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Sample records for chromosome-linked dystrophin abnormalities

  1. Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy.

    PubMed Central

    Beggs, A H; Neumann, P E; Arahata, K; Arikawa, E; Nonaka, I; Anderson, M S; Kunkel, L M

    1992-01-01

    Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, we propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1. Images PMID:1731332

  2. Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy

    SciTech Connect

    Beggs, A.H.; Neumann, P.E.; Anderson, M.S.; Kunkel, L.M. ); Arahata, Kiichi; Arikawa, Eri; Nonaka, Ikuya )

    1992-01-15

    Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3,500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, the authors propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1.

  3. Abnormalities of dystrophin, the sarcoglycans, and laminin alpha2 in the muscular dystrophies.

    PubMed Central

    Jones, K J; Kim, S S; North, K N

    1998-01-01

    Abnormalities of dystrophin, the sarcoglycans, and laminin alpha2 are responsible for a subset of the muscular dystrophies. In this study we aim to characterise the nature and frequency of abnormalities of these proteins in an Australian population and to formulate an investigative algorithm to aid in approaching the diagnosis of the muscular dystrophies. To reduce ascertainment bias, biopsies with dystrophic (n=131) and non-dystrophic myopathic (n=71) changes were studied with antibodies to dystrophin, alpha, beta, and gamma sarcoglycan, beta dystroglycan, and laminin alpha2, and results were correlated with clinical phenotype. Abnormalities of dystrophin, the sarcoglycans, or laminin alpha2 were present in 61/131 (47%) dystrophic biopsies and in 0/71 myopathic biopsies, suggesting that immunocytochemical study of dystrophin, the sarcoglycans, and laminin alpha2 may, in general, be restricted to patients with dystrophic biopsies. Two patients with mutations identified in gamma sarcoglycan had abnormal dystrophin (by immunocytochemistry and immunoblot), showing that abnormalities of dystrophin may be a secondary phenomenon. Therefore, biopsies should not be excluded from sarcoglycan analysis on the basis of abnormal dystrophin alone. The diagnostic yield was highest in those with severe, rapidly progressive limb-girdle weakness (92%). Laminin alpha2 deficiency was identified in 5/131 (4%) patients; 215 patients presented after infancy, indicating that abnormalities of laminin alpha2 are not limited to the congenital muscular dystrophy phenotype. Overall patterns of immunocytochemistry and immunoblotting provided a guide to mutation analysis and, on the basis of this study, we have formulated a diagnostic algorithm to guide the investigation of patients with muscular dystrophy. Images PMID:9610800

  4. Drastic reduction of sarcalumenin in Dp427 (dystrophin of 427 kDa)-deficient fibres indicates that abnormal calcium handling plays a key role in muscular dystrophy.

    PubMed Central

    Dowling, Paul; Doran, Philip; Ohlendieck, Kay

    2004-01-01

    Although the primary abnormality in dystrophin is the underlying cause for mdx (X-chromosome-linked muscular dystrophy), abnormal Ca2+ handling after sarcolemmal microrupturing appears to be the pathophysiological mechanism leading to muscle weakness. To develop novel pharmacological strategies for eliminating Ca2+-dependent proteolysis, it is crucial to determine the fate of Ca2+-handling proteins in dystrophin-deficient fibres. In the present study, we show that a key luminal Ca2+-binding protein SAR (sarcalumenin) is affected in mdx skeletal-muscle fibres. One- and two-dimensional immunoblot analyses revealed the relative expression of the 160 kDa SR (sarcoplasmic reticulum) protein to be approx. 70% lower in mdx fibres when compared with normal skeletal muscles. This drastic reduction in SAR was confirmed by immunofluorescence microscopy. Patchy internal labelling of SAR in dystrophic fibres suggests an abnormal formation of SAR domains. Differential co-immunoprecipitation experiments and chemical cross-linking demonstrated a tight linkage between SAR and the SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1) isoform of the SR Ca2+-ATPase. However, the relative expression of the fast Ca2+ pump was not decreased in dystrophic membrane preparations. This implies that the reduction in SAR and calsequestrin-like proteins plays a central role in the previously reported impairment of Ca2+ buffering in the dystrophic SR [Culligan, Banville, Dowling and Ohlendieck (2002) J. Appl. Physiol. 92, 435-445]. Impaired Ca2+ shuttling between the Ca2+-uptake SERCA units and calsequestrin clusters via SAR, as well as an overall decreased luminal ion-binding capacity, might indirectly amplify the Ca2+-leak-channel-induced increase in cytosolic Ca2+ levels. This confirms the idea that abnormal Ca2+ cycling is involved in Ca2+-induced myonecrosis. Hence, manipulating disturbed Ca2+ handling might represent new modes of abolishing proteolytic degradation in muscular dystrophy

  5. Dystrophin quantification

    PubMed Central

    Anthony, Karen; Arechavala-Gomeza, Virginia; Taylor, Laura E.; Vulin, Adeline; Kaminoh, Yuuki; Torelli, Silvia; Feng, Lucy; Janghra, Narinder; Bonne, Gisèle; Beuvin, Maud; Barresi, Rita; Henderson, Matt; Laval, Steven; Lourbakos, Afrodite; Campion, Giles; Straub, Volker; Voit, Thomas; Sewry, Caroline A.; Morgan, Jennifer E.; Flanigan, Kevin M.

    2014-01-01

    Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval. PMID:25355828

  6. Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities.

    PubMed

    Galbiati, F; Engelman, J A; Volonte, D; Zhang, X L; Minetti, C; Li, M; Hou, H; Kneitz, B; Edelmann, W; Lisanti, M P

    2001-06-15

    Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an approximately 95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 -/-) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level. PMID:11259414

  7. A quantitative ELISA for dystrophin.

    PubMed

    Morris, G E; Ellis, J M; Nguyen, T M

    1993-05-01

    A novel approach to the quantitation of the muscular dystrophy protein, dystrophin, in muscle extracts is described. The two-site ELISA uses two monoclonal antibodies against dystrophin epitopes which lie close together in the rod domain of the dystrophin molecule in order to minimize the effects of dystrophin degradation. Dystrophin is assayed in its native form by extracting with non-ionic detergents and avoiding the use of SDS. PMID:8486926

  8. Dystrophin: The dead calm of a dogma.

    PubMed

    Górecki, Dariusz C

    2016-01-01

    Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease leading to severe disability and death of young men. Current interventions are palliative as no treatment improves the long-term outcome. Therefore, new therapeutic modalities with translational potential are urgently needed and abnormalities downstream from the absence of dystrophin are realistic targets. It has been shown that DMD mutations alter extracellular ATP (eATP) signaling via P2RX7 purinoceptor upregulation, which leads to autophagic death of dystrophic muscle cells. Furthermore, the eATP-P2RX7 axis contributes to DMD pathology by stimulating harmful inflammatory responses. We demonstrated recently that genetic ablation or pharmacological inhibition of P2RX7 in the mdx mouse model of DMD produced functional attenuation of both muscle and non-muscle symptoms, establishing this receptor as an attractive therapeutic target. Central to the argument presented here, this purinergic phenotype affects dystrophic myoblasts. Muscle cells were believed not to be affected at this stage of differentiation, as they do not produce detectable dystrophin protein. Our findings contradict the central hypothesis stating that aberrant dystrophin expression is inconsequential in myoblasts and the DMD pathology results from effects such as sarcolemma fragility, due to the absence of dystrophin, in differentiated myofibres. However, we discuss here the evidence that, already in myogenic cells, DMD mutations produce a plethora of abnormalities, including in cell proliferation, differentiation, energy metabolism, Ca(2+) homeostasis and death, leading to impaired muscle regeneration. We hope that this discussion may bring to light further results that will help re-evaluating the established belief. Clearly, understanding how DMD mutations alter such a range of functions in myogenic cells is vital for developing effective therapies. PMID:27141413

  9. Dystrophin: The dead calm of a dogma

    PubMed Central

    Górecki, Dariusz C.

    2016-01-01

    ABSTRACT Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease leading to severe disability and death of young men. Current interventions are palliative as no treatment improves the long-term outcome. Therefore, new therapeutic modalities with translational potential are urgently needed and abnormalities downstream from the absence of dystrophin are realistic targets. It has been shown that DMD mutations alter extracellular ATP (eATP) signaling via P2RX7 purinoceptor upregulation, which leads to autophagic death of dystrophic muscle cells. Furthermore, the eATP-P2RX7 axis contributes to DMD pathology by stimulating harmful inflammatory responses. We demonstrated recently that genetic ablation or pharmacological inhibition of P2RX7 in the mdx mouse model of DMD produced functional attenuation of both muscle and non-muscle symptoms, establishing this receptor as an attractive therapeutic target. Central to the argument presented here, this purinergic phenotype affects dystrophic myoblasts. Muscle cells were believed not to be affected at this stage of differentiation, as they do not produce detectable dystrophin protein. Our findings contradict the central hypothesis stating that aberrant dystrophin expression is inconsequential in myoblasts and the DMD pathology results from effects such as sarcolemma fragility, due to the absence of dystrophin, in differentiated myofibres. However, we discuss here the evidence that, already in myogenic cells, DMD mutations produce a plethora of abnormalities, including in cell proliferation, differentiation, energy metabolism, Ca2+ homeostasis and death, leading to impaired muscle regeneration. We hope that this discussion may bring to light further results that will help re-evaluating the established belief. Clearly, understanding how DMD mutations alter such a range of functions in myogenic cells is vital for developing effective therapies. PMID:27141413

  10. Identification and characterization of a novel retinal isoform of dystrophin

    SciTech Connect

    D`Souza, V.N.; Sigesmund, D.A.; Man, N.

    1994-09-01

    We have shown that dystrophin is required for normal function of the retina as measured by electroretinography (ERG). In these studies a genotype/phenotype correlation was found in which DMD/BMD patients with deletions in the central to distal region of the gene had abnormal ERGs, while patients with deletions in the 5{prime} end of the gene had a mild or normal retinal phenotype. A similar correlation was also observed in the mouse in which the mdx mouse having a mutation in exon 23 had a normal retinal phenotype, whereas the mdx{sup Cv3} mouse (mutation in intron 65) had an abnormal phenotype. Molecular analysis of both human and mouse retina indicated that at least two isoforms of dystrophin are expressed in the retina and localize to the outer plexiform layer, the synaptic junction between the photoreceptors, the bipolar cells, and the horizontal cells. Using a panel of monoclonal dystrophin antisera to analyze mdx mouse retina which does not contain full length dystrophin antisera, we showed that a shorter dystrophin isoform (approximately 260 kDa) was present and contained part of the rod, the cysteine-rich and C-terminal domains. The 5{prime} end of the transcript giving rise to this isoform was characterized and cloned using 5{prime}RACE. Sequence analysis indicated that this transcript contained a novel exon 1 consisting of 240 nucleotides and coded for a unique N-terminus of 13 amino acids. This isoform is distinct from the DP116 dystrophin isoform identified in peripheral nerve. From the functional analysis of DMD patients and dystrophic mice we conclude that this 260 kDa dystrophin isoform is required for normal retinal electrophysiology.

  11. Alterations of dystrophin-associated glycoproteins in the heart lacking dystrophin or dystrophin and utrophin.

    PubMed

    Sharpe, Katharine M; Premsukh, Monica D; Townsend, DeWayne

    2013-12-01

    Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD). Patients with DMD lack the protein dystrophin, which is widely expressed in striated muscle. In skeletal muscle, the loss of dystrophin results in dramatically decreased expression of the dystrophin associated glycoprotein complex (DGC). Interestingly, in the heart the DGC is normally expressed without dystrophin; this has been attributed to presence of the dystrophin homologue utrophin. We demonstrate here that neither utrophin nor dystrophin are required for the expression of the cardiac DGC. However, alpha-dystroglycan (α-DG), a major component of the DGC, is differentially glycosylated in dystrophin-(mdx) and dystrophin-/utrophin-(dko) deficient mouse hearts. In both models the altered α-DG retains laminin binding activity, but has an altered localization at the sarcolemma. In hearts lacking both dystrophin and utrophin, the alterations in α-DG glycosylation are even more dramatic with changes in gel migration equivalent to 24 ± 3 kDa. These data show that the absence of dystrophin and utrophin alters the processing of α-DG; however it is not clear if these alterations are a consequence of the loss of a direct interaction with dystrophin/utrophin or results from an indirect response to the presence of severe pathology. Recently there have been great advances in our understanding of the glycosylation of α-DG regarding its role as a laminin receptor. Here we present data that alterations in glycosylation occur in the hearts of animal models of DMD, but these changes do not affect laminin binding. The physiological consequences of these alterations remain unknown, but may have significant implications for the development of therapies for DMD. PMID:24096570

  12. Immobility reduces muscle fiber necrosis in dystrophin deficient muscular dystrophy.

    PubMed

    Kimura, S; Ikezawa, M; Nomura, K; Ito, K; Ozasa, S; Ueno, H; Yoshioka, K; Yano, S; Yamashita, T; Matuskura, M; Miike, T

    2006-08-01

    Duchenne/Becker muscular dystrophy is a progressive muscle disease, which is caused by the abnormality of dystrophin. Spina bifida is characterized by paralysis of the feet, with most of the upper extremities not being affected. We report here on the first case of Becker muscular dystrophy coinciding with spina bifida. The muscle biopsy specimens of the patient showed dystrophic changes in upper extremities, but clearly less in lower extremities. The results show that the restriction of excessive exercise is important for dystrophin deficiency disease. PMID:16516424

  13. Alterations of dystrophin associated glycoproteins in the heart lacking dystrophin or dystrophin and utrophin

    PubMed Central

    Sharpe, Katharine M.; Premsukh, Monica D.; Townsend, DeWayne

    2013-01-01

    Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD). Patients with DMD lack the protein dystrophin, which is widely expressed in striated muscle. In skeletal muscle, the loss of dystrophin results in dramatically decreased expression of the dystrophin associated glycoprotein complex (DGC). Interestingly, in the heart the DGC is normally expressed without dystrophin; this has been attributed to presence of the dystrophin homologue utrophin. We demonstrate here that neither utrophin nor dystrophin are required for the expression of the cardiac DGC. However, alpha-dystroglycan (α-DG), a major component of the DGC, is differentially glycosylated in dystrophin-(mdx) and dystrophin−/utrophin− (dko) deficient mouse hearts. In both models the altered α-DG retains laminin binding activity, but has an altered localization at the sarcolemma. In hearts lacking both dystrophin and utrophin, the alterations in α-DG glycosylation are even more dramatic with changes in gel migration equivalent to 24 ± 3 kDa. These data show that the absence of dystrophin and utrophin alters the processing of α-DG; however it is not clear if these alterations are a consequence of the loss of a direct interaction with dystrophin/utrophin, or results from an indirect response to the presence of severe pathology. Recently there have been great advances in our understanding of the glycosylation of α-DG regarding its role as a laminin receptor. Here we present data that alterations in glycosylation occurs in the hearts of animal models of DMD, but these changes do not affect laminin binding. The physiological consequences of these alterations remain to be determined, but may have significant implications for the development of therapies for DMD. PMID:24096570

  14. Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene.

    PubMed

    Matsumura, K; Lee, C C; Caskey, C T; Campbell, K P

    1993-04-12

    Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extracellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex. PMID:8462697

  15. Dystrophin-Deficient Cardiomyopathy.

    PubMed

    Kamdar, Forum; Garry, Daniel J

    2016-05-31

    Dystrophinopathies are a group of distinct neuromuscular diseases that result from mutations in the structural cytoskeletal Dystrophin gene. Dystrophinopathies include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, as well as DMD and BMD female carriers. The primary presenting symptom in most dystrophinopathies is skeletal muscle weakness. However, cardiac muscle is also a subtype of striated muscle and is similarly affected in many of the muscular dystrophies. Cardiomyopathies associated with dystrophinopathies are an increasingly recognized manifestation of these neuromuscular disorders and contribute significantly to their morbidity and mortality. Recent studies suggest that these patient populations would benefit from cardiovascular therapies, annual cardiovascular imaging studies, and close follow-up with cardiovascular specialists. Moreover, patients with DMD and BMD who develop end-stage heart failure may benefit from the use of advanced therapies. This review focuses on the pathophysiology, cardiac involvement, and treatment of cardiomyopathy in the dystrophic patient. PMID:27230049

  16. Dystrophin analysis using a panel of anti-dystrophin antibodies in Duchenne and Becker muscular dystrophy.

    PubMed

    Muntoni, F; Mateddu, A; Cianchetti, C; Marrosu, M G; Clerk, A; Cau, M; Congiu, R; Cao, A; Melis, M A

    1993-01-01

    Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in 19 patients with Xp21 disorders and in 25 individuals with non-Xp21 muscular dystrophy. Antibodies raised to seven different regions spanning most of the protein were used for immunocytochemistry. In all patients specific dystrophin staining anomalies were detected and correlated with clinical severity and also gene deletion. In patients with Becker muscular dystrophy (BMD) the anomalies detected ranged from inter- and intra-fibre variation in labelling intensity with the same antibody or several antibodies to general reduction in staining and discontinuous staining. In vitro evidence of abnormal dystrophin breakdown was observed reanalysing the muscle of patients, with BMD and not that of non-Xp21 dystrophies, after it has been stored for several months. A number of patients with DMD showed some staining but this did not represent a diagnostic problem. Based on the data presented, it was concluded that immunocytochemistry is a powerful technique in the prognostic diagnosis of Xp21 muscular dystrophies. PMID:8429320

  17. Monoclonal antibodies against the muscle-specific N-terminus of dystrophin: Characterization of dystrophin in a muscular dystrophy patient with a frameshift deletion of Exons 3-7

    SciTech Connect

    Thanh, L. T.; Man, N. thi; Morris, G.E. ); Love, D.R.; Davies, K.E. ); Helliwell, T.R. )

    1993-07-01

    The first three exons of the human muscle dystrophin gene were expressed as a [beta]-galactosidase fusion protein. 1-his protein was then used to prepare two monoclonal antibodies (mAbs) which react with native dystrophin on frozen muscle sections and with denatured dystrophin on western blots but which do not cross-react with the distrophin-related protein, utrophin. Both mAbs recognized dystrophin in muscular dystrophy (MD) patients with deletions of exon 3, and further mapping with 11 overlapping synthetic peptides showed that they both recognize an epitope encoded by the muscle-specific exon 1. Neither mAb recognizes the brain dystrophin isoform, confirming the prediction from mRNA data that this has a different N-terminus. One Becker MD patient with a frameshift deletion of exons 3-7 is shown to produce dystrophin which reacts with the N-terminal mAbs, as well as with mAbs which bind on the C-terminal side of the deletion. The data suggest that transcription begins at the normal muscle dystrophin promoter and that the normal reading frame is restored after the deletion. A number of mechanisms have been proposed for restoration of the reading frame after deletion of exons 3-7, but those which predict dystrophin with an abnormal N-terminus do not appear to be major mechanisms in this patient. 27 refs., 6 figs.

