RESUMO
Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a dramatic neuromuscular disease in which crippling muscle weakness is evident from birth. Here, we use the dyW mouse model for human MDC1A to trace the onset of the disease during development in utero. We find that myotomal and primary myogenesis proceed normally in homozygous dyW-/- embryos. Fetal dyW-/- muscles display the same number of myofibers as wildtype (WT) muscles, but by E18.5 dyW-/- muscles are significantly smaller and muscle size is not recovered post-natally. These results suggest that fetal dyW-/- myofibers fail to grow at the same rate as WT myofibers. Consistent with this hypothesis between E17.5 and E18.5 dyW-/- muscles display a dramatic drop in the number of Pax7- and myogenin-positive cells relative to WT muscles, suggesting that dyW-/- muscles fail to generate enough muscle cells to sustain fetal myofiber growth. Gene expression analysis of dyW-/- E17.5 muscles identified a significant increase in the expression of the JAK-STAT target gene Pim1 and muscles from 2-day and 3-week old dyW-/- mice demonstrate a dramatic increase in pSTAT3 relative to WT muscles. Interestingly, myotubes lacking integrin α7ß1, a laminin-receptor, also show a significant increase in pSTAT3 levels compared with WT myotubes, indicating that α7ß1 can act as a negative regulator of STAT3 activity. Our data reveal for the first time that dyW-/- mice exhibit a myogenesis defect already in utero. We propose that overactivation of JAK-STAT signaling is part of the mechanism underlying disease onset and progression in dyW-/- mice.
Assuntos
Desenvolvimento Muscular/fisiologia , Distrofias Musculares/metabolismo , Animais , Modelos Animais de Doenças , Janus Quinase 1/metabolismo , Laminina/metabolismo , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Distrofia Muscular Animal/embriologia , Distrofia Muscular Animal/metabolismo , Miogenina/metabolismo , Fator de Transcrição PAX7/metabolismo , Receptores de Laminina , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Lamina-associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that has been implicated in striated muscle maintenance. Mutations in its gene have been linked to muscular dystrophy and cardiomyopathy. As germline deletion of the gene encoding LAP1 is perinatal lethal, we explored its potential role in myogenic differentiation and development by generating a conditional knockout mouse in which the protein is depleted from muscle progenitors at embryonic day 8.5 (Myf5-Lap1CKO mice). Although cultured myoblasts lacking LAP1 demonstrated defective terminal differentiation and altered expression of muscle regulatory factors, embryonic myogenesis and formation of skeletal muscle occurred in both mice with a Lap1 germline deletion and Myf5-Lap1CKO mice. However, skeletal muscle fibres were hypotrophic and their nuclei were morphologically abnormal with a wider perinuclear space than normal myonuclei. Myf5-Lap1CKO mouse skeletal muscle contained fewer satellite cells than normal and these cells had evidence of reduced myogenic potential. Abnormalities in signalling pathways required for postnatal hypertrophic growth were also observed in skeletal muscles of these mice. Our results demonstrate that early embryonic depletion of LAP1 does not impair myogenesis but that it is necessary for postnatal skeletal muscle growth.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Distrofias Musculares/embriologia , Mioblastos/citologia , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Fatores de Regulação MiogênicaRESUMO
Myofibrillar myopathies are a group of muscle disorders characterized by the disintegration of skeletal muscle fibers and formation of sarcomeric protein aggregates. All the proteins known to be involved in myofibrillar myopathies localize to a region of the sarcomere known as the Z-disk, the site at which defects are first observed. Given the common cellular phenotype observed in this group of disorders, it is thought that there is a common mechanism of pathology. Mutations in filamin C, which has several proposed roles in the development and function of skeletal muscle, can result in filamin-related myofibrillar myopathy. The lack of a suitable animal model system has limited investigation into the mechanism of pathology in this disease and the role of filamin C in muscle development. Here, we characterize stretched out (sot), a zebrafish filamin Cb mutant, together with targeted knockdown of zebrafish filamin Ca, revealing fiber dissolution and formation of protein aggregates strikingly similar to those seen in filamin-related myofibrillar myopathies. Through knockdown of both zebrafish filamin C homologues, we demonstrate that filamin C is not required for fiber specification and that fiber damage is a consequence of muscle activity. The remarkable similarities in the myopathology between our models and filamin-related myofibrillar myopathy makes them suitable for the study of these diseases and provides unique opportunities for the investigation of the function of filamin C in muscle and development of therapies.
