente caracterizou-se os movimentos utilizados na passagem da postura de bipedestação para sedestação. Encontrou-se duas formas características de realização desta manobra, com cinco variações cada. Observou-se relação entre a idade da criança e a forma adotada para realizar a mudança de postura, não havendo relação com o tempo de realização

Correlação entre o tempo de realização de diferentes atividades físicas por portadores de distrofia muscular de Duchenne

DMD is a disease determined by genetic disturbance of recessive character linked to the chromosome X, that determines the deficiency or absence in the distrofin production in the muscular cellular membrane, carting a progressive and irreversible dysfunction, mainly of the skeletal musculature. The functional evaluation of the children carriers of DMD is extensive and exhaustive for child and for physical therapist. The search of information and methods that promote the facilitation or simplification of the routines of functional evaluation has been conducting to the research of correlations among the time of development of functional activities. The goal of this study was to determine and to research possible correlations among the time used to realize physical activities (to go up stairway, to go down stairway, to lift of the chair and to pass from sitting on the floor to standing through the Maneuver of Gowers) in 15 children (6 to 10 years old) with DMD. The children were filmed accomplishing the proposed physical activities, in five serial evaluations during one year, separated by a period of three months. The time for each activity was measured for each child. We confirmed the heterogeneity of the evolution of the disease, in the studied activities, as expected. We found lineal relationship among the functional activities and the age, and the presence of direct lineal correlation between the accomplishment of the Maneuver of Gowers and the functional activities. Up stairway was the activity of larger correlation.A DMD é uma doença determinada por um distúrbio genético de caráter recessivo ligada ao cromossomo X, que determina a deficiência ou ausência na produção de distrofina na membrana celular muscular, acarretando uma disfunção progressiva e irreversível, principalmente da musculatura esquelética. A avaliação funcional das crianças portadoras de DMD é extensa e exaustiva, tanto para criança, quanto para o fisioterapeuta. A procura de informações e métodos que promovam a facilitação ou simplificação das rotinas de avaliação fisioterápica tem conduzido à pesquisa de correlações entre o tempo de desenvolvimento de atividades funcionais. O objetivo desse estudo foi determinar e pesquisar possíveis correlações entre o tempo utilizado na realização de diferentes atividades físicas, a saber, subir escada, descer escada, levantar da cadeira e passar da postura de sedestação no solo para bipedestação (através da Manobra de Gowers) em 15 crianças (de 6 a 10 anos) portadoras de Distrofia Muscular de Duchenne. As crianças foram filmadas realizando as atividades físicas propostas, em cinco avaliações consecutivas durante um ano, separadas por um período de três meses. Posteriormente cronometrou-se o tempo para realização das atividades. Confirmou-se a heterogeneidade da evolução da doença, para as atividades estudadas, como esperado. Encontrou-se relação linear entre as atividades funcionais estudadas e a idade, e a presença de correlação linear direta entre a realização da Manobra de Gowers e as atividades funcionais, sendo o subir escada a atividade de maior correlação

Preclinical Studies with Umbilical Cord Mesenchymal Stromal Cells in Different Animal Models for Muscular Dystrophy

Umbilical cord mesenchymal stromal cells (MSC) have been widely investigated for cell-based therapy studies as an alternative source to bone marrow transplantation. Umbilical cord tissue is a rich source of MSCs with potential to derivate at least muscle, cartilage, fat, and bone cells in vitro. The possibility to replace the defective muscle cells using cell therapy is a promising approach for the treatment of progressive muscular dystrophies (PMDs), independently of the specific gene mutation. Therefore, preclinical studies in different models of muscular dystrophies are of utmost importance. The main objective of the present study is to evaluate if umbilical cord MSCs have the potential to reach and differentiate into muscle cells in vivo in two animal models of PMDs. In order to address this question we injected (1) human umbilical cord tissue (hUCT) MSCs into the caudal vein of SJL mice; (2) hUCT and canine umbilical cord vein (cUCV) MSCs intra-arterially in GRMD dogs. Our results here reported support the safety of the procedure and indicate that the injected cells could engraft in the host muscle in both animal models but could not differentiate into muscle cells. These observations may provide important information aiming future therapy for muscular dystrophies

Silent polymorphisms in the RYR1 gene do not\ud modify the phenotype of the p.4898 I>T\ud pathogenic mutation in central core disease:\ud a case report