  18. Mutation of the proteolipid protein gene PLP in a human X chromosome-linked myelin disorder.

    PubMed

    Hudson, L D; Puckett, C; Berndt, J; Chan, J; Gencic, S

    1989-10-01

    Myelin is a highly specialized membrane unique to the nervous system that ensheaths axons to permit the rapid saltatory conduction of impulses. The elaboration of a compact myelin sheath is disrupted in a diverse spectrum of human disorders, many of which are of unknown etiology. The X chromosome-linked human disorder Pelizaeus-Merzbacher disease is a clinically and pathologically heterogeneous group of disorders that demonstrate a striking failure of oligodendrocyte differentiation. This disease appears pathologically and genetically to be similar to the disorder seen in the dysmyelinating mouse mutant jimpy, which has a point mutation in the gene encoding an abundant myelin protein, proteolipid protein (PLP). We report that the molecular defect in one Pelizaeus-Merzbacher family is likewise a point mutation in the PLP gene. A single T----C transition results in the substitution of a charged amino acid residue, arginine, for tryptophan in one of the four extremely hydrophobic domains of the PLP protein. The identification of a mutation in this Pelizaeus-Merzbacher family should facilitate the molecular classification and diagnosis of these X chromosome-linked human dysmyelinating disorders. PMID:2479017

  19. HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

    PubMed

    Nishida, Atsushi; Yasuno, Sato; Takeuchi, Atsuko; Awano, Hiroyuki; Lee, Tomoko; Niba, Emma Tabe Eko; Fujimoto, Takahiro; Itoh, Kyoko; Takeshima, Yasuhiro; Nishio, Hisahide; Matsuo, Masafumi

    2016-09-01

    The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5' part of the dystrophin cDNA. In contrast, fragments covering exons 64-79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells. PMID:27109495

  20. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

    PubMed Central

    Dumont, Nicolas A.; Wang, Yu Xin; von Maltzahn, Julia; Pasut, Alessandra; Bentzinger, C. Florian; Brun, Caroline E.; Rudnicki, Michael A.

    2016-01-01

    Dystrophin is expressed in differentiated myofibers where it is required for sarcolemmal integrity, and loss-of-function mutations in its gene result in Duchenne Muscular Dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells) where it associates with the Ser/Thr kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to polarize Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, while also displaying a loss of polarity, abnormal division patterns including centrosome amplification, impaired mitotic spindle orientation, and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD is not only caused by myofiber fragility, but is also exacerbated by impaired regeneration due to intrinsic satellite cell dysfunction. PMID:26569381

  1. DNA linkage analysis of X chromosome-linked chronic granulomatous disease.

    PubMed Central

    Baehner, R L; Kunkel, L M; Monaco, A P; Haines, J L; Conneally, P M; Palmer, C; Heerema, N; Orkin, S H

    1986-01-01

    Chronic granulomatous disease (CGD) is a disorder of phagocytes that is usually inherited as an X chromosome-linked trait. Previous family studies suggested that the CGD locus resides on the distal short arm (Xp22-Xpter). Using cloned, polymorphic DNA probes we have performed a linkage analysis within CGD families that suggests a more proximal location (Xp21). In addition, the CGD locus is proximal to the Duchenne muscular dystrophy locus and lies within a broad region of Xp in which recombination appears to be greater than anticipated on the basis of physical distance between markers. Regional localization of the X chromosome CGD locus should facilitate molecular cloning of the CGD gene and molecular dissection of the phagocyte oxidase system. Images PMID:3010296

  2. Detection of New Paternal Dystrophin Gene Mutations in Isolated Cases of Dystrophinopathy in Females

    PubMed Central

    Pegoraro, Elena; Schimke, R. Neil; Arahata, Kiichi; Hayashi, Yukiko; Stern, Harvey; Marks, Harold; Glasberg, Mark R.; Carroll, James E.; Taber, Joseph W.; Wessel, Henry B.; Bauserman, Steven C.; Marks, Warren A.; Toriello, Helga V.; Higgins, James V.; Appleton, Staci; Schwartz, Lisa; Garcia, Carlos A.; Hoffman, Eric P.

    1994-01-01

    Duchenne muscular dystrophy is one of the most common lethal monogenic disorders and is caused by dystrophin deficiency. The disease is transmitted as an X-linked recessive trait; however, recent biochemical and clinical studies have shown that many girls and women with a primary myopathy have an underlying dystrophinopathy, despite a negative family history for Duchenne dystrophy. These isolated female dystrophinopathy patients carried ambiguous diagnoses with presumed autosomal recessive inheritance (limbgirdle muscular dystrophy) prior to biochemical detection of dystrophin abnormalities in their muscle biopsy. It has been assumed that these female dystrophinopathy patients are heterozygous carriers who show preferential inactivation of the X chromosome harboring the normal dystrophin gene, although this has been shown for only a few X:autosome translocations and for two cases of discordant monozygotic twin female carriers. Here we study X-inactivation patterns of 13 female dystrophinopathy patients—10 isolated cases and 3 cases with a positive family history for Duchenne dystrophy in males. We show that all cases have skewed X-inactivation patterns in peripheral blood DNA. Of the nine isolated cases informative in our assay, eight showed inheritance of the dystrophin gene mutation from the paternal germ line. Only a single case showed maternal inheritance. The 10-fold higher incidence of paternal transmission of dystrophin gene mutations in these cases is at 30-fold variance with Bayesian predictions and gene mutation rates. Thus, our results suggest some mechanistic interaction between new dystrophin gene mutations, paternal inheritance, and skewed X inactivation. Our results provide both empirical risk data and a molecular diagnostic test method, which permit genetic counseling and prenatal diagnosis of this new category of patients. ImagesFigure 1 PMID:8198142

  3. Detection of new paternal dystrophin gene mutations in isolated cases of dystrophinopathy in females

    SciTech Connect

    Pegoraro, E.; Wessel, H.B.; Schwartz, L.; Hoffman, E.P. ); Schimke, R.N. ); Arahata, Kiichi; Hayashi, Yukiko ); Stern, H. ); Marks, H. ); Glasberg, M.R. )

    1994-06-01

    Duchenne muscular dystrophy is one of the most common lethal monogenic disorders and is caused by dystrophin deficiency. The disease is transmitted as an X-linked recessive trait; however, recent biochemical and clinical studies have shown that many girls and women with a primary myopathy have an underlying dystrophinopathy, despite a negative family history for Duchenne dystrophy. These isolated female dystrophinopathy patients carried ambiguous diagnoses with presumed autosomal recessive inheritance (limb-girdle muscular dystrophy) prior to biochemical detection of dystrophin abnormalities in their muscle biopsy. It has been assumed that these female dystrophinopathy patients are heterozygous carries who show preferential inactivation of the X chromosome harboring the normal dystrophin gene, although this has been shown for only a few X:autosome translocations and for two cases of discordant monozygotic twin female carriers. Here the authors study X-inactivation patterns of 13 female dystrophinopathy patients - 10 isolated cases and 3 cases with a positive family history for Duchenne dystrophy in males. They show that all cases have skewed X-inactivation patterns in peripheral blood DNA. Of the nine isolated cases informative in the assay, eight showed inheritance of the dystrophin gene mutation from the paternal germ line. Only a single case showed maternal inheritance. The 10-fold higher incidence of paternal transmission of dystrophin gene mutations in these cases is at 30-fold variance with Bayesian predictions and gene mutation rates. Thus, the results suggest some mechanistic interaction between new dystrophin gene mutations, paternal inheritance, and skewed X inactivation. The results provide both empirical risk data and a molecular diagnostic test method, which permit genetic counseling and prenatal diagnosis of this new category of patients. 58 refs., 7 figs., 2 tabs.

  4. Localizing multiple X chromosome-linked retinitis pigmentosa loci using multilocus homogeneity tests

    SciTech Connect

    Ott, J.; Terwilliger, J.D. ); Bhattacharya, S. ); Chen, J.D.; Denton, J.; Donald, J. ); Dubay, C.; Litt, M.; Weleber, R.G. ); Farrar, G.J.; Humphries, P. ); Fishman, G.A.; Wong, F. ); Frey, D.; Maechler, M. )

    1990-01-01

    Multilocus linkage analysis of 62 family pedigrees with X chromosome-linked retinitis pigmentosa (XLRP) was undertaken to determine the presence of possible multiple disease loci and to reliability estimate their map location. Multilocus homogeneity tests furnish convincing evidence for the presence of two XLRP loci, the likelihood ratio being 6.4 {times} 10{sup 9}:1 in a favor of two versus a single XLRP locus and gave accurate estimates for their map location. In 60-75% of the families, location of an XLRP gene was estimated at 1 centimorgan distal to OTC, and in 25-40% of the families, an XLRP locus was located halfway between DXS14 (p58-1) and DXZ1 (Xcen), with an estimated recombination fraction of 25% between the two XLRP loci. There is also good evidence for third XLRP locus, midway between DXS28 (C7) and DXS164 (pERT87), supported by a likelihood ratio of 293:1 for three versus two XLRP loci.

  5. X-chromosome-linked inheritance of the variant thyroxine-binding globulin in Australian aborigines.

    PubMed

    Refetoff, S; Murata, Y

    1985-02-01

    The inheritance of quantitative changes in serum T4-binding globulin (TBG; reduced or elevated serum levels) and electrophoretic variants of TBG have been shown to be X-chromosome linked. However, it recently was suggested that another TBG variant, widely distributed in the Australian Aborigine population, may be inherited as an autosomal dominant trait. This communication deals with studies directed to the elucidation of the mode of inheritance of the Aboriginal variant TBG. By measuring the rate of denaturation of TBG at 56 C, we identified three distinct types of TBG in Australian Aborigines. One was a relatively heat-stable TBG (mean t1/2, 58.0 min; range, 68-53 min; group A), indistinguishable from TBG in caucasians (mean t1/2, 55.1; range, 67-43); another was a heat-labile TBG (mean t1/2, 20.8 min; range, 23.7-18.4 min; group C); and a third had intermediate values (mean t1/2, 35.7 min; range, 39.5-30.6 min; group B). Serum samples from the latter group belonged exclusively to women. Assuming that individuals from group A were homozygous for the caucasian type TBG (TBGCC), those from group C were homozygous for the Aboriginal variant of TBG (TBGAA), and individuals from group B were heterozygous (TBGCA), gene frequencies were calculated for the product of TBGC and TBGA, and the incidence of expected genotypes was compared to that observed. The results are compatible with X-chromosome, but not autosomal, inheritance, with a gene frequency of TBGC of 0.4118 and of TBGA of 0.5882. The ability to identify individuals who are heterozygous for the Aboriginal variant TBG confirmed that the structural gene of TBG in man is located on the X-chromosome. PMID:3917459

  6. Molecular patterns of X chromosome-linked color vision genes among 134 men of European ancestry.

    PubMed

    Drummond-Borg, M; Deeb, S S; Motulsky, A G

    1989-02-01

    We used Southern blot hybridization to study X chromosome-linked color vision genes encoding the apoproteins of red and green visual pigments in 134 unselected Caucasian men. One hundred and thirteen individuals (84.3%) had a normal arrangement of their color vision pigment genes. All had one red pigment gene; the number of green pigment genes ranged from one to five with a mode of two. The frequency of molecular genotypes indicative of normal color vision (84.3%) was significantly lower than had been observed in previous studies of color vision phenotypes. Color vision defects can be due to deletions of red or green pigment genes or due to formation of hybrid genes comprising portions of both red and green pigment genes [Nathans, J., Piantanida, T.P., Eddy, R.L., Shows, T.B., Jr., & Hogness, D.S. (1986) Science 232, 203-210]. Characteristic anomalous patterns were seen in 15 (11.2%) individuals: 7 (5.2%) had patterns characteristic of deuteranomaly (mild defect in green color perception), 2 (1.5%) had patterns characteristic of deuteranopia (severe defect in green color perception), and 6 (4.5%) had protan patterns (the red perception defects protanomaly and protanopia cannot be differentiated by current molecular methods). Previously undescribed hybrid gene patterns consisting of both green and red pigment gene fragments in addition to normal red and green genes were observed in another 6 individuals (4.5%). Only 2 of these patterns were considered as deuteranomalous. Thus, DNA testing detected anomalous color vision pigment genes at a higher frequency than expected from phenotypic color vision tests. Some color vision gene arrays associated with hybrid genes are likely to mediate normal color vision. PMID:2915991

  7. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice.

    PubMed

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  8. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  9. A family with a dystrophin gene mutation specifically affecting dystrophin expression in the heart

    SciTech Connect

    Muntoni, F.; Davies, K.; Dubowitz, V.

    1994-09-01

    We recently described a family with X-linked dilated cardiomyopathy where a large deletion in the muscle promoter region of the dystrophin gene was associated with a severe dilated cardiomyopathy in absence of clinical skeletal muscle involvement. The deletion removed the entire muscle promoter region, the first muscle exon and part of intron 1. The brain and Purkinje cell promoters were not affected by the deletion. Despite the lack of both the muscle promoter and the first muscle exon, dystrophin was detected immunocytochemically in relative high levels in the skeletal muscle of the affected males. We have now found that both the brain and Purkinje cell promoters were transcribed at high levels in the skeletal muscle of these individuals. This phenomenon, that does not occur in normal skeletal muscle, indicates that these two isoforms, physiologically expressed mainly in the central nervous system, can be transcribed and be functionally active in skeletal muscle under specific circumstances. Contrary to what is observed in skeletal muscle, dystrophin was not detected in the heart of one affected male using immunocytochemistry and an entire panel of anti-dystrophin antibodies. This was most likely the cause for the pronounced cardiac fibrosis observed and eventually responsible for the severe cardiac involvement invariably seen in seven affected males. In conclusion, the mutation of the muscle promoter, first muscle exon and part of intron 1 specifically affected expression of dystrophin in the heart. We believe that this deletion removes sequences involved in regulation of dystrophin expression in the heart and are at the moment characterizing other families with X-linked cardiomyopathy secondary to a dystrophinopathy.

  10. Dystrophin delivery in dystrophin-deficient DMDmdx skeletal muscle by isogenic muscle-derived stem cell transplantation.

    PubMed

    Ikezawa, Makoto; Cao, Baohong; Qu, Zhuqing; Peng, Hairong; Xiao, Xiao; Pruchnic, Ryan; Kimura, Shigemi; Miike, Teruhisa; Huard, Johnny

    2003-11-01

    Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology. PMID:14577915

  11. Laryngeal Muscles Are Spared in the Dystrophin Deficient "mdx" Mouse

    ERIC Educational Resources Information Center

    Thomas, Lisa B.; Joseph, Gayle L.; Adkins, Tracey D.; Andrade, Francisco H.; Stemple, Joseph C.

    2008-01-01

    Purpose: "Duchenne muscular dystrophy (DMD)" is caused by the loss of the cytoskeletal protein, dystrophin. The disease leads to severe and progressive skeletal muscle wasting. Interestingly, the disease spares some muscles. The purpose of the study was to determine the effects of dystrophin deficiency on 2 intrinsic laryngeal muscles, the…

  12. Disease-proportional proteasomal degradation of missense dystrophins

    PubMed Central

    Talsness, Dana M.; Belanto, Joseph J.; Ervasti, James M.

    2015-01-01

    The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin. PMID:26392559

  13. Disease-proportional proteasomal degradation of missense dystrophins.

    PubMed

    Talsness, Dana M; Belanto, Joseph J; Ervasti, James M

    2015-10-01

    The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin. PMID:26392559

  14. Rescue of a dystrophin-like protein by exon skipping normalizes synaptic plasticity in the hippocampus of the mdx mouse.

    PubMed

    Dallérac, Glenn; Perronnet, Caroline; Chagneau, Carine; Leblanc-Veyrac, Pascale; Samson-Desvignes, Nathalie; Peltekian, Elise; Danos, Olivier; Garcia, Luis; Laroche, Serge; Billard, Jean-Marie; Vaillend, Cyrille

    2011-09-01

    Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, a protein that fulfills important functions in both muscle and brain. The mdx mouse model of DMD, which also lacks dystrophin, shows a marked reduction in γ-aminobutyric acid type A (GABA(A))-receptor clustering in central inhibitory synapses and enhanced long-term potentiation (LTP) at CA3-CA1 synapses of the hippocampus. We have recently shown that U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors are able to induce skipping of the mutated exon 23 and to rescue expression of a functional dystrophin-like product both in the muscle and nervous tissue in vivo. In the brain, this rescue was accompanied by restoration of both the size and number of hippocampal GABA(A)-receptor clustering. Here, we report that 25.2±8% of re-expression two months after intrahippocampal injection of rAAV reverses the abnormally enhanced LTP phenotype at CA3-CA1 synapses of mdx mice. These results suggests that dystrophin expression indirectly influences synaptic plasticity through modulation of GABA(A)-receptor clustering and that re-expression of the otherwise deficient protein in the adult can significantly alleviate alteration of neural functions in DMD. PMID:21624465

  15. Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency while Becker muscular dystrophy (BMD) is caused by a dystrophin insufficiency or expression of a partially functional protein product. Both of these dystrophinopathies are most commonly studied using the mdx mouse and a golden r...

  16. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    SciTech Connect

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H.

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  17. X chromosome-linked and mitochondrial gene control of Leber hereditary optic neuropathy: evidence from segregation analysis for dependence on X chromosome inactivation.

    PubMed Central

    Bu, X D; Rotter, J I

    1991-01-01

    Leber hereditary optic neuropathy (LHON) has been shown to involve mutation(s) of mitochondrial DNA, yet there remain several confusing aspects of its inheritance not explained by mitochondrial inheritance alone, including male predominance, reduced penetrance, and a later age of onset in females. By extending segregation analysis methods to disorders that involve both a mitochondrial and a nuclear gene locus, we show that the available pedigree data for LHON are most consistent with a two-locus disorder, with one responsible gene being mitochondrial and the other nuclear and X chromosome-linked. Furthermore, we have been able to extend the two-locus analytic method and demonstrate that a proportion of affected females are likely heterozygous at the X chromosome-linked locus and are affected due to unfortunate X chromosome inactivation, thus providing an explanation for the later age of onset in females. The estimated penetrance for a heterozygous female is 0.11 +/- 0.02. The calculated frequency of the X chromosome-linked gene for LHON is 0.08. Among affected females, 60% are expected to be heterozygous, and the remainder are expected to be homozygous at the responsible X chromosome-linked locus. PMID:1896469

  18. X chromosome-linked and mitochondrial gene control of Leber hereditary optic neuropathy: Evidence from segregation analysis for dependence on X chromosome inactivation

    SciTech Connect

    Xiangdong Bu; Rotter, J.I. Univ. of California, Los Angeles )

    1991-09-15

    Leber hereditary optic neuropathy (LHON) has been shown to involve mutation(s) of mitochondrial DNA, yet there remain several confusing aspects of its inheritance not explained by mitochondrial inheritance alone, including male predominance, reduced penetrance, and a later age of onset in females. By extending segregation analysis methods to disorders that involve both a mitochondrial and a nuclear gene locus, the authors show that the available pedigree data for LHON are most consistent with a two-locus disorder, with one responsible gene being mitochondrial and the other nuclear and X chromosome-linked. Furthermore, they have been able to extend the two-locus analytic method and demonstrate that a proportion of affected females are likely heterozygous at the X chromosome-linked locus and are affected due to unfortunate X chromosome inactivation, thus providing an explanation for the later age of onset in females. The estimated penetrance for a heterozygous female is 0.11{plus minus}0.02. The calculated frequency of the X chromosome-linked gene for LHON is 0.l08. Among affected females, 60% are expected to be heterozygous, and the remainder are expected to be homozygous at the responsible X chromosome-linked locus.