Assuntos
Modelos Animais de Doenças , Proteínas dos Microfilamentos/genética , Distrofias Musculares/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Padronização Corporal/genética , Códon sem Sentido , Análise Mutacional de DNA , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Filaminas , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ligação Genética , Humanos , Funções Verossimilhança , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Contração Muscular , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/patologia , Distrofias Musculares/embriologia , Mutagênese , Miosinas/metabolismo , Fenótipo , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
1. Evidence is accumulating for a role for Ca²âº signalling in the differentiation and development of embryonic skeletal muscle. 2. Imaging of intact, normally developing transgenic zebrafish that express the protein component of the Ca²âº-sensitive complex aequorin, specifically in skeletal muscle, show that two distinct periods of spontaneous synchronised Ca²âº transients occur in the trunk: one at approximately 17.5-19.5 h post-fertilization (h.p.f.; termed signalling period SP1) and the other after approximately 23 h.p.f. (termed SP2). These periods of intense Ca²âº signalling activity are separated by a quiet period. 3. Higher-resolution confocal imaging of embryos loaded with the fluorescent Ca²âº reporter calcium green-1 dextran shows that the Ca²âº signals are generated almost exclusively in the slow muscle cells, the first muscle cells to differentiate, with distinct nuclear and cytoplasmic components. 4. Here, we show that coincidental with the SP1 Ca²âº signals, dystrophin becomes localized to the vertical myoseptae of the myotome. Introduction of a dmd morpholino (dmd-MO) resulted in no dystrophin being expressed in the vertical myoseptae, as well as a disruption of myotome morphology and sarcomere organization. In addition, the Ca²âº signalling signatures of dmd-MO-injected embryos or homozygous sapje mutant embryos were abnormal such that the frequency, amplitude and timing of the Ca²âº signals were altered compared with controls. 5. Our new data suggest that, in addition to a structural role, dystrophin may function in the regulation of [Ca²âº](i) during the early stages of slow muscle cell differentiation when the Ca²âº signals generated in these cells coincide with the first spontaneous contractions of the trunk.
Assuntos
Sinalização do Cálcio , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Desenvolvimento Muscular , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/patologia , Distrofias Musculares/metabolismo , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Citoplasma/efeitos dos fármacos , Citoplasma/patologia , Desenvolvimento Embrionário/efeitos dos fármacos , Medições Luminescentes/métodos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Microscopia de Fluorescência/métodos , Morfolinos/farmacologia , Contração Muscular/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofias Musculares/embriologia , Distrofias Musculares/patologia , Mutação , Especificidade de Órgãos , Transporte Proteico/efeitos dos fármacos , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Sarcômeros/patologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Allelic mutations in putative glycosyltransferase genes, fukutin and fukutin-related protein (fkrp), lead to a wide range of muscular dystrophies associated with hypoglycosylation of α-dystroglycan, commonly referred to as dystroglycanopathies. Defective glycosylation affecting dystroglycan-ligand interactions is considered to underlie the disease pathogenesis. We have modelled dystroglycanopathies in zebrafish using a novel loss-of-function dystroglycan allele and by inhibition of Fukutin family protein activities. We show that muscle pathology in embryos lacking Fukutin or FKRP is different from loss of dystroglycan. In addition to hypoglycosylated α-dystroglycan, knockdown of Fukutin or FKRP leads to a notochord defect and a perturbation of laminin expression before muscle degeneration. These are a consequence of endoplasmic reticulum stress and activation of the unfolded protein response (UPR), preceding loss of dystroglycan-ligand interactions. Together, our results suggest that Fukutin family proteins may play important roles in protein secretion and that the UPR may contribute to the phenotypic spectrum of some dystroglycanopathies in humans.