Background: Central core disease is a congenital myopathy, characterized by presence of central core-like areas in\ud muscle fibers. Patients have mild or moderate weakness, hypotonia and motor developmental delay. The disease is\ud caused by mutations in the human ryanodine receptor gene (RYR1), which encodes a calcium-release channel.\ud Since the RYR1 gene is huge, containing 106 exons, mutation screening has been limited to three ‘hot spots’, with\ud particular attention to the C-terminal region. Recent next- generation sequencing methods are now identifying\ud multiple numbers of variants in patients, in which interpretation and phenotype prevision is difficult.\ud Case presentation: In a Brazilian Caucasian family, clinical, histopathological and molecular analysis identified a\ud new case of central core disease in a 48-year female. Sanger sequencing of the C-terminal region of the RYR1\ud gene identified two different missense mutations: c.14256 A > C polymorphism in exon 98 and c.14693 T > C in\ud exon 102, which have already been described as pathogenic. Trans-position of the 2 mutations was confirmed\ud because patient’s daughter, mother and sister carried only the exon 98’s mutation, a synonymous variant that was\ud subsequently found in the frequency of 013–0,05 of alleles. Further next generation sequencing study of the whole\ud RYR1 gene in the patient revealed the presence of additional 5 common silent polymorphisms in homozygosis and\ud 8 polymorphisms in heterozygosis.\ud Conclusions: Considering that patient’s relatives showed no pathologic phenotype, and the phenotype presented\ud by the patient is within the range observed in other central core disease patients with the same mutation, it was\ud concluded that the c.14256 A > C polymorphism alone is not responsible for disease, and the associated additional\ud silent polymorphisms are not acting as modifiers of the primary pathogenic mutation in the affected patient. The\ud case described above illustrates the present reality where new methods for wide genome screening are becoming\ud more accessible and able to identify a great variety of mutations and polymorphisms of unknown function in\ud patients and their families.Fundação de Amparo a Pesquisa do Estado de São Paulo - Centro de Pesquisa, Inovação e Difusão (FAPESP-CEPID)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-INCT)Associação Brasileira de Distrofia Muscular (ABDIM)CAPES-COFECU

Telethonin protein expression in neuromuscular disorders

Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb-girdle muscular dystrophy type 2G (LGMD2G). We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, alpha-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. the possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression. (C) 2002 Elsevier Science B.V. All rights reserved.Univ São Paulo, Ctr Study Human Genome, Dept Biol, IBUSP, BR-05508900 São Paulo, BrazilInt Ctr Genet Engn & Biotechnol, Tieste, ItalyUniv Padua, CRIBI Biotechnol Ctr, I-35121 Padua, ItalyHarvard Univ, Childrens Hosp, Sch Med, Div Genet, Boston, MA 02115 USAUniv Helsinki, Helsinki, FinlandUNIFESP, Dept Neurol, São Paulo, BrazilFMUSP, Dept Neurol, São Paulo, BrazilFMUSP, Dept Pathol, São Paulo, BrazilUNIFESP, Dept Neurol, São Paulo, BrazilWeb of Scienc

Reviewing Large LAMA2 Deletions and Duplications in Congenital MuscularDystrophy Patients

Background: Congenital muscular dystrophy (CMD) type 1A (MDC1A) is caused by recessive mutations in laminin-α2 (LAMA2) gene. Laminin-211, a heterotrimeric glycoprotein that contains the α2 chain, is crucial for muscle stability establishing a bond between the sarcolemma and the extracellular matrix. More than 215 mutations are listed in the locus specific database (LSDB) for LAMA2 gene (May 2014). Objective: A limited number of large deletions/duplications have been reported in LAMA2. Our main objective was the identification of additional large rearrangements in LAMA2 found in CMD patients and a systematic review of cases in the literature and LSDB. Methods: In four of the fifty-two patients studied over the last 10 years, only one heterozygous mutation was identified, after sequencing and screening for a frequent LAMA2 deletion. Initial screening of large mutations was performed by multiplex ligation-dependent probe application (MLPA). Further characterization implied several techniques: long-range PCR, cDNA and Southern-blot analysis. Results: Three novel large deletions in LAMA2 and the first pathogenic large duplication were successfully identified, allowing a definitive molecular diagnosis, carrier screening and prenatal diagnosis. A total of fifteen deletions and two duplications previously reported were also reviewed. Two possible mutational “hotspots” for deletions may exist, the first encompassing exons 3 and 4 and second in the 3′ region (exons 56 to 65) of LAMA2. Conclusions: Our findings show that this type of mutation is fairly frequent (18.4% of mutated alleles) and is underestimated in the literature. It is important to include the screening of large deletions/duplications as part of the genetic diagnosis strategy.The authors would like to thank all referring clini-cians.UMIB is funded by National Funds through FCT-Foundation for Science and Technology, under thePest-OE/SAU/U10215/2014.MV is founded by FAPESP-CEPID, and CNPq-INCT

Dmdmdx/Largemyd: a new mouse model of neuromuscular diseases useful for studying physiopathological mechanisms and testing therapies

Although muscular dystrophies are among the most common human genetic disorders, there are few treatment options available. Animal models have become increasingly important for testing new therapies prior to entering human clinical trials. The Dmdmdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD), presenting the same molecular and protein defect as seen in humans with the disease. However, this mouse is not useful for clinical trials because of its very mild phenotype. The mouse model for congenital myodystrophy type 1D, Largemyd, harbors a mutation in the glycosyltransferase Large gene and displays a severe phenotype. To help elucidate the role of the proteins dystrophin and LARGE in the organization of the dystrophin-glycoprotein complex in muscle sarcolemma, we generated double-mutant mice for the dystrophin and LARGE proteins. The new Dmdmdx/Largemyd mouse model is viable and shows a severe phenotype that is associated with the lack of dystrophin in muscle. We tested the usefulness of our new mouse model for cell therapy by systemically injecting them with normal murine mesenchymal adipose stem cells (mASCs). We verified that the mASCs were hosted in the dystrophic muscle. The new mouse model has proven to be very useful for the study of several other therapies, because injected cells can be screened both through DNA and protein analysis. Study of its substantial muscle weakness will also be very informative in the evaluation of functional benefits of these therapies.FAPESP - CEPIDInstituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular (INCTC) - CNPqFINEPABDIMCAPES / COFECU