  19. Differential expression of dystrophin, utrophin, and dystrophin-associated proteins in human muscle culture.

    PubMed

    Radojevic, V; Lin, S; Burgunder, J M

    2000-06-01

    The dystrophin-associated protein complex (DAP) plays an important role in sarcolemmal function. Mutations of DAP elements lead to diverse forms of muscular dystrophies, among them Duchenne muscular dystrophy, one of the most severe neuromuscular diseases. Strategies in gene therapy are being assessed to restore DAP stability. However, the relationship between DAP elements and time-course of the DAP formation are still not known in detail. In order to better understand the relationship among DAP proteins, we therefore studied their expression during development in human muscle culture in comparison with developmentally regulated muscle proteins. Desmin immunoreactivity (IR) was detected by 3 days in vitro (DIV3), IR for developmental heavy-chain myosin, vimentin, utrophin, and beta-dystroglycan, as well as alpha-, beta-, and gamma-sarcoglycan, a day later. delta-Sarcoglycan was found by DIV7; dystrophin could be detected only by DIV11. In general, DAP proteins were first located in the perinuclear region, later diffusely in the cytoplasm, and finally exclusively at the membrane. This sequence of events during muscle development gives further support to our suggestion that utrophin could be a precursor of dystrophin during development and regeneration. These data also suggest that utrophin alone is sufficient to anchor the complex, which is important when utrophin replacement strategies are being investigated for the treatment of dystrophinopathies. In this study we demonstrated the establishment of a culture technique that should allow the close study of DAP expression in diseased muscle, including its use after gene modulatory strategies. PMID:10928275

  20. Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity.

    PubMed

    Eghtesad, S; Zheng, H; Nakai, H; Epperly, M W; Clemens, P R

    2010-09-01

    Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600 rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900 rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle. PMID:20827278

  1. Isolated dystrophin molecules as seen by electron microscopy.

    PubMed

    Pons, F; Augier, N; Heilig, R; Léger, J; Mornet, D; Léger, J J

    1990-10-01

    Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in striated and smooth muscles as well as in non-muscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 +/- 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel alpha-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function. PMID:2236001

  2. Neural integrity is maintained by dystrophin in C. elegans

    PubMed Central

    Zhou, Shan

    2011-01-01

    The dystrophin protein complex (DPC), composed of dystrophin and associated proteins, is essential for maintaining muscle membrane integrity. The link between mutations in dystrophin and the devastating muscle failure of Duchenne’s muscular dystrophy (DMD) has been well established. Less well appreciated are the accompanying cognitive impairment and neuropsychiatric disorders also presented in many DMD patients, which suggest a wider role for dystrophin in membrane–cytoskeleton function. This study provides genetic evidence of a novel role for DYS-1/dystrophin in maintaining neural organization in Caenorhabditis elegans. This neuronal function is distinct from the established role of DYS-1/dystrophin in maintaining muscle integrity and regulating locomotion. SAX-7, an L1 cell adhesion molecule (CAM) homologue, and STN-2/γ-syntrophin also function to maintain neural integrity in C. elegans. This study provides biochemical data that show that SAX-7 associates with DYS-1 in an STN-2/γ-syntrophin–dependent manner. These results reveal a recruitment of L1CAMs to the DPC to ensure neural integrity is maintained. PMID:21242290

  3. Nonrandon X chromosome inactivation in B cells from carriers of X chromosome-linked severe combined immunodeficiency

    SciTech Connect

    Conley, M.E.; Lavoie, A.; Briggs, C.; Brown, P.; Guerra, C.; Puck, J.M.

    1988-05-01

    X chromosome-linked sever combined immunodeficiency (XSCID) is characterized by markedly reduced numbers of T cells, the absence of proliferative responses to mitogens, and hypogammaglobulinemia but normal or elevated number of B cells. To determine if the failure of the B cells to produce immunoglobulin might be due to expression of the XSCID gene defect in B-lineage cells as well as T cells, the authors analyzed patterns of X chromosome inactivation in B cells from nine obligate carriers of this disorder. A series of somatic cell hybrids that selectively retained the active X chromosome was produced from Epstein-Barr virus-stimulated B cells from each woman. To distinguish between the two X chromosome, the hybrids from each woman were analyzed using an X-linked restriction fragment length polymorphism for which the woman in question was heterozygous. In all obligate carriers of XSCID, the B-cell hybrids demonstrated preferential use of a single X chromosome, the nonmutant X, as the active X. To determine if the small number of B-cell hybrids that contained the mutant X were derived from an immature subset of B cells, lymphocytes from three carriers were separated into surface IgM positive and surface IgM negative B cells prior to exposure to Epstein-Barr virus and production of B-cell hybrids. The results demonstrated normal random X chromosome inactivation in B-cell hybrids derived from the less mature surface IgM positive B cells. These results suggest that the XSCID gene product has a direct effect on B cells as well as T cells and is required during B-cell maturation.

  4. Characterization of Dystrophin Deficient Rats: A New Model for Duchenne Muscular Dystrophy

    PubMed Central

    Tesson, Laurent; Remy, Séverine; Thepenier, Virginie; François, Virginie; Le Guiner, Caroline; Goubin, Helicia; Dutilleul, Maéva; Guigand, Lydie; Toumaniantz, Gilles; De Cian, Anne; Boix, Charlotte; Renaud, Jean-Baptiste; Cherel, Yan; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio; Huchet, Corinne

    2014-01-01

    A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD. PMID:25310701

  5. Metabolic and Signaling Alterations in Dystrophin-Deficient Hearts Precede Overt Cardiomyopathy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cytoskeletal protein dystrophin has been implicated in hereditary and acquired forms of cardiomyopathy. However, much remains to be learned about the role of dystrophin in the heart. We hypothesized that the dystrophin-deficient heart displays early alterations in energy metabolism that precede ...

  6. Dystrophin insufficiency causes a Becker muscular dystrophy-like phenotype in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency while Becker MD is caused by a dystrophin insufficiency or expression of a partially functional dystrophin protein. Deficiencies in existing mouse and dog models necessitate the development of a novel large animal model. Our pu...

  7. How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse.

    PubMed

    Godfrey, Caroline; Muses, Sofia; McClorey, Graham; Wells, Kim E; Coursindel, Thibault; Terry, Rebecca L; Betts, Corinne; Hammond, Suzan; O'Donovan, Liz; Hildyard, John; El Andaloussi, Samir; Gait, Michael J; Wood, Matthew J; Wells, Dominic J

    2015-08-01

    Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model. PMID:25935000

  8. Dystrophin is a tumor suppressor in human cancers with myogenic programs.

    PubMed

    Wang, Yuexiang; Marino-Enriquez, Adrian; Bennett, Richard R; Zhu, Meijun; Shen, Yiping; Eilers, Grant; Lee, Jen-Chieh; Henze, Joern; Fletcher, Benjamin S; Gu, Zhizhan; Fox, Edward A; Antonescu, Cristina R; Fletcher, Christopher D M; Guo, Xiangqian; Raut, Chandrajit P; Demetri, George D; van de Rijn, Matt; Ordog, Tamas; Kunkel, Louis M; Fletcher, Jonathan A

    2014-06-01

    Many common human mesenchymal tumors, including gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS), feature myogenic differentiation. Here we report that intragenic deletion of the dystrophin-encoding and muscular dystrophy-associated DMD gene is a frequent mechanism by which myogenic tumors progress to high-grade, lethal sarcomas. Dystrophin is expressed in the non-neoplastic and benign counterparts of GIST, RMS and LMS tumors, and DMD deletions inactivate larger dystrophin isoforms, including 427-kDa dystrophin, while preserving the expression of an essential 71-kDa isoform. Dystrophin inhibits myogenic sarcoma cell migration, invasion, anchorage independence and invadopodia formation, and dystrophin inactivation was found in 96%, 100% and 62% of metastatic GIST, embryonal RMS and LMS samples, respectively. These findings validate dystrophin as a tumor suppressor and likely anti-metastatic factor, suggesting that therapies in development for muscular dystrophies may also have relevance in the treatment of cancer. PMID:24793134

  9. Dystrophin, utrophin and {beta}-dystroglycan expression in skeletal muscle from patients with Becker muscular dystrophy

    SciTech Connect

    Kawajiri, Masakazu; Mitsui, Takao; Kawai, Hisaomi

    1996-08-01

    The precise localization and semiquantitative correlation of dystrophin, utrophin and {beta}-dystroglycan expression on the sarcolemma of skeletal muscle cells obtained from patients with Becker muscular dystrophy (BMD) was studied using three types of double immunofluorescence. Staining intensity was measured using a confocal laser microscope. Each of these proteins was identified at the same locus on the sarcolemma. The staining intensities of dystrophin and utrophin were approximately reciprocal at sarcolemmal sites where dystrophin expression was obviously observed. The staining intensity of {beta}-dystroglycan was strong in areas where dystrophin staining was also strong and utrophin expression was weak. Quantitative analysis revealed that the staining intensity of {beta}-dystroglycan minus that of dystrophin approximated the staining intensity of utrophin, indicating that the sum of dystrophin and utrophin expression corresponds to that of {beta}-dystroglycan. These results suggest that utrophin may compensate for dystrophin deficiency found in BMD by binding to {beta}-dystroglycan. 35 refs., 3 figs., 1 tab.

  10. Ocular and neurodevelopmental features of Duchenne muscular dystrophy: a signature of dystrophin function in the central nervous system.

    PubMed

    Ricotti, Valeria; Jägle, Herbert; Theodorou, Maria; Moore, Anthony T; Muntoni, Francesco; Thompson, Dorothy A

    2016-04-01

    Multiple isoforms of dystrophin (Dp427, Dp260, Dp140, Dp71) are expressed differentially in the central nervous system (CNS) including the retinal layers. Disruption of these protein products is responsible for cognitive dysfunction, electroretinogram (ERG) abnormalities and behavioural disorders in Duchenne muscular dystrophy (DMD). We studied the ocular characteristics and neuropsychiatric profile of 16 DMD boys. The ISCEV standard, full-field flash ERGs were assessed. Intellectual ability and behavioural disturbances were measured. All genotypes were associated with mildly abnormal photopic ERG a:b-wave amplitude ratios. In addition, we identified the following genotype/phenotype correlations: boys with mutations upstream of exon 30 (ie, isolated Dp427 altered expression) showed normal scotopic a:b ratios, abnormal photopic oscillatory potential OP2 and normal scotopic OP2. Conversely, all boys with DMD mutations downstream of exon 30 showed profoundly 'negative' scotopic ERGs (a:b ratios >1). In these patients, the involvement of Dp260 isoform resulted in the absence of slow rod pathway signalling in15 Hz scotopic flicker ERGs. These boys had abnormal scotopic OP2 and normal photopic OP2. Finally, children with mutations also affecting Dp71 were associated with more pronounced electronegative ERGs. When correlating ERGs to neurodevelopmental outcome, we found a positive correlation between negative scotopic ERGs and neurodevelopmental disturbances, and the most severe findings were in boys with Dp71 disruption. These findings suggest a strong association between DMD mutations affecting different DMD isoforms with characteristically abnormal scotopic ERGs and severe neurodevelopmental problems. The role of the ERG as a potential biomarker for dystrophin function in the CNS and response to novel genetic therapies warrants further exploration. PMID:26081639

  11. Dystrophin Distribution and Expression in Human and Experimental Temporal Lobe Epilepsy

    PubMed Central

    Hendriksen, Ruben G. F.; Schipper, Sandra; Hoogland, Govert; Schijns, Olaf E. M. G.; Dings, Jim T. A.; Aalbers, Marlien W.; Vles, Johan S. H.

    2016-01-01

    Objective: Dystrophin is part of a protein complex that connects the cytoskeleton to the extracellular matrix. In addition to its role in muscle tissue, it functions as an anchoring protein within the central nervous system such as in hippocampus and cerebellum. Its presence in the latter regions is illustrated by the cognitive problems seen in Duchenne Muscular Dystrophy (DMD). Since epilepsy is also supposed to constitute a comorbidity of DMD, it is hypothesized that dystrophin plays a role in neuronal excitability. Here, we aimed to study brain dystrophin distribution and expression in both, human and experimental temporal lobe epilepsy (TLE). Method: Regional and cellular dystrophin distribution was evaluated in both human and rat hippocampi and in rat cerebellar tissue by immunofluorescent colocalization with neuronal (NeuN and calbindin) and glial (GFAP) markers. In addition, hippocampal dystrophin levels were estimated by Western blot analysis in biopsies from TLE patients, post-mortem controls, amygdala kindled (AK)-, and control rats. Results: Dystrophin was expressed in all hippocampal pyramidal subfields and in the molecular-, Purkinje-, and granular cell layer of the cerebellum. In these regions it colocalized with GFAP, suggesting expression in astrocytes such as Bergmann glia (BG) and velate protoplasmic astrocytes. In rat hippocampus and cerebellum there were neither differences in dystrophin positive cell types, nor in the regional dystrophin distribution between AK and control animals. Quantitatively, hippocampal full-length dystrophin (Dp427) levels were about 60% higher in human TLE patients than in post-mortem controls (p < 0.05), whereas the level of the shorter Dp71 isoform did not differ. In contrast, AK animals showed similar dystrophin levels as controls. Conclusion: Dystrophin is ubiquitously expressed by astrocytes in the human and rat hippocampus and in the rat cerebellum. Hippocampal full-length dystrophin (Dp427) levels are upregulated

  12. Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin.

    PubMed

    Nguyen, T M; Ellis, J M; Ginjaar, I B; van Paassen, M M; van Ommen, G J; Moorman, A F; Cartwright, A J; Morris, G E

    1990-10-15

    A monoclonal antibody, MANDYS141, binds to both dystrophin and actinin on Western blots (SDS-denatured), but only to actinin in frozen sections of human muscle (native conformation). It differs from a polyclonal cross-reacting antiserum in that it binds to several muscle isoforms of actinin (smooth, fast and slow) from man, mouse and chicken and recognises a quite different part of the proposed triple-helical region of dystrophin (amino acids 1750-2248). The results suggest that structural homologies between actinin and dystrophin occur more than once in their central helical regions and provide experimental support for an actinin-like central rod model for dystrophin. PMID:1699800

  13. Evolutionary study of vertebrate and invertebrate members of the dystrophin and utrophin gene family

    SciTech Connect

    Roberts, R.G.; Nicholson, L.; Bobrow, M.

    1994-09-01

    Vertebrates express two members of the dystrophin gene family. The prototype, dystrophin, is expressed in muscle and neural tissue, and is defective in the human disorders Duchenne and Becker muscular dystrophy (DMD, BMD). The dystrophin homologue utrophin is more generally expressed but has not yet been associated with a genetic disorder. The function of neither protein is clear. A comparison of human utrophin with the known dystrophins (human, mouse, chicken, Torpedo) suggests that dystrophin and utrophin diverged before the vertebrate radiation. We have used reverse-transcript PCR (RT-PCR) directed by degenerate primers to characterize dystrophin and utrophin transcripts from a range of vertebrate and invertebrate animals. Our results suggest that the duplication leading to distinct dystrophin and utrophin genes occurred close to the point of divergence of urochordates from the cephalochordate-vertebrate lineage. This divergence may have occurred to fulfill a novel role which arose at this point, or may reflect a need for separate regulation of the neuromuscular and other functions of the ancient dystrophin. Our data include sequences of the first non-human utrophins to be characterized, and show these to be substantially more divergent than their cognate dystrophins. In addition, our results provide a large body of information regarding the tolerance of amino acid positions in the cysteine-rich and C-terminal domains to substitution. This will aid the interpretations of DMD and BMD missense mutations in these regions.

  14. Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy

    PubMed Central

    Meng, Jinhong; Counsell, John R.; Reza, Mojgan; Laval, Steven H.; Danos, Olivier; Thrasher, Adrian; Lochmüller, Hanns; Muntoni, Francesco; Morgan, Jennifer E.

    2016-01-01

    Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation. PMID:26813695

  15. Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy.

    PubMed

    Meng, Jinhong; Counsell, John R; Reza, Mojgan; Laval, Steven H; Danos, Olivier; Thrasher, Adrian; Lochmüller, Hanns; Muntoni, Francesco; Morgan, Jennifer E

    2016-01-01

    Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation. PMID:26813695

  16. Dystrophin in frameshift deletion patients with Becker Muscular Dystrophy

    SciTech Connect

    Gangopadhyay, S.B.; Ray, P.N.; Worton, R.G.; Sherratt, T.G.; Heckmatt, J.Z.; Dubowitz, V.; Strong, P.N.; Miller, G. ); Shokeir, M. )

    1992-09-01

    In a previous study the authors identified 14 cases with Duchenne muscular dystrophy (DMD) or its milder variant, Becker muscular dystrophy (BMD), with a deletion of exons 3-7, a deletion that would be expected to shift the translational reading frame of the mRNA and give a severe phenotype. They have examined dystrophin and its mRNA from muscle biopsies of seven cases with either mild or intermediate phenotypes. In all cases they detected slightly lower-molecular-weight dystrophin in 12%-15% abundance relative to the normal. By sequencing amplified mRNA they have found that exon 2 is spliced to exon 8, a splice that produces a frameshifted mRNA, and have found no evidence for alternate splicing that might be involved in restoration of dystrophin mRNA reading frame in the patients with a mild phenotype. Other transcriptional and posttranscriptional mechanisms such as cryptic promoter, ribosomal frameshifting, and reinitiation are suggested that might play some role in restoring the reading frame. 34 refs., 5 figs. 1 tab.

  17. Spectrum of small mutations in the dystrophin coding region

    SciTech Connect

    Prior, T.W.; Bartolo, C.; Pearl, D.K.

    1995-07-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5` and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened {approximately} 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3` of exon 55. The extent of protein truncation caused by the 3` mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications. 71 refs., 2 figs., 2 tabs.

  18. In Vivo Fusion of Circulating Fluorescent Cells with Dystrophin-Deficient Myofibers Results in Extensive Sarcoplasmic Fluorescence Expression but Limited Dystrophin Sarcolemmal Expression

    PubMed Central

    Chretien, Fabrice; Dreyfus, Patrick A.; Christov, Christo; Caramelle, Philippe; Lagrange, Jean-Léon; Chazaud, Bénédicte; Gherardi, Romain K.