Assuntos
Modelos Animais de Doenças , Glicosiltransferases/metabolismo , Distrofias Musculares/metabolismo , Resposta a Proteínas não Dobradas , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Feminino , Técnicas de Silenciamento de Genes , Glicosilação , Glicosiltransferases/genética , Humanos , Masculino , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Transporte Proteico , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Gene delivery to the fetal muscles is a potential strategy for the early treatment of muscular dystrophies. In utero muscle gene therapy can also be used to treat other genetic disorders such as hemophilia, where the missing clotting proteins may be secreted from the treated muscle. In the past few years, studies in small animal models have raised the hopes that a phenotypic cure can be obtained after fetal application of gene therapy. Studies of efficacy and safety in large animals are, however, essential before clinical application can be considered in the human fetus. For this reason, the development of clinically applicable strategies for the delivery of gene therapy to the fetal muscles is of prime importance. In this chapter, we describe the protocols for in utero ultrasound-guided gene delivery to the ovine fetal muscle in early gestation. In particular, procedures to inject skeletal muscle groups such as the thigh and thoracic musculature and targeting the diaphragm in the fetus are described in detail.
Assuntos
Terapias Fetais/métodos , Doenças Genéticas Inatas/terapia , Terapia Genética/métodos , Músculo Esquelético/embriologia , Distrofias Musculares/terapia , Animais , Modelos Animais de Doenças , Feminino , Feto , Técnicas de Transferência de Genes , Doenças Genéticas Inatas/embriologia , Doenças Genéticas Inatas/genética , Hemofilia A/embriologia , Hemofilia A/genética , Hemofilia A/terapia , Humanos , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Gravidez , Ovinos , Ultrassonografia DopplerRESUMO
Type II lissencephaly (type II LIS) is a group of autosomal recessive congenital muscular dystrophies (CMD) associated with defects in alpha-DG O-glycosylation, which comprises Walker-Warburg syndrome, Fukuyama cerebral and muscular dystrophy, or muscle-eye-brain disease. The most severe forms of these diseases often have a fetal presentation and lead to a pregnancy termination. We report here the first molecular study on fetal type II LIS in a series of 47 fetuses from 41 unrelated families. Sequencing of the different genes known to be involved in alpha-DG O-glycosylation allowed the molecular diagnosis in 22 families: involvement of POMT1 was demonstrated in 32% of cases, whereas POMGNT1 and POMT2 were incriminated in 15% and in 7% of cases, respectively. We found 30 different mutations in these three genes, 25 were described herein for the first time, 15 in POMT1, and five in POMT2 and POMGNT1. Despite sequencing of FKRP, FCMD, and LARGE, no definitive molecular diagnosis could be made for the other half of our cases. Preliminary results concerning genotype-phenotype correlations show that the choice of the first gene sequenced should depend on the clinical severity of the type II LIS; POMT1 and POMT2 for severest clinical picture and POMGNT1 for milder disease. The other genes, FKRP, FCMD, and LARGE, seem not to be implicated in the fetal form of CMD.
Assuntos
Regulação da Expressão Gênica , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Alelos , Distroglicanas/metabolismo , Feminino , Genótipo , Idade Gestacional , Humanos , Masculino , Manosiltransferases/genética , Repetições de Microssatélites , Modelos Genéticos , Mutação , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Neuronal overmigration is the underlying cellular mechanism of cerebral cortical malformations in syndromes of congenital muscular dystrophies caused by defects in O-mannosyl glycosylation. Overmigration involves multiple developmental abnormalities in the brain surface basement membrane, Cajal-Retzius cells, and radial glia. We tested the hypothesis that breaches in basement membrane and the underlying glia limitans are the key initial events of the cellular pathomechanisms by carrying out a detailed developmental study with a mouse model of muscle-eye-brain disease, mice deficient in O-mannose beta31,2-N-acetylglucosaminyltransferase 1 (POMGnT1). The pial basement membrane was normal in the knockout mouse at E11.5. It was breached during rapid cerebral cortical expansion at E13.5. Radial glial endfeet, which comprise glia limitans, grew out of the neural boundary. Neurons moved out of the neural boundary through these breaches. The overgrown radial glia and emigrated neurons disrupted the overlying pia mater. The overmigrated neurons did not participate in cortical plate (CP) development; rather they formed a diffuse cell zone (DCZ) outside the original cortical boundary. Together, the DCZ and the CP formed the knockout cerebral cortex, with disappearance of the basement membrane and the glia limitans. These results suggest that disappearance of the basement membrane and the glia limitans at the cerebral cortical surface during development underlies cortical lamination defects in congenital muscular dystrophies and a cellular mechanism of cortical malformation distinct from that of the reeler mouse, double cortex syndrome, and periventricular heterotopia.
Assuntos
Córtex Cerebral/embriologia , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Mutação , N-Acetilglucosaminiltransferases/genética , Neuroglia/patologia , Pia-Máter/embriologia , Animais , Membrana Basal/ultraestrutura , Bromodesoxiuridina , Movimento Celular , Córtex Cerebral/patologia , Modelos Animais de Doenças , Desenvolvimento Embrionário , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Crescimento , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Neurônios/patologia , Pia-Máter/patologiaRESUMO
The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.
Assuntos
Distrofias Musculares/genética , Membrana Nuclear/metabolismo , Membrana Nuclear/patologia , Proteínas Nucleares/biossíntese , Proteínas Nucleares/deficiência , Animais , Fibroblastos/patologia , Marcação de Genes , Triagem de Portadores Genéticos , Homozigoto , Humanos , Imuno-Histoquímica , Laminas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distrofias Musculares/embriologia , Distrofias Musculares/patologia , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Deleção de Sequência , TransfecçãoAssuntos
Testes Genéticos , Distrofias Musculares/diagnóstico , Diagnóstico Pré-Natal , Universidades , Desenvolvimento Embrionário , Revisão Ética , Comitês de Ética em Pesquisa , Ética Médica , Feminino , Humanos , Japão , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Defesa do Paciente , GravidezRESUMO
alpha5-deficient mice die early in embryogenesis (). To study the functions of alpha5 integrin later in mouse embryogenesis and during adult life we generated alpha5 -/-;+/+ chimeric mice. These animals contain alpha5-negative and positive cells randomly distributed. Analysis of the chimerism by glucose- 6-phosphate isomerase (GPI) assay revealed that alpha5 -/- cells contributed to all the tissues analyzed. High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was lower than for the controls, however the subsequent life span of the survivors was only slightly reduced compared with controls (). Histological analysis of alpha5 -/-;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle degeneration-regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an increase in the number of mitochondria in some muscle fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant muscle fibers. All the alterations were already visible at late stages of embryogenesis. The number of altered muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric animals. Differentiation of alpha5 -/- ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was impaired. Our data suggest that a novel form of muscle dystrophy in mice is alpha5-integrin-dependent.