    2005-01-01

    To investigate the therapeutic potential of bone marrow transplantation in Duchenne muscular dystrophy, green fluorescent protein-positive (GFP+) bone marrow cells were transplanted into irradiated wild-type and dystrophin-deficient mdx mice. Tibialis anterior muscles showed fivefold to sixfold more GFP+ mononucleated cells and threefold to fourfold more GFP+ myofibers in mdx than in wild-type mice. In contrast, dystrophin expression in mdx mice remained within the level of nontransplanted mdx mice, and co-expression with GFP was rare. Longitudinal sections of 5000 myofibers showed 160 GFP+ fibers, including 9 that co-expressed dystrophin. GFP was always visualized as full-length sarcoplasmic fluorescence that exceeded the span of sample length (up to 1500 μm), whereas dystrophin expression was restricted to 11 to 28% of this length. Dystrophin expression span was much shorter in GFP+ fibers (116 ± 46 μm) than in revertant fibers (654 ± 409 μm). These data suggest that soluble GFP diffuses far from the fusion site with a pre-existing dystrophin− myofiber whereas dystrophin remains mainly expressed close to the site of fusion. Because restoration of dystrophin in whole muscle fiber length is required to expect functional improvement and clinical benefits for Duchenne muscular dystrophy, future applications of cell therapies to neuromuscular disorders could be more appropriately envisaged for replacement of defective soluble sarcoplasmic proteins. PMID:15920159

  19. A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells

    SciTech Connect

    Singer-Sam, J.; Dai, A.; Riggs, A.D. ); Goldstein, L.; Gartler, S.M. )

    1992-02-15

    Hpa II site H8 is in the CpG-rich 5{prime} untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG island' whose methylation pattern is perfectly correlated with transcriptional silence of this gene. The authors measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. They conclude that site H8 is the unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.

  20. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy

    PubMed Central

    Yue, Yongping; Wasala, Nalinda B; Bostick, Brian; Duan, Dongsheng

    2016-01-01

    Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity. PMID:27419194

  1. 100-fold but not 50-fold dystrophin overexpression aggravates electrocardiographic defects in the mdx model of Duchenne muscular dystrophy.

    PubMed

    Yue, Yongping; Wasala, Nalinda B; Bostick, Brian; Duan, Dongsheng

    2016-01-01

    Dystrophin gene replacement holds the promise of treating Duchenne muscular dystrophy. Supraphysiological expression is a concern for all gene therapy studies. In the case of Duchenne muscular dystrophy, Chamberlain and colleagues found that 50-fold overexpression did not cause deleterious side effect in skeletal muscle. To determine whether excessive dystrophin expression in the heart is safe, we studied two lines of transgenic mdx mice that selectively expressed a therapeutic minidystrophin gene in the heart at 50-fold and 100-fold of the normal levels. In the line with 50-fold overexpression, minidystrophin showed sarcolemmal localization and electrocardiogram abnormalities were corrected. However, in the line with 100-fold overexpression, we not only detected sarcolemmal minidystrophin expression but also observed accumulation of minidystrophin vesicles in the sarcoplasm. Excessive minidystrophin expression did not correct tachycardia, a characteristic feature of Duchenne muscular dystrophy. Importantly, several electrocardiogram parameters (QT interval, QRS duration and the cardiomyopathy index) became worse than that of mdx mice. Our data suggests that the mouse heart can tolerate 50-fold minidystrophin overexpression, but 100-fold overexpression leads to cardiac toxicity. PMID:27419194

  2. Mini-dystrophin Expression Down-regulates IP3-mediated Calcium Release Events in Resting Dystrophin-deficient Muscle Cells

    PubMed Central

    Balghi, Haouaria; Sebille, Stéphane; Mondin, Ludivine; Cantereau, Anne; Constantin, Bruno; Raymond, Guy; Cognard, Christian

    2006-01-01

    We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling. PMID:16847098

  3. In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

    PubMed Central

    Bajanca, Fernanda; Gonzalez-Perez, Vinicio; Gillespie, Sean J; Beley, Cyriaque; Garcia, Luis; Theveneau, Eric; Sear, Richard P; Hughes, Simon M

    2015-01-01

    Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics. DOI: http://dx.doi.org/10.7554/eLife.06541.001 PMID:26459831

  4. Expression of recombinant dystrophin and its localization to the cell membrane.

    PubMed

    Lee, C C; Pearlman, J A; Chamberlain, J S; Caskey, C T

    1991-01-24

    Duchenne's muscular dystrophy (DMD) is an X-linked progressive myopathy caused by a defect in the DMD gene locus. The gene corresponding to the DMD locus produces a 14-kilobase (kb) messenger RNA that codes for a large cytoskeletal membrane protein, dystrophin. DMD and Becker's muscular dystrophy are the consequences of dystrophin mutations. The exact biological function of dystrophin remains unknown but it has been demonstrated that it is localized to the cytoplasmic face of the cell membrane and has direct interaction with several other membrane proteins. We report here the synthesis of a 14-kb full-length complementary DNA for the mouse muscle dystrophin mRNA and the expression of this cDNA in COS cells. The recombinant dystrophin is indistinguishable from mouse muscle dystrophin by western blot analysis with anti-dystrophin antibodies and was shown by an immunofluorescent technique to be localized in the cell membrane. Our successful construction of a functional full-length cDNA opens opportunities for the study of structure and function of dystrophin and provides an opportunity to initiate gene therapy studies. PMID:1824797

  5. Role of Dystrophin in Airway Smooth Muscle Phenotype, Contraction and Lung Function

    PubMed Central

    Sharma, Pawan; Basu, Sujata; Mitchell, Richard W.; Stelmack, Gerald L.; Anderson, Judy E.; Halayko, Andrew J.

    2014-01-01

    Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD) and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC) and calponin and reduced Ca2+ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3β and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh) when compared to genetic control BL10ScSnJ mice (wild-type). In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM. PMID:25054970

  6. Structural Basis of Neuronal Nitric-oxide Synthase Interaction with Dystrophin Repeats 16 and 17.

    PubMed

    Molza, Anne-Elisabeth; Mangat, Khushdeep; Le Rumeur, Elisabeth; Hubert, Jean-François; Menhart, Nick; Delalande, Olivier

    2015-12-01

    Duchenne muscular dystrophy is a lethal genetic defect that is associated with the absence of dystrophin protein. Lack of dystrophin protein completely abolishes muscular nitric-oxide synthase (NOS) function as a regulator of blood flow during muscle contraction. In normal muscles, nNOS function is ensured by its localization at the sarcolemma through an interaction of its PDZ domain with dystrophin spectrin-like repeats R16 and R17. Early studies suggested that repeat R17 is the primary site of interaction but ignored the involved nNOS residues, and the R17 binding site has not been described at an atomic level. In this study, we characterized the specific amino acids involved in the binding site of nNOS-PDZ with dystrophin R16-17 using combined experimental biochemical and structural in silico approaches. First, 32 alanine-scanning mutagenesis variants of dystrophin R16-17 indicated the regions where mutagenesis modified the affinity of the dystrophin interaction with the nNOS-PDZ. Second, using small angle x-ray scattering-based models of dystrophin R16-17 and molecular docking methods, we generated atomic models of the dystrophin R16-17·nNOS-PDZ complex that correlated well with the alanine scanning identified regions of dystrophin. The structural regions constituting the dystrophin interaction surface involve the A/B loop and the N-terminal end of helix B of repeat R16 and the N-terminal end of helix A' and a small fraction of helix B' and a large part of the helix C' of repeat R17. The interaction surface of nNOS-PDZ involves its main β-sheet and its specific C-terminal β-finger. PMID:26378238

  7. Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin.

    PubMed

    Zatz, M; Pavanello, R C M; Lazar, M; Yamamoto, G L; Lourenço, N C V; Cerqueira, A; Nogueira, L; Vainzof, M

    2014-11-01

    Duchenne muscular dystrophy (DMD), a severe and lethal condition, is caused by the absence of muscle dystrophin. Therapeutic trials aiming at the amelioration of muscle function have been targeting the production of muscle dystrophin in affected Duchenne patients. However, how much dystrophin is required to rescue the DMD phenotype remains an open question. We have previously identified two exceptional golden retriever muscular dystrophy (GRMD) dogs with a milder course despite the total absence of muscle dystrophin. Here we report two unusual patients carrying nonsense mutations in the DMD gene and dystrophin deficiency but with an unexpectedly mild phenotype. Three reported polymorphisms, respectively in genes LTBP4, SPP1 and ACTN3 were excluded as possible DMD genetic modifiers in our patients. Finding the mechanisms that protect some rare patients and dogs from the deleterious effect of absent muscle dystrophin is of utmost importance and may lead to new avenues for treatment. Importantly, these observations indicate that it is possible to have a functional large muscle even without dystrophin. PMID:25047667

  8. Exon skipping and translation in patients with frameshift deletions in the dystrophin gene

    SciTech Connect

    Sherratt, T.G.; Dubowitz, V.; Sewry, C.A.; Strong, P.N. ); Vulliamy, T. )

    1993-11-01

    Although many Duchenne muscular dystrophy patients have a deletion in the dystrophin gene which disrupts the translational reading frame, they express dystrophin in a small proportion of skeletal muscle fibers ([open quotes]revertant fibers[close quotes]). Antibody studies have shown, indirectly, that dystrophin synthesis in revertant fibers is facilitated by a frame-restoring mechanism; in the present study, the feasibility of mRNA splicing was investigated. Dystrophin transcripts were analyzed in skeletal muscle from individuals possessing revertant fibers and a frameshift deletion in the dystrophin gene. In each case a minor in-frame transcript was detected, in which exons adjacent to those deleted from the genome had been skipped. There appeared to be some correlation between the levels of in-frame transcripts and the predicted translation products. Low levels of alternatively spliced transcripts were also present in normal muscle. The results provide further evidence of exon skipping in the dystrophin gene and indicate that this may be involved in the synthesis of dystrophin by revertant fibers. 44 refs., 12 figs.

  9. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  10. Expression of human dystrophin following the transplantation of genetically modified mdx myoblasts.

    PubMed

    Moisset, P A; Gagnon, Y; Karpati, G; Tremblay, J P

    1998-10-01

    Transplantation of genetically modified autologous myoblasts has been proposed as a possible solution to avoid long-term use of immunosuppressive drugs. To determine the conditions to be used in this kind of approach for possible treatment of dystrophin deficiency, mdx myoblasts were infected at different multiplicities of infection (MOI or 0.01-1000) with an adenoviral vector containing a CMV promoter/enhancer driven 6.3 kb human dystrophin cDNA (minigene) and tested in vitro for transgene expression. In these cultures, dystrophin mRNA was found to be proportionate with increasing MOI. Primary myoblast cultures derived from transgenic mdx mice expressing beta-Gal under a muscle-specific promoter and showing high expression of the human mini-dystrophin transgene introduced by the adenoviral vector were grafted into anterior tibialis muscles of SCID mice. Ten and 24 days after transplantation, numerous muscle fibers expressing both human dystrophin and beta-Gal were detected throughout the mouse muscles by immunohistochemistry using an antibody specific for human dystrophin. The presence of the human mini-dystrophin mRNA was also detected by RT-PCR. These results demonstrate that three essential conditions in autologous myoblast transplantation can be achieved: (1) in vivo survival of at least some of the transduced myoblasts; (2) efficient fusion of these cells with the host muscle fibers; and (3) the high expression of the dystrophin transgene in situ. Furthermore, this article provides a novel RT-PCR-based technique to quantify the human dystrophin minigene expression in vitro and in vivo. PMID:9930339

  11. Organization of the human CD40L gene: Implications for molecular defects in X chromosome-linked hyper-IgM syndrome and prenatal diagnosis

    SciTech Connect

    Villa, A.; Macchi, P.P.; Strina, D.; Frattini, A.; Lucchini, F.; Patrosso, C.M.; Vezzoni, P.; Notarangelo, L.D.; Giliani, S.; Mantuano, E.

    1994-03-15

    Recently, CD40L has been identified as the gene responsible for X chromosome-linked hyper-IgM syndrome (HIGM1). CD40L on activated T cells from HIGM1 patients fails to bind B-cell CD40 molecules, and subsequent analysis of CD40L transcripts by reverse transcription PCR demonstrated coding region mutations in these patients. This approach, however, is of limited use for prenatal diagnosis of HIGM1 in the early-gestation fetus. In this report, the authors have defined the genomic structure of the CD40L gene, which is composed of five exons and four intervening introns. With this information, the authors have defined at the genomic level the CD40L coding region. These different deletions arose from three distinct mechanisms, including (i) a splice donor mutation with exon skipping, (ii) a splice acceptor mutation with utilization of a cryptic splice site, and (iii) a deletion/insertion event with the creation of a new splice acceptor site. In addition, they have performed prenatal evaluation of an 11-week-old fetus at risk for HIGM1. CD40L genomic clones provide a starting point for further studies of the genetic elements that control CD40L expression. Knowledge of the CD40L gene structure will prove useful for the identification of additional mutations in HIGM1 and for performing genetic counseling about this disease. 54 refs., 4 figs., 1 tab.

  12. Close linkage of the locus for X chromosome-linked severe combined immunodeficiency to polymorphic DNA markers in Xq11-q13

    SciTech Connect

    de Saint Basile, G.; Arveiler, B.; Oberle, I.; Malcolm, S.; Levinsky, R.J.; Lau, Y.L.; Hofker, M.; Debre, M.; Fischer, A.; Griscelli, C.; Mandel, J.L.

    1987-11-01

    The gene for X chromosome-linked severe combined immunodeficiency (SCID), a disease characterized by a block in early T-cell differentiation, has been mapped to the region Xq11-q13 by linkage analysis with restriction fragment length polymorphisms. High logarithm of odds (lod) scores were obtained with the marker 19.2 (DXS3) and with the marker cpX73 (DXS159) that showed complete cosegregation with the disease locus in the informative families analyzed. Other significant linkages were obtained with several markers from Xq11 to q22. With the help of a recently developed genetic map of the region, it was possible to perform multipoint linkage analysis, and the most likely genetic order is DXS1-(SCID, DXS159)-DXYS1-DXYS12-DXS3, with a maximum multipoint logarithm of odds score of 11.0. The results demonstrate that the SCID locus (gene symbol IMD4) is not closely linked to the locus of Bruton's agammaglobulinemia (a defect in B-cell maturation). They also provide a way for a better estimation of risk for carrier and antenatal diagnosis.

  13. Overexpression of X chromosome-linked inhibitor of apoptosis by inhibiting microRNA-24 protects periodontal ligament cells against hydrogen peroxide-induced cell apoptosis.

    PubMed

    Liu, C; Chen, Z; Wang, J; Hu, H

    2016-01-01

    Hydrogen peroxide (H2O2), a common oral clinical drug for the tooth bleaching, induces severe cell apoptosis of periodontal ligament cells (PDLCs). The excessive cell apoptosis of PDLCs impairs periodontal tissue damage and repair. However, the underlying mechanism is incompletely understood. Here, we showed that microRNA-24 (miR-24) played an important role in regulating H2O2-induced cell apoptosis of PDLCs. We found that miR-24 expression was increased in PDLCs in response to H2O2 treatment. Down-regulation of miR-24 obviously rescued H2O2-induced cell apoptosis in PDLCs. By bioinformatic analysis, X chromosome-linked inhibitor of apoptosis (XIAP) was identified as a candidate target gene of miR-24, which was further verified by the dual-luciferase reporter assay. Furthermore, the protein expression level of phosphatase and tensin homolog deleted on chromosome ten was significantly decreased by miR-24 silencing, whereas the phosphorylation of Akt was remarkably increased by miR-24 silencing. In addition, the gene silencing of XIAP significantly reduced Akt activity and blocked the protective effect of the miR-24 inhibitor against H2O2-induced cell apoptosis. Overall, our findings suggest that miR-24 plays an important role in regulating the cell survival of PDLCs through targeting XIAP. PMID:27188727

  14. Metabolic profiles of dystrophin and utrophin expression in mouse models of Duchenne muscular dystrophy.

    PubMed

    Griffin, J L; Sang, E; Evens, T; Davies, K; Clarke, K

    2002-10-23

    Metabolic profiles from (1)H nuclear magnetic resonance spectroscopy have been used to describe both one and two protein systems in four mouse models related to Duchenne muscular dystrophy using the pattern recognition technique partial least squares. Robust statistical models were built for extracts and intact cardiac tissue, distinguishing mice according to expression of dystrophin. Using metabolic profiles of diaphragm, models were built describing dystrophin and utrophin, a dystrophin related protein, expression. Increased utrophin expression counteracted some of the deficits associated with dystrophic tissue. This suggests the method may be ideal for following treatment regimes such as gene therapy. PMID:12387876

  15. Craniofacial Abnormalities

    MedlinePlus

    ... of the skull and face. Craniofacial abnormalities are birth defects of the face or head. Some, like cleft ... palate, are among the most common of all birth defects. Others are very rare. Most of them affect ...

  16. Walking abnormalities

    MedlinePlus

    ... include: Arthritis of the leg or foot joints Conversion disorder (a psychological disorder) Foot problems (such as a ... injuries. For an abnormal gait that occurs with conversion disorder, counseling and support from family members are strongly ...

  17. Congenital Abnormalities

    MedlinePlus

    ... serious health problems (e.g. Down syndrome ). Single-Gene Abnormalities Sometimes the chromosomes are normal in number, ... blood flow to the fetus impair fetal growth. Alcohol consumption and certain drugs during pregnancy significantly increase ...

  18. Chromosome Abnormalities

    MedlinePlus

    ... decade, newer techniques have been developed that allow scientists and doctors to screen for chromosomal abnormalities without using a microscope. These newer methods compare the patient's DNA to a normal DNA ...

  19. Nail abnormalities

    MedlinePlus

    Nail abnormalities are problems with the color, shape, texture, or thickness of the fingernails or toenails. ... Fungus or yeast cause changes in the color, texture, and shape of the nails. Bacterial infection may ...