Assuntos
Antígenos CD/fisiologia , Distrofias Musculares/etiologia , Animais , Antígenos CD/genética , Linhagem Celular , Quimera , Feminino , Integrina alfa5 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/embriologia , Músculo Esquelético/patologia , Distrofias Musculares/embriologiaRESUMO
The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of muscle disorders with onset in utero or during the first year of life. Several forms of CMD show various types of brain involvement in addition to a muscular dystrophy. Two forms are defined at the molecular level: merosin deficient-CMD caused by mutations in the LAMA2-gene on chromosome 6q2. Fukuyama congenital muscular dystrophy (FCMD) is prevalent in Japan and caused by an as yet unidentified gene on chromosome 9q31. At least two further forms of CMD with brain involvement are nosologically well defined: Walker--Warburg-CMD is characterized by lissencephaly type 11, eye dysgenesis and muscular dystrophy. This autosomal recessive disorder is fatal or results in complete lack of development. A similar but much milder phenotype with pachygyria of the brain, various degrees of eye changes and milder muscular dystrophy that is compatible with achievement of simple motor milestones has been described under the name of muscle-eye-brain disease (MEB) in Finland. A number of nosologically less distinct forms of muscular dystrophy have been outlined such as 'pure' CMD without brain involvement, CMD with cerebellar hypoplasia or CMD type Ullrich with hyperelasticity of the distal joints. Several other CMD phenotypes are known, some of which are suggestive of more distinctly separate nosological entities due to their occurrence in siblings or due to a characteristic pattern of clinical, histopathological and imaging features, and await further clarification.
Assuntos
Distrofias Musculares/classificação , Criança , Mapeamento Cromossômico , Cromossomos Humanos Par 9 , Feminino , Genes Recessivos , Humanos , Recém-Nascido , Deficiência Intelectual/classificação , Laminina/deficiência , Masculino , Distrofias Musculares/congênito , Distrofias Musculares/embriologia , SíndromeRESUMO
After studies which have mapped the gamma-sarcoglycan deficient limb-girdle muscular dystrophy (LGMD2C) to chromosome 13q12 and recent identification of mutations within this gene, prenatal diagnosis has become possible. The deletion of exon 5 in the gamma-sarcoglycan gene was found in a consanguineous family and prenatal diagnosis was successfully provided. This is the first prenatal diagnosis of LGMD2C.
Assuntos
Aberrações Cromossômicas/diagnóstico , Proteínas do Citoesqueleto/genética , Distrofina/genética , Glicoproteínas de Membrana/genética , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Diagnóstico Pré-Natal , Adulto , Criança , Aberrações Cromossômicas/genética , Transtornos Cromossômicos , Cromossomos Humanos Par 13/genética , Consanguinidade , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/deficiência , Análise Mutacional de DNA , Distrofina/análise , Distrofina/deficiência , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/deficiência , Músculo Esquelético/química , Músculo Esquelético/patologia , Distrofias Musculares/embriologia , Linhagem , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SarcoglicanasRESUMO
Complete or partial deficiency of the laminin alpha2 chain of merosin has been demonstrated in a proportion of children with classical congenital muscular dystrophy and linkage to the laminin alpha2 chain gene (LAMA2) on chromosome 6q2 has been established. As the laminin alpha2 chain is also expressed in the trophoblast, its detection and linkage analysis are useful tools for prenatal diagnosis. We report our experience of seven prenatal diagnoses in families with partial deficiency or total absence of the laminin alpha2 chain in the muscle of the propositi. In five instances, expression of the laminin alpha2 chain in the trophoblast was normal and linkage data suggested that the fetuses were unaffected. In one family, the immunocytochemical studies of the trophoblast showed the absence of laminin alpha2, suggesting that the fetus was affected. Linkage analysis confirmed that the fetus had inherited the two at-risk haplotypes. In one family with partial laminin alpha2 chain deficiency, the haplotype analysis was hampered by maternal DNA contamination. Immunocytochemical analysis of chorionic villus sampling showed a reduction in laminin alpha2 expression. The pregnancy was presumed to be at high-risk and terminated. However, subsequent analysis of fetal DNA indicated that the fetus was probably heterozygous. Our data suggest that immunocytochemical analysis of the trophoblast can detect abnormalities in affected fetuses and gives normal results in unaffected and carrier fetuses. Nevertheless, we recommend that linkage analysis to the LAMA2 locus is also studied in all cases.
Assuntos
Laminina/deficiência , Laminina/genética , Distrofias Musculares/diagnóstico , Diagnóstico Pré-Natal , Feminino , Ligação Genética , Humanos , Imuno-Histoquímica , Masculino , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Linhagem , Gravidez , TrofoblastosRESUMO
A branch of a highly inbred family was referred for prenatal counseling with an initial misdiagnosis of Becker Muscular Dystrophy (BMD) due to the limited clinical and laboratory data obtained in pre-dystrophin era and hidden family information. In a second branch of the family with a diagnosis of limb-girdle muscular dystrophy type 2A (LGMD2A) molecular studies revealed a homozygous 550 delta A mutation in the calcium-activated neutral protease 3 (calpain 3, CANP3) gene in the affected members. Finally, in the third branch of the family, it turned out that both parents were heterozygous for the 550 delta A mutation and the 13-week-old fetus was homozygous. The same mutation subsequently also was found in the first branch of the family. The parents were informed that the risk of their child of developing the disease would be very high given that he was carrying the same homozygous mutation of the other affected members. They were informed also that in another population (in Reunion Island) the same disease does not necessarily follow such a simple pattern of inheritance. After counseling the parents decided to terminate the pregnancy.
Assuntos
Calpaína/genética , Inibidores de Cisteína Proteinase/genética , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Adulto , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Consanguinidade , DNA/sangue , Éxons , Feminino , Feto , Aconselhamento Genético , Heterozigoto , Homozigoto , Humanos , Masculino , Distrofias Musculares/embriologia , Gravidez , Diagnóstico Pré-NatalRESUMO
We developed a method that allows prenatal diagnosis of Duchenne muscular dystrophy using a single nucleated erythrocyte (NRBC) isolated from maternal blood. Maternal blood was obtained at 8 to 20 weeks of gestation. NRBCs were separated with Percoll using a discontinuous density gradient method and then collected by micromanipulator under microscopic observation. The entire genome of a single cell was amplified by primer extension preamplification (PEP). Sex was determined from a small aliquot of the PEP reaction. After an NRBC was determined to be male and confirmed to be of fetal origin, dystrophin exons 4, 8, 12, 45, 48, 50, and 51 were determined from the same PEP reaction. This diagnostic method using maternal blood is safer than amniocentesis or cordocentesis and can be applied to other X-linked diseases.
Assuntos
Distrofina/genética , Eritrócitos/patologia , Deleção de Genes , Distrofias Musculares/diagnóstico , Diagnóstico Pré-Natal/métodos , Sequência de Bases , Núcleo Celular/patologia , Separação Celular , Centrifugação Zonal , Primers do DNA , Distrofina/sangue , Embrião de Mamíferos , Feminino , Feto , Idade Gestacional , Humanos , Masculino , Dados de Sequência Molecular , Distrofias Musculares/sangue , Distrofias Musculares/embriologia , Reação em Cadeia da Polimerase/métodos , Gravidez , Análise para Determinação do Sexo , Cromossomo X , Cromossomo YRESUMO
A light and electron microscopic study of the brain of an 18-week fetus with a prenatal genetic diagnosis of Fukuyama-type congenital muscular dystrophy revealed a widespread mantle of abnormal neurogliomesenchymal tissue that covered a dysplastic cerebral cortex. In this area alone, the glia limitans that adjoined the abnormal mantle via one or two layers of basal lamina had frequent breaches, through which neuroglial elements extruded. In the most severely affected cortical region, which had only a rudimentary and fragmentary glia limitans, the majority of cortical neurons had migrated into the neurogliomesenchymal tissue. The massive overmigrated neurons still maintained a somewhat columnar arrangement, and the marked dysplasia abruptly shifted to a neurogliomesenchymal tissue-free normal cortical structure with an intact glia limitans, thus indicating essentially vertical overmigration of neurons without significant tangential migration of them. Together the above findings imply that breaches in the glia limitans may be the primary cause of the micropolygyria seen in this genetic disorder.