  20. Lack of Dystrophin Affects Bronchial Epithelium in mdx Mice.

    PubMed

    Morici, Giuseppe; Rappa, Francesca; Cappello, Francesco; Pace, Elisabetta; Pace, Andrea; Mudò, Giuseppa; Crescimanno, Grazia; Belluardo, Natale; Bonsignore, Maria R

    2016-10-01

    Mild exercise training may positively affect the course of Duchenne Muscular Dystrophy (DMD). Training causes mild bronchial epithelial injury in both humans and mice, but no study assessed the effects of exercise in mdx mice, a well known model of DMD. The airway epithelium was examined in mdx (C57BL/10ScSn-Dmdmdx) mice, and in wild type (WT, C57BL/10ScSc) mice either under sedentary conditions (mdx-SD, WT-SD) or during mild exercise training (mdx-EX, WT-EX). At baseline, and after 30 and 45 days of training (5 d/wk for 6 weeks), epithelial morphology and markers of regeneration, apoptosis, and cellular stress were assessed. The number of goblet cells in bronchial epithelium was much lower in mdx than in WT mice under all conditions. At 30 days, epithelial regeneration (PCNA positive cells) was higher in EX than SD animals in both groups; however, at 45 days, epithelial regeneration decreased in mdx mice irrespective of training, and the percentage of apoptotic (TUNEL positive) cells was higher in mdx-EX than in WT-EX mice. Epithelial expression of HSP60 (marker of stress) progressively decreased, and inversely correlated with epithelial apoptosis (r = -0.66, P = 0.01) only in mdx mice. Lack of dystrophin in mdx mice appears associated with defective epithelial differentiation, and transient epithelial regeneration during mild exercise training. Hence, lack of dystrophin might impair repair in bronchial epithelium, with potential clinical consequences in DMD patients. J. Cell. Physiol. 231: 2218-2223, 2016. © 2016 Wiley Periodicals, Inc. PMID:26868633

  1. Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

    PubMed

    Remmelink, E; Aartsma-Rus, A; Smit, A B; Verhage, M; Loos, M; van Putten, M

    2016-07-01

    Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients. PMID:27220066

  2. The ZZ domain of dystrophin in DMD: making sense of missense mutations.

    PubMed

    Vulin, Adeline; Wein, Nicolas; Strandjord, Dana M; Johnson, Eric K; Findlay, Andrew R; Maiti, Baijayanta; Howard, Michael T; Kaminoh, Yuuki J; Taylor, Laura E; Simmons, Tabatha R; Ray, Will C; Montanaro, Federica; Ervasti, Jim M; Flanigan, Kevin M

    2014-02-01

    Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with β-dystroglycan and other members of the transmembrane dystrophin-associated protein complex. The ZZ domain, a cysteine-rich zinc-finger domain near the dystrophin C-terminus, is implicated in forming a stable interaction between dystrophin and β-dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β-dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with β-dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β-dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains. PMID:24302611

  3. Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragm

    PubMed Central

    Doran, Philip; Wilton, Steve D.; Fletcher, Sue; Ohlendieck, Kay

    2009-01-01

    The disintegration of the dystrophin-glycoprotein complex represents the initial pathobiochemical insult in Duchenne muscular dystrophy. However, secondary changes in signalling, energy metabolism and ion homeostasis are probably the main factors that eventually cause progressive muscle wasting. Thus, for the proper evaluation of novel therapeutic approaches, it is essential to analyse the reversal of both primary and secondary abnormalities in treated muscles. Antisense oligomer-mediated exon skipping promises functional restoration of the primary deficiency in dystrophin. In this study, an established phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide was employed for the specific removal of exon 23 in the mutated mouse dystrophin gene transcript. Using DIGE analysis, we could show the reversal of secondary pathobiochemical abnormalities in the dystrophic diaphragm following exon-23 skipping. In analogy to the restoration of dystrophin, β-dystroglycan and neuronal nitric oxide synthase, the muscular dystrophy-associated differential expression of calsequestrin, adenylate kinase, aldolase, mitochondrial creatine kinase and cvHsp was reversed in treated muscle fibres. Hence, the re-establishment of Dp427 coded by the transcript missing exon 23 has counter-acted dystrophic alterations in Ca2+-handling, nucleotide metabolism, bioenergetic pathways and cellular stress response. This clearly establishes the exon-skipping approach as a realistic treatment strategy for diminishing diverse downstream alterations in dystrophinopathy. PMID:19132684

  4. Phosphorylation within the cysteine-rich region of dystrophin enhances its association with β-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting.

    PubMed

    Swiderski, Kristy; Shaffer, Scott A; Gallis, Byron; Odom, Guy L; Arnett, Andrea L; Scott Edgar, J; Baum, Dale M; Chee, Annabel; Naim, Timur; Gregorevic, Paul; Murphy, Kate T; Moody, James; Goodlett, David R; Lynch, Gordon S; Chamberlain, Jeffrey S

    2014-12-20

    Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin-glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin-dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin. PMID:25082828

  5. Ex vivo gene editing of the dystrophin gene in muscle stem cells mediated by peptide nucleic acid single stranded oligodeoxynucleotides induces stable expression of dystrophin in a mouse model for Duchenne muscular dystrophy.

    PubMed

    Nik-Ahd, Farnoosh; Bertoni, Carmen

    2014-07-01

    Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level. In this study, we show that PNA-ssODNs can target and correct muscle satellite cells (SCs), a population of stem cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SCs transfected with correcting PNA-ssODNs were able to engraft and to restore dystrophin expression. The number of dystrophin-positive fibers was shown to significantly increase over time. Expression was confirmed to be the result of the activation of a subpopulation of SCs that had undergone repair as demonstrated by immunofluorescence analyses of engrafted muscles using antibodies specific to full-length dystrophin transcripts and by genomic DNA analysis of dystrophin-positive fibers. Furthermore, the increase in dystrophin expression detected over time resulted in a significant improvement in muscle morphology. The ability of transplanted cells to return into quiescence and to activate upon demand was confirmed in all engrafted muscles following injury. These results demonstrate the feasibility of using gene editing strategies to target and correct SCs and further establish the therapeutic potential of this approach to permanently restore dystrophin expression into muscle of DMD patients. PMID:24753122

  6. Parental source effect of inherited mutations in the dystrophin gene of mice and men

    SciTech Connect

    Kress, W.; Grimm, T.; Mueller, C.R.; Bittner, R.

    1994-09-01

    Skewed X-inactivation has been suspected the genetic cause for some manifesting female carriers of BMD and DMD. To test whether a parental source effect on the protein expression of the dystrophin gene exists, we have set up backcrosses of mdx mice to wild type strains, enabling us to study the effect of the well-defined origin of the mutation on the dystrophin expression. In skeletal muscle sections the immunohistological staining patterns of dystrophin antibodies were showing a significant difference in the proportion of dystrophin positive versus negative fibers, suggesting a lower expression of paternally inherited mdx mutations. These data are in concordance with the pyruvate kinase (PK) levels in the serum: PK levels were much higher when the mutation was of maternal origin as compared to PK levels in paternally derived mutations. In order to test this {open_quotes}paternal source effect{close_quotes} in humans, we checked obligatory carriers of Becker muscular dystrophy (BMD) for the origin of their mutations. Creatin kinase (CK) levels in 21 carriers with maternally derived mutations were compared to CK values from 8 heterozygotes with mutations of paternal origin: CK (mat) = 140.3 IU/1 versus CK (pat) = 48.6 IU/I. The difference is statistically significant at the 5% level. These observations suggest either a differential X-inactivation or an imprinting of the dystrophin gene in mice and men.

  7. Genomic organization of the mouse dystrobrevin gene: Comparative analysis with the dystrophin gene

    SciTech Connect

    Ambrose, H.J.; Blake, D.J.; Nawrotzki, R.A.; Davies, K.E.

    1997-02-01

    Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a member of the dystrophin gene family with homology to the cysteine-rich carboxy-terminal domain of dystrophin. Torpedo dystrobrevin copurifies with the acetylcholine receptors and is thought to form a complex with dystrophin and syntrophin. This complex is also found at the sarcolemma in vertebrates and defines the cytoplasmic component of the dystrophin-associated protein complex. Previously we have cloned several dystrobrevin isoforms from mouse brain and muscle. Here we show that these transcripts are the products of a single gene located on proximal mouse chromosome 18. To investigate the diversity of dystrobrevin transcripts we have determined that the mouse dystrobrevin gene is organized into 24 coding exons that span between 130 and 170 kb at the genomic level. The gene encodes at least three distinct protein isoforms that are expressed in a tissue-specific manner. Interestingly, although there is only 27% amino acid identity between the homologous regions of dystrobrevin and dystrophin, the positions of 8 of the 15 exon-intron junctions are identical. 47 refs., 4 figs., 2 tabs.

  8. The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle

    PubMed Central

    Murphy, Sandra; Ohlendieck, Kay

    2015-01-01

    The development of advanced mass spectrometric methodology has decisively enhanced the analytical capabilities for studies into the composition and dynamics of multi-subunit protein complexes and their associated components. Large-scale complexome profiling is an approach that combines the systematic isolation and enrichment of protein assemblies with sophisticated mass spectrometry-based identification methods. In skeletal muscles, the membrane cytoskeletal protein dystrophin of 427 kDa forms tight interactions with a variety of sarcolemmal, cytosolic and extracellular proteins, which in turn associate with key components of the extracellular matrix and the intracellular cytoskeleton. A major function of this enormous assembly of proteins, including dystroglycans, sarcoglycans, syntrophins, dystrobrevins, sarcospan, laminin and cortical actin, is postulated to stabilize muscle fibres during the physical tensions of continuous excitation-contraction-relaxation cycles. This article reviews the evidence from recent proteomic studies that have focused on the characterization of the dystrophin-glycoprotein complex and its central role in the establishment of the cytoskeleton-sarcolemma-matrisome axis. Proteomic findings suggest a close linkage of the core dystrophin complex with a variety of protein species, including tubulin, vimentin, desmin, annexin, proteoglycans and collagens. Since the almost complete absence of dystrophin is the underlying cause for X-linked muscular dystrophy, a more detailed understanding of the composition, structure and plasticity of the dystrophin complexome may have considerable biomedical implications. PMID:26793286

  9. Our trails and trials in the subsarcolemmal cytoskeleton network and muscular dystrophy researches in the dystrophin era.

    PubMed

    Ozawa, Eijiro

    2010-01-01

    In 1987, about 150 years after the discovery of Duchenne muscular dystrophy (DMD), its responsible gene, the dystrophin gene, was cloned by Kunkel. This was a new substance. During these 20 odd years after the cloning, our understanding on dystrophin as a component of the subsarcolemmal cytoskeleton networks and on the pathomechanisms of and experimental therapeutics for DMD has been greatly enhanced. During this paradigm change, I was fortunately able to work as an active researcher on its frontiers for 12 years. After we discovered that dystrophin is located on the cell membrane in 1988, we studied the architecture of dystrophin and dystrophin-associated proteins (DAPs) complex in order to investigate the function of dystrophin and pathomechanism of DMD. During the conduct of these studies, we came to consider that the dystrophin-DAP complex serves to transmembranously connect the subsarcolemmal cytoskeleton networks and basal lamina to protect the lipid bilayer. It then became our working hypothesis that injury of the lipid bilayer upon muscle contraction is the cause of DMD. During this process, we predicted that subunits of the sarcoglycan (SG) complex are responsible for respective types of DMD-like muscular dystrophy with autosomal recessive inheritance. Our prediction was confirmed to be true by many researchers including ourselves. In this review, I will try to explain what we observed and how we considered concerning the architecture and function of the dystrophin-DAP complex, and the pathomechanisms of DMD and related muscular dystrophies. PMID:20948175

  10. Physiological Characterization of Muscle Strength With Variable Levels of Dystrophin Restoration in mdx Mice Following Local Antisense Therapy

    PubMed Central

    Sharp, Paul S; Bye-a-Jee, Hema; Wells, Dominic J

    2011-01-01

    Antisense-induced exon skipping can restore the open reading frame, and thus correct the dystrophin deficiency that causes Duchenne muscular dystrophy (DMD), a lethal muscle wasting condition. Successful proof-of-principle in preclinical models has led to human clinical trials. However, it is still not known what percentage of dystrophin-positive fibers and what level of expression is necessary for functional improvement. This study directly address these key questions in the mdx mouse model of DMD. To achieve a significant variation in dystrophin expression, we locally administered into tibialis anterior muscles various doses of a phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 from the mRNA of murine dystrophin. We found a highly significant correlation between the number of dystrophin-positive fibers and resistance to contraction-induced injury, with a minimum of 20% of dystrophin-positive fibers required for meaningful improvement. Furthermore, our results also indicate that a relatively low level of dystrophin expression in muscle fibers may have significant clinical benefits. In contrast, improvements in muscle force were not correlated with either the number of positive fibers or total dystrophin levels, which highlight the need to conduct appropriate functional assessments in preclinical testing using the mdx mouse. PMID:20924363

  11. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

    PubMed

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis

    2015-01-01

    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies. PMID:26159373

  12. Effective Dystrophin Restoration by a Novel Muscle-Homing Peptide–Morpholino Conjugate in Dystrophin-Deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Zhao, Jingwen; Han, Gang; Zhang, Yajie; Dong, Xue; Cao, Limin; Wang, Qingsong; Moulton, Hong M; Yin, HaiFang

    2014-01-01

    Antisense oligonucleotide (AO)–mediated splice correction therapy for Duchenne muscular dystrophy has shown huge promise from recent phase 2b clinical trials, however high doses and costs are required and targeted delivery can lower both of these. We have previously demonstrated the feasibility of targeted delivery of AOs by conjugating a chimeric peptide, consisting of a muscle-specific peptide and a cell-penetrating peptide, to AOs in mdx mice. Although increased uptake in muscle was observed, the majority of peptide–AO conjugate was found in the liver. To search for more effective muscle-homing peptides, we carried out in vitro biopanning in myoblasts and identified a novel 12-mer peptide (M12) showing preferential binding to skeletal muscle compared to the liver. When conjugated to phosphorodiamidate morpholino oligomers, ~25% of normal level of dystrophin expression was achieved in body-wide skeletal muscles in mdx mice with significant recovery in grip strength, whereas <2% in corresponding tissues treated with either muscle-specific peptide–phosphorodiamidate morpholino oligomer or unmodified phosphorodiamidate morpholino oligomer under identical conditions. Our data provide evidences for the first time that a muscle-homing peptide alone can enhance AO delivery to muscle without appreciable toxicity at 75 mg/kg, suggesting M12-phosphorodiamidate morpholino oligomer can be an alternative option to current AOs. PMID:24732757

  13. Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

    SciTech Connect

    Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R.; Moxley, R.T.

    1994-03-01

    Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

  14. The sarcoglycan-sarcospan complex localization in mouse retina is independent from dystrophins

    PubMed Central

    Fort, Patrice; Estrada, Francisco-Javier; Bordais, Agnès; Mornet, Dominique; Sahel, José-Alain; Picaud, Serge; Vargas, Haydeé Rosas; Coral-Vázquez, Ramón M.; Rendon, Alvaro

    2005-01-01

    The sarcoglycan–sarcospan (SG–SSPN) complex is part of the dystrophin-glycoprotein complex that has been extensively characterized in muscle. To establish the framework for functional studies of sarcoglycans in retina here, we quantified sarcoglycans mRNA levels with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and performed immunohistochemistry to determine their cellular and subcellular distribution. We showed that the β-, δ-, γ-, ε-sarcoglycans and sarcospan are expressed in mouse retina. They are localized predominantly in the outer and the inner limiting membranes, probably in the Müller cells and also in the ganglion cells axons where the expression of dystrophins have never been reported. We also investigated the status of the sarcoglycans in the retina of mdx3cv mutant mice for all Duchene Muscular Dystrophy (DMD) gene products. The absence of dystrophin did not produce any change in the sarcoglycan–sarcospan components expression and distribution. PMID:15993965

  15. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.

    PubMed

    Long, Chengzu; Amoasii, Leonela; Mireault, Alex A; McAnally, John R; Li, Hui; Sanchez-Ortiz, Efrain; Bhattacharyya, Samadrita; Shelton, John M; Bassel-Duby, Rhonda; Olson, Eric N

    2016-01-22

    CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus-9 (AAV9) to deliver gene-editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intraperitoneal at postnatal day 1 (P1), intramuscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after injection. Postnatal gene editing also enhanced skeletal muscle function, as measured by grip strength tests 4 weeks after injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth. PMID:26721683

  16. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy.

    PubMed Central

    Nguyen, T M; Morris, G E

    1993-01-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random "libraries" of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25-60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1-41, and we now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. Images Figure 4 Figure 1 PMID:7684887

  17. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    SciTech Connect

    Nguyen thi Man; Morris, G.E. )

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  18. The dystrophin gene and cognitive function in the general population

    PubMed Central

    Vojinovic, Dina; Adams, Hieab HH; van der Lee, Sven J; Ibrahim-Verbaas, Carla A; Brouwer, Rutger; van den Hout, Mirjam CGN; Oole, Edwin; van Rooij, Jeroen; Uitterlinden, Andre; Hofman, Albert; van IJcken, Wilfred FJ; Aartsma-Rus, Annemieke; van Ommen, GertJan B; Ikram, M Arfan; van Duijn, Cornelia M; Amin, Najaf

    2015-01-01

    The aim of our study is to investigate whether single-nucleotide dystrophin gene (DMD) variants associate with variability in cognitive functions in healthy populations. The study included 1240 participants from the Erasmus Rucphen family (ERF) study and 1464 individuals from the Rotterdam Study (RS). The participants whose exomes were sequenced and who were assessed for various cognitive traits were included in the analysis. To determine the association between DMD variants and cognitive ability, linear (mixed) modeling with adjustment for age, sex and education was used. Moreover, Sequence Kernel Association Test (SKAT) was used to test the overall association of the rare genetic variants present in the DMD with cognitive traits. Although no DMD variant surpassed the prespecified significance threshold (P<1 × 10−4), rs147546024:A>G showed strong association (β=1.786, P-value=2.56 × 10−4) with block-design test in the ERF study, while another variant rs1800273:G>A showed suggestive association (β=−0.465, P-value=0.002) with Mini-Mental State Examination test in the RS. Both variants are highly conserved, although rs147546024:A>G is an intronic variant, whereas rs1800273:G>A is a missense variant in the DMD which has a predicted damaging effect on the protein. Further gene-based analysis of DMD revealed suggestive association (P-values=0.087 and 0.074) with general cognitive ability in both cohorts. In conclusion, both single variant and gene-based analyses suggest the existence of variants in the DMD which may affect cognitive functioning in the general populations. PMID:25227141

  19. An intronic LINE-1 element insertion in the dystrophin gene aborts dystrophin expression and results in Duchenne-like muscular dystrophy in the corgi breed.

    PubMed

    Smith, Bruce F; Yue, Yongping; Woods, Philip R; Kornegay, Joe N; Shin, Jin-Hong; Williams, Regina R; Duan, Dongsheng

    2011-02-01

    Duchenne muscular dystrophy (DMD) is a dystrophin-deficient lethal muscle disease. To date, the catastrophic muscle wasting phenotype has only been seen in dystrophin-deficient humans and dogs. Although Duchenne-like symptoms have been observed in more than a dozen dog breeds, the mutation is often not known and research colonies are rarely established. Here, we report an independent canine DMD model originally derived from the Pembroke Welsh corgi breed. The affected dogs presented clinical signs of muscular dystrophy. Immunostaining revealed the absence of dystrophin and upregulation of utrophin. Histopathologic examination showed variable fiber size, central nucleation, calcification, fibrosis, neutrophil and macrophage infiltration and cardiac focal vacuolar degeneration. Carrier dogs also displayed mild myopathy. The mutation was identified as a long interspersed repetitive element-1 (LINE-1) insertion in intron 13, which introduced a new exon containing an in-frame stop codon. Similar mutations have been seen in human patients. A colony was generated by crossing carrier females with normal males. Affected puppies had a normal birth weight but they experienced a striking growth delay in the first 5 days. In summary, the new corgi DMD model offers an excellent opportunity to study DMD pathogenesis and to develop novel therapies. PMID:20714321

  20. Absence of Glial α-Dystrobrevin Causes Abnormalities of the Blood-Brain Barrier and Progressive Brain Edema*

    PubMed Central

    Lien, Chun Fu; Mohanta, Sarajo Kumar; Frontczak-Baniewicz, Malgorzata; Swinny, Jerome D.; Zablocka, Barbara; Górecki, Dariusz C.

    2012-01-01

    The blood-brain barrier (BBB) plays a key role in maintaining brain functionality. Although mammalian BBB is formed by endothelial cells, its function requires interactions between endotheliocytes and glia. To understand the molecular mechanisms involved in these interactions is currently a major challenge. We show here that α-dystrobrevin (α-DB), a protein contributing to dystrophin-associated protein scaffolds in astrocytic endfeet, is essential for the formation and functioning of BBB. The absence of α-DB in null brains resulted in abnormal brain capillary permeability, progressively escalating brain edema, and damage of the neurovascular unit. Analyses in situ and in two-dimensional and three-dimensional in vitro models of BBB containing α-DB-null astrocytes demonstrated these abnormalities to be associated with loss of aquaporin-4 water and Kir4.1 potassium channels from glial endfeet, formation of intracellular vacuoles in α-DB-null astrocytes, and defects of the astrocyte-endothelial interactions. These caused deregulation of tight junction proteins in the endothelia. Importantly, α-DB but not dystrophins showed continuous expression throughout development in BBB models. Thus, α-DB emerges as a central organizer of dystrophin-associated protein in glial endfeet and a rare example of a glial protein with a role in maintaining BBB function. Its abnormalities might therefore lead to BBB dysfunction. PMID:23043099

  1. Dystrophin and Dysferlin Double Mutant Mice: A Novel Model For Rhabdomyosarcoma

    PubMed Central

    Hosur, Vishnu; Kavirayani, Anoop; Riefler, Jennifer; Carney, Lisa M.B.; Lyons, Bonnie; Gott, Bruce; Cox, Gregory A.; Shultz, Leonard D.

    2012-01-01

    While researchers are yet to establish a link a between muscular dystrophy (MD) and sarcomas in human patients, literature suggests that MD genes dystrophin and dysferlin act as tumor suppressor genes in mouse models of MD. For instance, dystrophin deficient mdx and dysferlin deficient A/J mice, models of human Duchenne Muscular Dystrophy and Limb Girdle Muscular Dystrophy type 2B, respectively, develop mixed sarcomas with variable penetrance and latency. To further establish the correlation between MD and sarcoma development, and to test whether a combined deletion of dystrophin and dysferlin exacerbates MD and augments the incidence of sarcomas, we generated dystrophin and dysferlin double mutant mice (STOCK-Dysfprmd Dmdmdx-5Cv). Not surprisingly, the double mutant mice develop severe MD symptoms and moreover develop rhabdomyosarcoma at an average age of 12 months, with an incidence of > 90%. Histological and immunohistochemical analyses, using a panel of antibodies against skeletal muscle cell proteins, electron microscopy, cytogenetics, and molecular analysis reveal that the double mutant mice develop rhabdomyosarcoma. The present finding bolsters the correlation between MD and sarcomas, and provides a model not only to examine the cellular origins but also to identify mechanisms and signal transduction pathways triggering development of RMS. PMID:22682622

  2. Dystrophin and the two related genetic diseases, Duchenne and Becker muscular dystrophies

    PubMed Central

    Rumeur, Elisabeth Le

    2015-01-01

    Mutations of the dystrophin DMD gene, essentially deletions of one or several exons, are the cause of two devastating and to date incurable diseases, Duchenne (DMD) and Becker (BMD) muscular dystrophies. Depending upon the preservation or not of the reading frame, dystrophin is completely absent in DMD, or present in either a mutated or a truncated form in BMD. DMD is a severe disease which leads to a premature death of the patients. Therapy approaches are evolving with the aim to transform the severe DMD in the BMD form of the disease by restoring the expression of a mutated or truncated dystrophin. These therapies are based on the assumption that BMD is a mild disease. However, this is not completely true as BMD patients are more or less severely affected and no molecular basis of this heterogeneity of the BMD form of the disease is yet understood. The aim of this review is to report for the correlation between dystrophin structures in BMD deletions in view of this heterogeneity and to emphasize that examining BMD patients in details is highly relevant to anticipate for DMD therapy effects. PMID:26295289

  3. Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

    PubMed Central

    Jearawiriyapaisarn, Natee; Moulton, Hong M; Buckley, Brian; Roberts, Jennifer; Sazani, Peter; Fucharoen, Suthat; Iversen, Patrick L; Kole, Ryszard

    2009-01-01

    Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or β-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals. PMID:18545222

  4. Study about locomotory ability of dystrophin-defected C.elegans after spaceflight

    NASA Astrophysics Data System (ADS)

    Gao, Ying; Sun, Yeqing; Lei, Huang; Xu, Dan

    2012-07-01

    Space microgravity could induce a variety of biological changes such as muscular atrophy. Recent studies show that gravisensing is a key point in muscular atrophy process, but the molecular mechanism is still unknown. Dystrophin, a muscle-related protein, plays an important role in muscle development. It is reported that mutation of human dystrophin gene could cause muscular atrophy. In this study, we focus on whether dystrophin gene acts as a gravisensing factor and observe locomotory ability of dystrophin-defected Caenorhabditis elegans (C.elegans) after spaceflight. We used wild-type (WT) and dystrophin-defected (dys-1) mutant of C.elegans, which were cultured to dauer stage and sent to space by Shenzhou 8 spacecraft (from Nov 1st to 17th, 2011). These worms were divided into three groups: space group (space radiation and microgravity conditions), space control group (space radiation and chmetcnvTCSC0NumberType1NegativeFalseHasSpaceFalseSourceValue1UnitNameg1g centrifuge force conditions) and ground control group.We already observed the progeny (generation F1 and F2) of worms which were sent to space, the movement of C. elegans is restricted to a two-dimensional sinusoidal pattern, and evaluated locomotory ability by the ratio (length/width) in crawl trace wave of C. elegans. The increased value of ratio indicates the decrease in locomotory ability of C. elegans. Our results from generation F1 showed that WT worms in space group(7.7±1.8) demonstrated the significant decrease in locomotory ability about 15%, compared with those in space control group(6.7±1.2). This finding indicates that locomotory ability of C. elegans progeny could be affected by microgravity in space environment. In comparison to the obvious difference in ratio between space group and space control group for WT worms, there is no significant difference between two space groups of generation F2 .For dys-1 mutant of C.elegans (generation F1 and F2), the results show that dystrophin deficiency

  5. Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

    PubMed Central

    Winnard, A V; Mendell, J R; Prior, T W; Florence, J; Burghes, A H

    1995-01-01

    Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7825572

  6. Our trails and trials in the subsarcolemmal cytoskeleton network and muscular dystrophy researches in the dystrophin era

    PubMed Central

    OZAWA, Eijiro

    2010-01-01

    In 1987, about 150 years after the discovery of Duchenne muscular dystrophy (DMD), its responsible gene, the dystrophin gene, was cloned by Kunkel. This was a new substance. During these 20 odd years after the cloning, our understanding on dystrophin as a component of the subsarcolemmal cytoskeleton networks and on the pathomechanisms of and experimental therapeutics for DMD has been greatly enhanced. During this paradigm change, I was fortunately able to work as an active researcher on its frontiers for 12 years. After we discovered that dystrophin is located on the cell membrane in 1988, we studied the architecture of dystrophin and dystrophin-associated proteins (DAPs) complex in order to investigate the function of dystrophin and pathomechanism of DMD. During the conduct of these studies, we came to consider that the dystrophin–DAP complex serves to transmembranously connect the subsarcolemmal cytoskeleton networks and basal lamina to protect the lipid bilayer. It then became our working hypothesis that injury of the lipid bilayer upon muscle contraction is the cause of DMD. During this process, we predicted that subunits of the sarcoglycan (SG) complex are responsible for respective types of DMD-like muscular dystrophy with autosomal recessive inheritance. Our prediction was confirmed to be true by many researchers including ourselves. In this review, I will try to explain what we observed and how we considered concerning the architecture and function of the dystrophin–DAP complex, and the pathomechanisms of DMD and related muscular dystrophies. PMID:20948175

  7. Phosphorylation within the cysteine-rich region of dystrophin enhances its association with β-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting

    PubMed Central

    Swiderski, Kristy; Shaffer, Scott A.; Gallis, Byron; Odom, Guy L.; Arnett, Andrea L.; Scott Edgar, J.; Baum, Dale M.; Chee, Annabel; Naim, Timur; Gregorevic, Paul; Murphy, Kate T.; Moody, James; Goodlett, David R.; Lynch, Gordon S.; Chamberlain, Jeffrey S.

    2014-01-01

    Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin–glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin–dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin. PMID:25082828

  8. Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

    PubMed Central

    Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Perez-Pinera, Pablo; Brown, Matthew T; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons. PMID:25492562

  9. Relatively low proportion of dystrophin gene deletions in Israeli Duchenne and Becker muscular dystrophy patients.

    PubMed

    Shomrat, R; Gluck, E; Legum, C; Shiloh, Y

    1994-02-15

    Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these to exons 44-45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. PMID:8160727

  10. Finding the sweet spot: Assembly and Glycosylation of the Dystrophin-Associated Glycoprotein Complex

    PubMed Central

    Townsend, DeWayne

    2014-01-01

    The dystrophin-associated glycoprotein complex (DGC) is a collection of glycoproteins that are essential for the normal function of striated muscle and many other tissues. Recent genetic studies have implicated the components of this complex in over a dozen forms of muscular dystrophy. Furthermore, disruption of the DGC has been implicated in many forms of acquired disease. This review aims to summarize the current state of knowledge regarding the processing and assembly of dystrophin associated proteins with a focus primarily on the dystroglycan heterodimer and the sarcoglycan complex. These proteins form the transmembrane portion of the DGC and undergo a complex multi-step processing with proteolytic cleavage, differential assembly, and both N- and O-glycosylation. The enzymes responsible for this processing and a model describing the sequence and subcellular localization of these events are discussed. PMID:25125182

  11. Relatively low proportion of dystrophin gene deletions in Israeili Duchenne and Becker muscular dystrophy patients

    SciTech Connect

    Shomrat, R.; Gluck, E.; Legum, C.; Shiloh, Y.

    1994-02-15

    Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these exons 44-45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. 43 refs., 1 fig., 1 tab.

  12. Restoration of half the normal dystrophin sequence in a double-deletion Duchenne muscular dystrophy family

    SciTech Connect

    Hoop, R.C.; Schwartz, L.S.; Hoffman, E.P.; Russo, L.S.; Riconda, D.L.

    1994-02-01

    Two male cousins with Duchenne muscular dystrophy were found to have different maternal dystrophin gene haplotypes and different deletion mutations. One propositus showed two noncontiguous deletions-one in the 5{prime}, proximal deletional hotspot region, and the other in the 3{prime}, more distal deletional hotspot region. The second propositus showed only the 5{prime} deletion. Using multiple fluorescent exon dosage and fluorescent multiplex CA repeat linkage analyses, the authors show that the mother of each propositus carries both deletions on the same grandmaternal X chromosome. This paradox is explained by a single recombinational event between the 2 deleted regions of one of the carrier`s dystrophin genes, giving rise to a son with a partially {open_quotes}repaired{close_quotes} gene retaining only the 5{prime} deletion. 20 refs., 4 figs.

  13. Dystrophin Gene Replacement and Gene Repair Therapy for Duchenne Muscular Dystrophy in 2016: An Interview.

    PubMed

    Duan, Dongsheng

    2016-03-01

    After years of relentless efforts, gene therapy has now begun to deliver its therapeutic promise in several diseases. A number of gene therapy products have received regulatory approval in Europe and Asia. Duchenne muscular dystrophy (DMD) is an X-linked inherited lethal muscle disease. It is caused by mutations in the dystrophin gene. Replacing and/or repairing the mutated dystrophin gene holds great promises to treated DMD at the genetic level. Last several years have evidenced significant developments in preclinical experimentations in murine and canine models of DMD. There has been a strong interest in moving these promising findings to clinical trials. In light of rapid progress in this field, the Parent Project Muscular Dystrophy (PPMD) recently interviewed me on the current status of DMD gene therapy and readiness for clinical trials. Here I summarized the interview with PPMD. PMID:27003751

  14. Intellectual ability in the duchenne muscular dystrophy and dystrophin gene mutation location.

    PubMed

    Milic Rasic, V; Vojinovic, D; Pesovic, J; Mijalkovic, G; Lukic, V; Mladenovic, J; Kosac, A; Novakovic, I; Maksimovic, N; Romac, S; Todorovic, S; Savic Pavicevic, D

    2014-12-01

    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD) gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation-dependent probe amplification (MLPA), polymerase chain reaction (PCR)] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale). In 37 patients with an estimated full scale intelligence quotient (FSIQ), six (16.22%) had borderline intelligence (70dystrophin isoforms and when mutations in the 5'-untranslated region (5'UTR) of Dp140 (exons 45-50) were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients. PMID:25937795

  15. Orphan drug development in muscular dystrophy: update on two large clinical trials of dystrophin rescue therapies.

    PubMed

    Hoffman, Eric P; Connor, Edward M

    2013-11-01

    Duchenne muscular dystrophy is a relatively common 'rare disorder,' with an incidence of about 1/5,000 males worldwide. The responsible gene and deficient protein (dystrophin) were identified in 1987, an early success of human molecular genetics and emerging genome projects. A rational approach to therapeutics is to replace dystrophin in patient muscle, thus addressing the primary biochemical defect. Fast forward 25 years, and two phase 2b/3 trials have been carried out with agents designed to induce de novo dystrophin production in DMD patient's muscle; ataluren (stop codon read through) with 174 patients, and drisapersen (exon skipping) with 186 patients. Both used a six minute walk test as the primary outcome measure. Neither drisapersen nor high dose ataluren showed any significant improvement in this outcome, whereas low dose ataluren is reported to show some possible improvement. Experience with ataluren and drisapersen has been disappointing and this is a good time to ask: What can we learn from these programs and how can this inform further drug development in DMD? At the times these two trials were started, there was a lack of existing data and infrastructure regarding both clinical and biochemical outcome measures. The recent publications of more extensive natural history data in multiple DMD cohorts, and ongoing efforts to define reliable and sensitive dystrophin assays are important. If the drisapersen and ataluren programs were instead begun today, new progress in biochemical and clinical endpoints may have triggered a re-design, with better de-risking in phase 2 studies prior to resource-intensive phase 3 trials. PMID:24229740

  16. Ex Vivo Stretch Reveals Altered Mechanical Properties of Isolated Dystrophin-Deficient Hearts

    PubMed Central

    Barnabei, Matthew S.; Metzger, Joseph M.

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD. PMID:22427904

  17. Intellectual Ability in the Duchenne Muscular Dystrophy and Dystrophin Gene Mutation Location

    PubMed Central

    Milic Rasic, V; Vojinovic, D; Pesovic, J; Mijalkovic, G; Lukic, V; Mladenovic, J; Kosac, A; Novakovic, I; Maksimovic, N; Romac, S; Todorovic, S; Savic Pavicevic, D

    2014-01-01

    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD) gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation-dependent probe amplification (MLPA), polymerase chain reaction (PCR)] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale). In 37 patients with an estimated full scale intelligence quotient (FSIQ), six (16.22%) had borderline intelligence (70dystrophin isoforms and when mutations in the 5′-untranslated region (5′UTR) of Dp140 (exons 45–50) were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients. PMID:25937795

  18. Gamma1- and gamma2-syntrophins, two novel dystrophin-binding proteins localized in neuronal cells.

    PubMed

    Piluso, G; Mirabella, M; Ricci, E; Belsito, A; Abbondanza, C; Servidei, S; Puca, A A; Tonali, P; Puca, G A; Nigro, V

    2000-05-26

    Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3' terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (alpha1) and two basic (beta1 and beta2) proteins of approximately 57-60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named gamma1- and gamma2-syntrophins. The human gamma1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human gamma2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the gamma1-syntrophin gene to human chromosome 8q11 and the gamma2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system. PMID:10747910

  19. Gene therapies that restore dystrophin expression for the treatment of Duchenne muscular dystrophy.

    PubMed

    Robinson-Hamm, Jacqueline N; Gersbach, Charles A

    2016-09-01

    Duchenne muscular dystrophy is one of the most common inherited genetic diseases and is caused by mutations to the DMD gene that encodes the dystrophin protein. Recent advances in genome editing and gene therapy offer hope for the development of potential therapeutics. Truncated versions of the DMD gene can be delivered to the affected tissues with viral vectors and show promising results in a variety of animal models. Genome editing with the CRISPR/Cas9 system has recently been used to restore dystrophin expression by deleting one or more exons of the DMD gene in patient cells and in a mouse model that led to functional improvement of muscle strength. Exon skipping with oligonucleotides has been successful in several animal models and evaluated in multiple clinical trials. Next-generation oligonucleotide formulations offer significant promise to build on these results. All these approaches to restoring dystrophin expression are encouraging, but many hurdles remain. This review summarizes the current state of these technologies and summarizes considerations for their future development. PMID:27542949

  20. Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy

    PubMed Central

    Risson, Valérie; Mazelin, Laetitia; Roceri, Mila; Sanchez, Hervé; Moncollin, Vincent; Corneloup, Claudine; Richard-Bulteau, Hélène; Vignaud, Alban; Baas, Dominique; Defour, Aurélia; Freyssenet, Damien; Tanti, Jean-François; Le-Marchand-Brustel, Yannick; Ferrier, Bernard; Conjard-Duplany, Agnès; Romanino, Klaas; Bauché, Stéphanie; Hantaï, Daniel; Mueller, Matthias; Kozma, Sara C.; Thomas, George; Rüegg, Markus A.; Ferry, Arnaud; Pende, Mario; Bigard, Xavier; Koulmann, Nathalie

    2009-01-01

    Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent. PMID:20008564

  1. CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice.

    PubMed

    Xu, Li; Park, Ki Ho; Zhao, Lixia; Xu, Jing; El Refaey, Mona; Gao, Yandi; Zhu, Hua; Ma, Jianjie; Han, Renzhi

    2016-03-01

    Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD. PMID:26449883

  2. Fate of the human Y chromosome linked genes and loci in prostate cancer cell lines DU145 and LNCaP

    PubMed Central

    2013-01-01

    Background Prostate cancer is a known cause of mortality in men worldwide although the risk factor varies among different ethnic groups. Loss of the Y chromosome is a common chromosomal abnormality observed in the human prostate cancer. Results We screened 51 standard sequence tagged sites (STSs) corresponding to a male-specific region of the Y chromosome (MSY), sequenced the coding region of the SRY gene and assessed the status of the DYZ1 arrays in the human prostate cancer cell lines DU145 and LNCaP. The MSY was found to be intact and coding region of SRY showed no sequence variation in both the cell lines. However, DYZ1 arrays showed sequence and copy number variations. DU145 and LNCaP cells were found to carry 742 and 1945 copies of the DYZ1, respectively per 3.3 pg of genomic DNA. The DYZ1 copies detected in these cell lines are much below the average of that reported in normal human males. Similarly, the number of “TTCCA” repeat and its derivatives within the DYZ1 arrays showed variation compared to those of the normal males. Conclusions Clearly, the DYZ1 is maximally affected in both the cell lines. Work on additional cell lines and biopsied samples would augment our understanding about the susceptibility of this region. Based on the present work, we construe that copy number status of the DYZ1 may be exploited as a supplementary prognostic tool to monitor the occurrence of prostate cancer using biopsied samples. PMID:23663454

  3. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

    PubMed Central

    Cação-Benedini, L.O.; Ribeiro, P.G.; Prado, C.M.; Chesca, D.L.; Mattiello-Sverzut, A.C.