Assuntos
Córtex Cerebral/patologia , Distrofias Musculares/congênito , Distrofias Musculares/patologia , Neuroglia/patologia , Córtex Cerebral/embriologia , Córtex Cerebral/ultraestrutura , Feto , Humanos , Distrofias Musculares/embriologia , Neuroglia/ultraestruturaRESUMO
In order to develop and validate methods for the preimplantation diagnosis of Duchenne muscular dystrophy (DMD), we have established and evaluated PCR assays for the analysis of four loci within the DMD gene and for two Y chromosome sequences in single cells. A model system using buccal cells picked from mouthwash samples has been used for an extensive evaluation of the sensitivity and specificity of the assays, and each assay has been tested in samples containing single cells, two cells, and three cells per tube. The four DMD and two Y assays have been combined in duplex and triplex reactions to enable simultaneous diagnosis of DMD and of fetal sex. One of the DMD markers is a highly polymorphic simple tandem repeat locus which produces a basic DNA profile, and provides a control for contamination by foreign DNA. Amplification of DMD or Y sequences was observed in 78 to 92% of single male cells, rising to 96% and 97% in tubes containing two or three male cells respectively. Coamplification of both a DMD and a Y sequence together occurred with a mean success of 74% in single male cells, increasing to 93% with two, and 95% with three cells per tube. With appropriate precautions, we believe that it is now possible to proceed to clinical application of these procedures.
Assuntos
Blastocisto , Doenças Fetais/diagnóstico , Marcadores Genéticos , Distrofias Musculares/diagnóstico , Reação em Cadeia da Polimerase , Diagnóstico Pré-Natal/métodos , Sequência de Bases , Epitélio/ultraestrutura , Feminino , Doenças Fetais/genética , Humanos , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Mucosa Bucal/citologia , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Distrofias Musculares/prevenção & controle , Polimorfismo Genético , Valor Preditivo dos Testes , Sequências Repetitivas de Ácido Nucleico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Cromossomo YRESUMO
The roles of innervation, muscle electrical activity, and muscle contraction in regulating the formation and survival of primary and secondary myotubes during embryonic and fetal development of skeletal muscle were studied using the mouse mutants peroneal muscular atrophy (pma) and muscular dysgenesis (mdg). The pma phenotype includes the absence of the peroneal division of the sciatic nerve, so muscles in the anterior compartment of the lower hindlimb are aneural throughout development. Muscles in mdg mice are paralyzed due to the absence of excitation-contraction coupling and hyperinnervated due to suppression of motoneuron death in consequence of their paralysis, but otherwise are electrically excitable and receive synaptic transmission. In a quantitative comparison between control and mutant extensor digitorum longus (EDL) muscles at E15, primary myotube numbers were depressed by 20-30% in both mutants and in paralyzed or denervated muscles from control strain animals. The number of secondary myotubes, however, was normal in pma mutants and two and a half times greater than normal in the hyperinnervated mdg EDL muscles, so that the ratio of secondary to primary myotubes was increased by 300% in the mutant with respect to heterozygous or -/- littermates. Chronic paralysis with tetrodotoxin (TTX) caused no further depression of primary myotube numbers in aneural pma muscles, but secondary myotube numbers were reduced by 40%, reducing the ratio of secondary to primary myotubes by 35%. We conclude that during normal development the generation of secondary myotubes depends on neurally evoked electrical activity in primary myotubes, which stimulates mitosis of secondary myoblasts. The effect of TTX shows that aneural pma primary myotubes discharge spontaneous myogenic action potentials, while mdg muscles may receive greater than normal electrical activation due to their hyperinnervation, explaining the presence and numbers of secondary myotubes in the mutant mouse muscles.