    2014-01-01

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres. PMID:24820070

  4. Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations that Cause Duchenne Muscular Dystrophy

    PubMed Central

    Ousterout, David G.; Kabadi, Ami M.; Thakore, Pratiksha I.; Majoros, William H.; Reddy, Timothy E.; Gersbach, Charles A.

    2015-01-01

    The CRISPR/Cas9 genome editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45–55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations. PMID:25692716

  5. Cognitive dysfunction in the dystrophin-deficient mouse model of Duchenne muscular dystrophy: A reappraisal from sensory to executive processes.

    PubMed

    Chaussenot, Rémi; Edeline, Jean-Marc; Le Bec, Benoit; El Massioui, Nicole; Laroche, Serge; Vaillend, Cyrille

    2015-10-01

    Duchenne muscular dystrophy (DMD) is associated with language disabilities and deficits in learning and memory, leading to intellectual disability in a patient subpopulation. Recent studies suggest the presence of broader deficits affecting information processing, short-term memory and executive functions. While the absence of the full-length dystrophin (Dp427) is a common feature in all patients, variable mutation profiles may additionally alter distinct dystrophin-gene products encoded by separate promoters. However, the nature of the cognitive dysfunctions specifically associated with the loss of distinct brain dystrophins is unclear. Here we show that the loss of the full-length brain dystrophin in mdx mice does not modify the perception and sensorimotor gating of auditory inputs, as assessed using auditory brainstem recordings and prepulse inhibition of startle reflex. In contrast, both acquisition and long-term retention of cued and trace fear memories were impaired in mdx mice, suggesting alteration in a functional circuit including the amygdala. Spatial learning in the water maze revealed reduced path efficiency, suggesting qualitative alteration in mdx mice learning strategy. However, spatial working memory performance and cognitive flexibility challenged in various behavioral paradigms in water and radial-arm mazes were unimpaired. The full-length brain dystrophin therefore appears to play a role during acquisition of associative learning as well as in general processes involved in memory consolidation, but no overt involvement in working memory and/or executive functions could be demonstrated in spatial learning tasks. PMID:26190833

  6. Systemic administration of micro-dystrophin restores cardiac geometry and prevents dobutamine-induced cardiac pump failure.

    PubMed

    Townsend, DeWayne; Blankinship, Michael J; Allen, James M; Gregorevic, Paul; Chamberlain, Jeffrey S; Metzger, Joseph M

    2007-06-01

    Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration resulting from the loss of the cytoskeletal protein dystrophin. Most patients develop significant cardiomyopathy, with heart failure being the second leading cause of death in DMD. Compared with the extensive studies on skeletal muscle defects and potential therapy in DMD, very little attention has been directed at the increasing incidence of heart failure in DMD. Here we show that a single systemic injection of recombinant adeno-associated virus (rAAV2/6) harboring micro-dystrophin leads to extensive cardiac transduction, with micro-dystrophin correctly localized at the periphery of the cardiac myocytes and functionally associated with the sarcolemmal membrane. Significantly, micro-dystrophin gene transfer corrected the baseline end-diastolic volume defect in the mdx mouse heart and prevented cardiac pump failure induced by dobutamine stress testing in vivo, although several parameters of systolic function were not corrected. These results demonstrate that systemic gene delivery of micro-dystrophin can restore ventricular distensibility and protect the mdx myocardium from pump dysfunction during adrenergic stimulation in vivo. PMID:17440445

  7. Exon skipping restores dystrophin expression, but fails to prevent disease progression in later stage dystrophic dko mice

    PubMed Central

    Wu, B; Cloer, C; Lu, P; Milazi, S; Shaban, M; Shah, SN; Marston-Poe, L; Moulton, HM; Lu, QL

    2014-01-01

    Antisense therapy with both chemistries of phosphorodiamidate morpholino oligomers (PMOs) and 2′-O-methyl phosphorothioate has demonstrated the capability to induce dystrophin expression in Duchenne muscular dystrophy (DMD) patients in phase II–III clinical trials with benefit in muscle functions. However, potential of the therapy for DMD at different stages of the disease progression is not understood. In this study, we examined the effect of peptide-conjugated PMO (PPMO)-mediated exon skipping on disease progression of utrophin-dystrophin-deficient mice (dko) of four age groups (21–29, 30–39, 40–49 and 50+ days), representing diseases from early stage to advanced stage with severe kyphosis. Biweekly intravenous (i.v.) administration of the PPMO restored the dystrophin expression in nearly 100% skeletal muscle fibers in all age groups. This was associated with the restoration of dystrophin-associated proteins including functional glycosylated dystroglycan and neuronal nitric synthase. However, therapeutic outcomes clearly depended on severity of the disease at the time the treatment started. The PPMO treatment alleviated the disease pathology and significantly prolonged the life span of the mice receiving treatment at younger age with mild phenotype. However, restoration of high levels of dystrophin expression failed to prevent disease progression to the mice receiving treatment when disease was already at advanced stage. The results could be critical for design of clinical trials with antisense therapy to DMD. PMID:24942628

  8. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin

    SciTech Connect

    Thanh, L.T.; Man, Nguyen Thi; Morris, G.E.

    1995-08-28

    We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immunohistochemistry or Western blotting with these {open_quotes}exon-specific{close_quotes} mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying {open_quotes}revertant{close_quotes} fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene. 27 refs., 7 figs., 3 tabs.

  9. Becker Muscular Dystrophy (BMD) caused by duplication of exons 3-6 of the dystrophin gene presenting as dilated cardiomyopathy

    SciTech Connect

    Tsai, A.C.; Allingham-Hawkins, D.J.; Becker, L.

    1994-09-01

    X-linked dilated cardiomyopathy (XLCM) is a progressive myocardial disease presenting with congestive heart failure in teenage males without clinical signs of skeletal myopathy. Tight linkage of XLCM to the DMD locus has been demonstrated; it has been suggested that, at least in some families, XLCM is a {open_quotes}dystrophinopathy.{close_quotes} We report a 14-year-old boy who presented with acute heart failure due to dilated cardiomyopathy. He had no history of muscle weakness, but physical examination revealed pseudohypertrophy of the calf muscles. He subsequently received a heart transplantation. Family history was negative. Serum CK level at the time of diagnosis was 10,416. Myocardial biopsy showed no evidence of carditis. Dystrophin staining of cardiac and skeletal muscle with anti-sera to COOH and NH{sub 2}termini showed a patchy distribution of positivity suggestive of Becker muscular dystrophy. Analysis of 18 of the 79 dystrophin exons detected a duplication that included exons 3-6. The proband`s mother has an elevated serum CK and was confirmed to be a carrier of the same duplication. A mutation in the muscle promotor region of the dystrophin gene has been implicated in the etiology of SLCM. However, Towbin et al. (1991) argued that other 5{prime} mutations in the dystrophin gene could cause selective cardiomyopathy. The findings in our patient support the latter hypothesis. This suggests that there are multiple regions in the dystrophin gene which, when disrupted, can cause isolated dilated cardiomyopathy.

  10. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.

    PubMed

    Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

    2015-01-01

    The CRISPR/Cas9 genome-editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene-editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations. PMID:25692716

  11. Myofibrillar myopathy with abnormal foci of desmin positivity. II. Immunocytochemical analysis reveals accumulation of multiple other proteins.

    PubMed

    De Bleecker, J L; Engel, A G; Ertl, B B

    1996-05-01

    The two major types of lesions in myofibrillar myopathy consist of hyaline spheroidal structures composed of compacted myofibrillar residues, and nonhyaline lesions that comprise foci of myofibrillar destruction. We employed immunocytochemical analysis to further characterize these abnormalities. The nonhyaline lesions are depleted of actin, alpha-actinin, myosin, and, less consistently, of titin and nebulin. Thus, each major component of the myofibrils is lost or decreased. These lesions also react strongly for both NCAM and desmin. By contrast, the hyaline structures are highly enriched in actin, are immunoreactive for fast and slow myosin, and show increased expression of titin, nebulin, and alpha-actinin. They fail to react for NCAM and react variably for desmin. Both types of lesion react, but with differing intensities, for gelsolin, dystrophin, beta-amyloid precursor protein (beta APP) epitopes amino-terminal to the alpha-secretase site, alpha 1-antichymotrypsin, and ubiquitin, and both can be congophilic. The increased expressions of desmin, dystrophin and gelsolin in muscle are also confirmed by immunoblot studies. The results, in harmony with the ultrastructural findings described in the companion paper, suggest that myofibrillar myopathy is conditioned by abnormal activation of a degradative process that primarily affects the myofibrils. A structural abnormality of desmin alone may not be sufficient to disrupt the myofibrillar architecture, but abnormal activation of a phosphorylating process could account for dissolution of the myofibrils. The cause and significance of the ectopic overexpression of desmin, dystrophin, NCAM, and beta APP components, and the chemical basis of the congophilia remain unknown. PMID:8627347

  12. Revisiting the dystrophin-ATP connection: How half a century of research still implicates mitochondrial dysfunction in Duchenne Muscular Dystrophy aetiology.

    PubMed

    Timpani, Cara A; Hayes, Alan; Rybalka, Emma

    2015-12-01

    Duchenne Muscular Dystrophy (DMD) is a fatal neuromuscular disease that is characterised by dystrophin-deficiency and chronic Ca(2+)-induced skeletal muscle wasting, which currently has no cure. DMD was once considered predominantly as a metabolic disease due to the myriad of metabolic insufficiencies evident in the musculature, however this aspect of the disease has been extensively ignored since the discovery of dystrophin. The collective historical and contemporary literature documenting these metabolic nuances has culminated in a series of studies that importantly demonstrate that metabolic dysfunction exists independent of dystrophin expression and a mild disease phenotype can be expressed even in the complete absence of dystrophin expression. Targeting and supporting metabolic pathways with anaplerotic and other energy-enhancing supplements has also shown therapeutic value. We explore the hypothesis that DMD is characterised by a systemic mitochondrial impairment that is central to disease aetiology rather than a secondary pathophysiological consequence of dystrophin-deficiency. PMID:26365249

  13. 5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.

    PubMed Central

    Hansen, R S; Gartler, S M

    1990-01-01

    Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5' CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region. Images PMID:1693431

  14. Dystrophin expression following the transplantation of normal muscle precursor cells protects mdx muscle from contraction-induced damage.

    PubMed

    Rousseau, Joel; Dumont, Nicolas; Lebel, Carl; Quenneville, Simon P; Côté, Claude H; Frenette, Jérome; Tremblay, Jacques P

    2010-01-01

    Duchenne muscular dystrophy (DMD) is the most frequent muscular dystrophy. Currently, there is no cure for the disease. The transplantation of muscle precursor cells (MPCs) is one of the possible treatments, because it can restore the expression of dystrophin in DMD muscles. In this study, we investigated the effects of myoblasts injected with cardiotoxin on the contractile properties and resistance to eccentric contractions of transplanted and nontransplanted muscles. We used the extensor digitorum longus (EDL) as a model for our study. We conclude that the sole presence of dystrophin in a high percentage of muscle fibers is not sufficient by itself to increase the absolute or the specific force in the EDL of transplanted mdx muscle. This lack of strength increase may be due to the extensive damage that was produced by the cardiotoxin, which was coinjected with the myoblasts. However, the dystrophin presence is sufficient to protect muscle from eccentric damage as indicated by the force drop results. PMID:20650035

  15. Abnormal Head Position

    MedlinePlus

    ... cause. Can a longstanding head turn lead to any permanent problems? Yes, a significant abnormal head posture could cause permanent ... occipitocervical synostosis and unilateral hearing loss. Are there any ... postures? Yes. Abnormal head postures can usually be improved depending ...

  16. Urine - abnormal color

    MedlinePlus

    ... straw-yellow. Abnormally colored urine may be cloudy, dark, or blood-colored. Causes Abnormal urine color may ... red blood cells, or mucus in the urine. Dark brown but clear urine is a sign of ...

  17. A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

    PubMed Central

    Beekman, Chantal; Sipkens, Jessica A.; Testerink, Janwillem; Giannakopoulos, Stavros; Kreuger, Dyonne; van Deutekom, Judith C.; Campion, Giles V.; de Kimpe, Sjef J.; Lourbakos, Afrodite

    2014-01-01

    Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound. PMID:25244123

  18. Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle fibers.

    PubMed

    Decrouy, A; Renaud, J M; Lunde, J A; Dickson, G; Jasmin, B J

    1998-01-01

    In normal skeletal muscle fibers, dystrophin accumulates at the cytoplasmic face of the sarcolemma where it associates with dystrophin-associated proteins (DAPs). Several studies have recently shown that the neuronal isoform of nitric oxide synthase (nNOS) is also located at the sarcolemma, and that this membrane localization is mediated through interactions of nNOS with one of the DAPs, namely alpha 1-syntrophin. Since the lack of dystrophin in muscle fibers from Duchenne muscular dystrophy patients and mdx mice is accompanied by an absence of sarcolemmal nNOS, we examined in the present study, whether dystrophin gene replacement would lead to the restoration of nNOS at its appropriate subcellular location. To this end, tibialis anterior muscles from mdx4cv mice were directly injected with plasmid DNA encoding either full-length (pRSV-dys) or mini-(pRSV-dyB; lacking exons 17-48) dystrophin. For these experiments, we chose to study 10-week-old mdx4cv mice since at this developmental stage, muscles from these mice have already undergone several cycles of degeneration-regeneration. Immunofluorescence experiments performed on serial cross-sections revealed that approximately 50% of the dystrophin-positive fibers also exhibited significant levels of nNOS at their sarcolemma 2 weeks following gene transfer with pRSV-dys. Similar results were obtained with pRSV-dyB indicating that exons 17-48 of the dystrophin gene are not essential for the correct localization of nNOS in skeletal muscle fibers. Taken together with the recent demonstration that dystrophin gene transfer leads to significant physiological benefits our results suggest that dystrophin gene therapy using full-length or truncated dystrophin, also induces a rapid recovery of biochemical functions. PMID:9536265

  19. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies.

    PubMed

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M; Krishnaswarmy, Sudarsan; Wong, Brenda L; Fletcher, Sue; Wilton, Steve D

    2016-01-01

    Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes. PMID:26745801

  20. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

    PubMed Central

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M.; Krishnaswarmy, Sudarsan; Wong, Brenda L.; Fletcher, Sue; Wilton, Steve D.

    2016-01-01

    Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes. PMID:26745801

  1. Abnormal coagulation factor VIII transcript in a Tennessee Walking Horse colt with hemophilia A.

    PubMed

    Norton, Elaine M; Wooldridge, Anne A; Stewart, Allison J; Cusimano, Layla; Schwartz, Dean D; Johnson, Calvin M; Boudreaux, Mary K; Christopherson, Pete W

    2016-03-01

    Hemophilia A is an X-chromosome-linked disorder caused by a deficiency in factor VIII (FVIII). Although foals have been diagnosed with hemophilia A based on deficiency in FVIII activity, causative gene mutations have not been identified. The genomic DNA and cDNA encoding FVIII of a Tennesee Walking Horse colt affected with hemophilia A and the genomic DNA of his dam and a normal unrelated horse were analyzed with no splice site or coding sequence abnormalities identified in any of the horses. Polymerase chain reactions (PCR) were then performed on hepatic cDNA from the affected colt and an unrelated normal horse, and no product was obtained for the sequence between and including exon 1 and exon 2 in the affected colt. Based on these results, suspected mutations were identified in the noncoding region of FVIII (intron 1), and genomic sequencing of intron 1 in the dam and the affected colt suggested maternal inheritance. PMID:26765501

  2. Screening of point mutations by multiple SSCP analysis in the dystrophin gene

    SciTech Connect

    Lasa, A.; Baiget, M.; Gallano, P.

    1994-09-01

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimal for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.

  3. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

    PubMed

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  4. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

    PubMed Central

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S.; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  5. Cardiac expression of a mini-dystrophin that normalizes skeletal muscle force only partially restores heart function in aged Mdx mice.

    PubMed

    Bostick, Brian; Yue, Yongping; Long, Chun; Marschalk, Nate; Fine, Deborah M; Chen, Jing; Duan, Dongsheng

    2009-02-01

    Duchenne muscular dystrophy (DMD) affects both skeletal and cardiac muscle. It is currently unclear whether the strategies developed for skeletal muscle can ameliorate cardiomyopathy. Synthetic mini-/micro-dystrophin genes have yielded impressive skeletal muscle protection in animal models. The 6-kb DeltaH2-R19 minigene is particularly promising because it completely restores skeletal muscle force to wild-type levels. Here, we examined whether expressing this minigene in the heart, but not skeletal muscle, could normalize cardiac function in the mdx model of DMD cardiomyopathy. Transgenic mdx mice were generated to express the DeltaH2-R19 minigene under the control of the alpha-myosin heavy-chain promoter. Heart structure and function were examined in adult and very old mice. The DeltaH2-R19 minigene enhanced cardiomyocyte sarcolemmal strength and prevented myocardial fibrosis. It also restored the dobutamine response and enhanced treadmill performance. Surprisingly, heart-restricted DeltaH2-R19 minigene expression did not completely normalize electrocardiogram and hemodynamic abnormalities. Overall, systolic function and ejection fraction were restored to normal levels but stroke volume and cardiac output remained suboptimal. Our results demonstrate that the skeletal muscle-proven DeltaH2-R19 minigene can correct cardiac histopathology but cannot fully normalize heart function. Novel strategies must be developed to completely restore heart function in DMD. PMID:19066599

  6. Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle.

    PubMed

    Brolin, Camilla; Shiraishi, Takehiko; Hojman, Pernille; Krag, Thomas O; Nielsen, Peter E; Gehl, Julie

    2015-01-01

    Peptide nucleic acid (PNA) is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m.) PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA) muscle of normal NMRI and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find that electroporation can enhance PNA antisense effects in muscle tissue. PMID:26623939

  7. Complex genomic rearrangements in the dystrophin gene due to replication-based mechanisms

    PubMed Central

    Baskin, Berivan; Stavropoulos, Dimitri J; Rebeiro, Paige A; Orr, Jennifer; Li, Martin; Steele, Leslie; Marshall, Christian R; Lemire, Edmond G; Boycott, Kym M; Gibson, William; Ray, Peter N

    2014-01-01

    Genomic rearrangements such as intragenic deletions and duplications are the most prevalent type of mutations in the dystrophin gene resulting in Duchenne and Becker muscular dystrophy (D/BMD). These copy number variations (CNVs) are nonrecurrent and can result from either nonhomologous end joining (NHEJ) or microhomology-mediated replication-dependent recombination (MMRDR). We characterized five DMD patients with complex genomic rearrangements using a combination of MLPA/mRNA transcript analysis/custom array comparative hybridization arrays (CGH) and breakpoint sequence analysis to investigate the mechanisms for these rearrangements. Two patients had complex rearrangements that involved microhomologies at breakpoints. One patient had a noncontiguous insertion of 89.7 kb chromosome 4 into intron 43 of DMD involving three breakpoints with 2–5 bp microhomology at the junctions. A second patient had an inversion of exon 44 flanked by intronic deletions with two breakpoint junctions each showing 2 bp microhomology. The third patient was a female with an inherited deletion of exon 47 in DMD on the maternal allele and a de novo noncontiguous duplication of exons 45–49 in DMD and MID1 on the paternal allele. The other two patients harbored complex noncontiguous duplications within the dystrophin gene. We propose a replication-based mechanisms for all five complex DMD rearrangements. This study identifies additional underlying mechanisms in DMD, and provides insight into the molecular bases of these genomic rearrangements. PMID:25614876

  8. Dystrophin-deficient large animal models: translational research and exon skipping

    PubMed Central

    Yu, Xinran; Bao, Bo; Echigoya, Yusuke; Yokota, Toshifumi

    2015-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder caused by mutations in the dystrophin gene. Affecting approximately 1 in 3,600-9337 boys, DMD patients exhibit progressive muscle degeneration leading to fatality as a result of heart or respiratory failure. Despite the severity and prevalence of the disease, there is no cure available. While murine models have been successfully used in illustrating the mechanisms of DMD, their utility in DMD research is limited due to their mild disease phenotypes such as lack of severe skeletal muscle and cardiac symptoms. To address the discrepancy between the severity of disease displayed by murine models and human DMD patients, dystrophin-deficient dog models with a splice site mutation in intron 6 were established. Examples of these are Golden Retriever muscular dystrophy and beagle-based Canine X-linked muscular dystrophy. These large animal models are widely employed in therapeutic DMD research due to their close resemblance to the severity of human patient symptoms. Recently, genetically tailored porcine models of DMD with deleted exon 52 were developed by our group and others, and can potentially act as a new large animal model. While therapeutic outcomes derived from these large animal models can be more reliably extrapolated to DMD patients, a comprehensive understanding of these models is still needed. This paper will discuss recent progress and future directions of DMD studies with large animal models such as canine and porcine models. PMID:26396664

  9. Fetal skeletal muscle progenitors have regenerative capacity after intramuscular engraftment in dystrophin deficient mice.

    PubMed

    Sakai, Hiroshi; Sato, Takahiko; Sakurai, Hidetoshi; Yamamoto, Takuya; Hanaoka, Kazunori; Montarras, Didier; Sehara-Fujisawa, Atsuko

    2013-01-01

    Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3(GFP/+) embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3(GFP/+) mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease. PMID:23671652

  10. Fetal Skeletal Muscle Progenitors Have Regenerative Capacity after Intramuscular Engraftment in Dystrophin Deficient Mice

    PubMed Central

    Sakai, Hiroshi; Sato, Takahiko; Sakurai, Hidetoshi; Yamamoto, Takuya; Hanaoka, Kazunori; Montarras, Didier; Sehara-Fujisawa, Atsuko

    2013-01-01

    Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3GFP/+ embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3GFP/+ mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease. PMID:23671652

  11. Nonmechanical Roles of Dystrophin and Associated Proteins in Exercise, Neuromuscular Junctions, and Brains

    PubMed Central

    Nichols, Bailey; Takeda, Shin’ichi; Yokota, Toshifumi

    2015-01-01

    Dystrophin-glycoprotein complex (DGC) is an important structural unit in skeletal muscle that connects the cytoskeleton (f-actin) of a muscle fiber to the extracellular matrix (ECM). Several muscular dystrophies, such as Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophies (dystroglycanopathies), and limb-girdle muscular dystrophies (sarcoglycanopathies), are caused by mutations in the different DGC components. Although many early studies indicated DGC plays a crucial mechanical role in maintaining the structural integrity of skeletal muscle, recent studies identified novel roles of DGC. Beyond a mechanical role, these DGC members play important signaling roles and act as a scaffold for various signaling pathways. For example, neuronal nitric oxide synthase (nNOS), which is localized at the muscle membrane by DGC members (dystrophin and syntrophins), plays an important role in the regulation of the blood flow during exercise. DGC also plays important roles at the neuromuscular junction (NMJ) and in the brain. In this review, we will focus on recently identified roles of DGC particularly in exercise and the brain. PMID:26230713

  12. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

    PubMed Central

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  13. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    PubMed

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  14. Losartan decreases cardiac muscle fibrosis and improves cardiac function in dystrophin-deficient mdx mice.

    PubMed

    Spurney, Christopher F; Sali, Arpana; Guerron, Alfredo D; Iantorno, Micaela; Yu, Qing; Gordish-Dressman, Heather; Rayavarapu, Sree; van der Meulen, Jack; Hoffman, Eric P; Nagaraju, Kanneboyina

    2011-03-01

    Recent studies showed that chronic administration of losartan, an angiotensin II type I receptor antagonist, improved skeletal muscle function in dystrophin-deficient mdx mice. In this study, C57BL/10ScSn-Dmd(mdx)/J female mice were either untreated or treated with losartan (n = 15) in the drinking water at a dose of 600 mg/L over a 6-month period. Cardiac function was assessed via in vivo high frequency echocardiography and skeletal muscle function was assessed using grip strength testing, Digiscan monitoring, Rotarod timing, and in vitro force testing. Fibrosis was assessed using picrosirius red staining and Image J analysis. Gene expression was evaluated using real-time polymerized chain reaction (RT-PCR). Percentage shortening fraction was significantly decreased in untreated (26.9% ± 3.5%) mice compared to losartan-treated (32.2% ± 4.2%; P < .01) mice. Systolic blood pressure was significantly reduced in losartan-treated mice (56 ± 6 vs 69 ± 7 mm Hg; P < .0005). Percentage cardiac fibrosis was significantly reduced in losartan-treated hearts (P < .05) along with diaphragm (P < .01), extensor digitorum longus (P < .05), and gastrocnemius (P < .05) muscles compared to untreated mdx mice. There were no significant differences in skeletal muscle function between treated and untreated groups. Chronic treatment with losartan decreases cardiac and skeletal muscle fibrosis and improves cardiac systolic function in dystrophin-deficient mdx mice. PMID:21304057

  15. mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration

    PubMed Central

    Roberts, Thomas C.; Blomberg, K. Emelie M.; Smith, C.I. Edvard; EL Andaloussi, Samir; Wood, Matthew J.A.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA) microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1) the determination of gene expression changes associated with dystrophic pathology, (2) identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3) investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis), and (4) prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE64420. PMID:26981371

  16. Combination antisense treatment for destructive exon skipping of myostatin and open reading frame rescue of dystrophin in neonatal mdx mice

    PubMed Central

    Lu-Nguyen, Ngoc B.; Jarmin, Susan A.; Saleh, Amer F.; Popplewell, Linda; Gait, Michael J.; Dickson, George

    2015-01-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  17. Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

    PubMed

    Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George

    2015-08-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  18. Adhalin, the 50 kD dystrophin associated protein, is not the locus for severe childhood autosomal recessive dystrophy (SCARMD)

    SciTech Connect

    McNally, E.M.; Selig, S.; Kunkel, L.M.

    1994-09-01

    Mutations in the carboxyl-terminus in dystrophin are normally sufficient to produce severely dystrophic muscle. This portion of dystrophin binds a complex of dystrophin-associated glycoproteins (DAGs). The genes encoding these DAGs are candidate genes for causing neuromuscular disease. Immunoreactivity for adhalin, the 50 kD DAG, is absent in muscle biopsies from patients with SCARMD, a form of dystrophy clinically similar Duchenne muscular dystrophy. Prior linkage analysis in SCARMD families revealed that the disease gene segregates with markers on chromosome 13. To determine the molecular role that adhalin may play in SCARMD, human cDNA and genomic sequences were isolated. Primers were designed based on predicted areas of conservation in rabbit adhalin and used in RT-PCR with human skeletal and cardiac muscle. RT-PCR products were confirmed by sequence as human adhalin and then used as probes for screening human cDNA and genomic libraries. Human and rabbit adhalin are 90% identical, and among the cDNAs, a novel splice form of adhalin was seen which may encode part of the 35 kD component of the dystrophin-glycoprotein complex. To our surprise, only human/rodent hybrids containing human chromosome 17 amplified adhalin sequences in a PCR analysis. FISH analysis with three overlapping genomic sequences confirmed the chromosome 17 location and further delineated the map position to 17q21. Therefore, adhalin is excluded as the gene causing SCARMD.

  19. mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration.

    PubMed

    Roberts, Thomas C; Blomberg, K Emelie M; Smith, C I Edvard; El Andaloussi, Samir; Wood, Matthew J A

    2016-03-01

    Duchenne muscular dystrophy (DMD) is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA) microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1) the determination of gene expression changes associated with dystrophic pathology, (2) identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3) investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis), and (4) prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE64420. PMID:26981371

  20. Tooth - abnormal shape

    MedlinePlus

    Hutchinson incisors; Abnormal tooth shape; Peg teeth; Mulberry teeth; Conical teeth ... from many different conditions. Specific diseases can affect tooth shape, tooth color, time of appearance, or absence ...

  1. Tooth - abnormal shape

    MedlinePlus

    Hutchinson incisors; Abnormal tooth shape; Peg teeth; Mulberry teeth; Conical teeth ... The appearance of normal teeth varies, especially the molars. ... conditions. Specific diseases can affect tooth shape, tooth ...

  2. Bortezomib (PS-341) Treatment Decreases Inflammation and Partially Rescues the Expression of the Dystrophin-Glycoprotein Complex in GRMD Dogs

    PubMed Central

    Araujo, Karla P. C.; Bonuccelli, Gloria; Duarte, Caio N.; Gaiad, Thais P.; Moreira, Dayson F.; Feder, David; Belizario, José E.; Miglino, Maria A.; Lisanti, Michael P.; Ambrosio, Carlos E.

    2013-01-01

    Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin

  3. Phase 2a Study of Ataluren-Mediated Dystrophin Production in Patients with Nonsense Mutation Duchenne Muscular Dystrophy

    PubMed Central

    Finkel, Richard S.; Flanigan, Kevin M.; Wong, Brenda; Bönnemann, Carsten; Sampson, Jacinda; Sweeney, H. Lee; Reha, Allen; Northcutt, Valerie J.; Elfring, Gary; Barth, Jay; Peltz, Stuart W.

    2013-01-01

    Background Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124) enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. Methods This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6) received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20) was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12) was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. Findings Twenty three of 38 (61%) subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. Interpretation Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. Trial Registration ClinicalTrials.gov NCT00264888 PMID:24349052

  4. Structurally abnormal human autosomes

    SciTech Connect

    1993-12-31

    Chapter 25, discusses structurally abnormal human autosomes. This discussion includes: structurally abnormal chromosomes, chromosomal polymorphisms, pericentric inversions, paracentric inversions, deletions or partial monosomies, cri du chat (cat cry) syndrome, ring chromosomes, insertions, duplication or pure partial trisomy and mosaicism. 71 refs., 8 figs.

  5. [Dystrophin gene expression in patients with Duchenne muscular dystrophy after myoblast transplantation].

    PubMed

    Shishkin, S S; Terekhov, S M; Krokhina, T B; Shakhovskaia, N I; Podobedova, A N; Linnaia, G F; Tarasov, V I; Ovchinnikov, V I; Krakhmaleva, I N; Zakharov, S F; Ershova, E S; Limborskaia, S A; Pogoda, T V; Zotikov, E A; Kut'ina, R M; Tarksh, M A; Sukhorukov, V S; Gerasimova, N L

    2001-08-01

    Based on originally designed technique of myoblast cultivation and in accordance with the approved by the Russian Ministry of Health "one muscle treatment" protocol of myoblast transplantation to the Duchenne muscular dystrophy patients, the first in Russia clinical trial of this gene correction method was carried out. Immonologically related myoblast cultures (30 to 90 million cells per patient) were injected after all preliminary procedures into tibialis anterior muscles of four boys selected from a group of volunteer recipients (Duchenne muscular dystrophy patients) based on the analysis of a number of surface antigens in donor-recipient pairs. The condition of the patients remained satisfactory during the whole period of post-transplantation follow-up (from 6 months to 1.5 years). Six months after myoblast transplantation the presence of donor DNA or dystrophin synthesis was demonstrated in muscle biopsies of three out of four patients. This result confirms efficacy and safety of the procedure used. PMID:11642111

  6. Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

    SciTech Connect

    Rodriguez-Munoz, Rafael; Villarreal-Silva, Marcela; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Mondragon, Monica; Mondragon, Ricardo; Cerna, Joel; Cisneros, Bulmaro

    2008-10-24

    The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in Center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation.

  7. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice

    PubMed Central

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J. A.; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose–fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  8. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice.

    PubMed

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J A; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose-fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  9. Restriction Factors Against Recombinant Adeno-associated Virus Vectormediated Gene Transfer in Dystrophin-deficient Muscles.

    PubMed

    Dupont, Jean-Baptiste

    2016-01-01

    Despite the unprecedented beneficial effects of rAAV gene therapy in animal models of Duchenne muscular dystrophy (DMD), the need to inject large amounts of vector in vivo to improve phenotype raises obvious biosafety concerns. While rAAV vectors generally exhibit a good safety profile, specific pathological phenotypes such as those observed in dystrophin-deficient muscles may promote immunotoxic/genotoxic effects. Increasing the therapeutic index of rAAV in DMD muscles by reducing the effective dose could be a pivotal means of ensuring efficient clinical translation. This requires a comprehensive understanding of the rAAV transduction process, which is almost always studied in non-pathological tissues or in vitro. In this review, we focus on the molecular fate of rAAV after injection, and how the individual stages of transduction could be affected in the context of DMD. PMID:27121109

  10. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

    SciTech Connect

    Fujimoto, Takahiro; Itoh, Kyoko Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    Highlights: • Identification of dystrophin (Dp) shortest isoform, Dp40, is a neuron-type Dp. • Dp40 expression is temporally and differentially regulated in comparison to Dp71. • Somatodendritic and nuclear localization of Dp40. • Dp40 is localized to excitatory postsynapses. • Dp40 might play roles in dendritic and synaptic functions. - Abstract: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH{sub 2}-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  11. Pathways of abnormal stress-induced Ca2+ influx into dystrophic mdx cardiomyocytes

    PubMed Central

    Fanchaouy, M.; Polakova, E.; Jung, C.; Ogrodnik, J.; Shirokova, N.; Niggli, E.

    2009-01-01

    In Duchenne muscular dystrophy, deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle, but also to dilated cardiomyopathy, accounting for about 20% of the mortality. Mechanisms leading to cardiomyocyte cell death and cardiomyopathy are not well understood. One hypothesis suggests that the lack of dystrophin leads to membrane instability during mechanical stress and to activation of Ca2+ entry pathways. Using cardiomyocytes isolated from dystrophic mdx mice we dissected the contribution of various putative Ca2+ influx pathways with pharmacological tools. Cytosolic Ca2+ and Na+ signals as well as uptake of membrane impermeant compounds were monitored with fluorescent indicators using confocal microscopy and photometry. Membrane stress was applied as moderate osmotic challenges while membrane current was quantified using the whole-cell patch-clamp technique. Our findings suggest a major contribution of two primary Ca2+ influx pathways, stretch-activated membrane channels and short-lived microruptures. Furthermore, we found evidence for a secondary Ca2+ influx pathway, the Na+-Ca2+ exchange (NCX), which in cardiac muscle has a large transport capacity. After stress it contributes to Ca2+ entry in exchange for Na+ which had previously entered via primary stress-induced pathways, representing a previously not recognized mechanism contributing to subsequent cellular damage. This complexity needs to be considered when targeting abnormal Ca2+ influx as a treatment option for dystrophy. PMID:19604578

  12. Abnormal Uterine Bleeding

    MedlinePlus

    ... Abnormal uterine bleeding is any bleeding from the uterus (through your vagina) other than your normal monthly ... or fibroids (small and large growths) in the uterus can also cause bleeding. Rarely, a thyroid problem, ...

  13. "Jeopardy" in Abnormal Psychology.

    ERIC Educational Resources Information Center

    Keutzer, Carolin S.

    1993-01-01

    Describes the use of the board game, Jeopardy, in a college level abnormal psychology course. Finds increased student interaction and improved application of information. Reports generally favorable student evaluation of the technique. (CFR)

  14. Abnormal Uterine Bleeding FAQ

    MedlinePlus

    ... as cancer of the uterus, cervix, or vagina • Polycystic ovary syndrome How is abnormal bleeding diagnosed? Your health care ... before the fetus can survive outside the uterus. Polycystic Ovary Syndrome: A condition characterized by two of the following ...

  15. Chromosomal Abnormalities and Schizophrenia

    PubMed Central

    BASSETT, ANNE S.; CHOW, EVA W.C.; WEKSBERG, ROSANNA

    2011-01-01

    Schizophrenia is a common and serious psychiatric illness with strong evidence for genetic causation, but no specific loci yet identified. Chromosomal abnormalities associated with schizophrenia may help to understand the genetic complexity of the illness. This paper reviews the evidence for associations between chromosomal abnormalities and schizophrenia and related disorders. The results indicate that 22q11.2 microdeletions detected by fluorescence in-situ hybridization (FISH) are significantly associated with schizophrenia. Sex chromosome abnormalities seem to be increased in schizophrenia but insufficient data are available to indicate whether schizophrenia or related disorders are increased in patients with sex chromosome aneuploidies. Other reports of chromosomal abnormalities associated with schizophrenia have the potential to be important adjuncts to linkage studies in gene localization. Advances in molecular cytogenetic techniques (i.e., FISH) have produced significant increases in rates of identified abnormalities in schizophrenia, particularly in patients with very early age at onset, learning difficulties or mental retardation, or dysmorphic features. The results emphasize the importance of considering behavioral phenotypes, including adult onset psychiatric illnesses, in genetic syndromes and the need for clinicians to actively consider identifying chromosomal abnormalities and genetic syndromes in selected psychiatric patients. PMID:10813803

  16. A PCR-based assay for the wild-type dystrophin gene transferred into the mdx mouse.

    PubMed

    Shrager, J B; Naji, A; Kelly, A M; Stedman, H H

    1992-10-01

    Myoblast transfer has emerged as a promising treatment for inherited myopathies such as Duchenne muscular dystrophy (DMD). Further development of the technique's therapeutic potential requires an experimental system in which issues of graft rejection can be clearly discriminated from those related to myoblast biology. Here we report the development and initial application of a quantitative assay for myogenic cells bearing a wild-type dystrophin gene following transfer into the mdx mouse. The technique relies upon the ability of a mutagenizing polymerase chain reaction (PCR) primer to create a new restriction site in the amplification production of the wild-type, but not the mdx dystrophin gene. The ratio of host to donor cells can be determined from muscle biopsies as small as 1 mg, regardless of donor H-2 background. This simple technique should allow a number of basic questions related to myoblast and direct gene transfer to be addressed using the mdx mouse model. PMID:1357549

  17. Tissue distribution of the dystrophin-related gene product and expression in the mdx and dy mouse

    SciTech Connect

    Love, D.R.; Marsden, R.F.; Bloomfield, J.F.; Davies, K.E. ); Morris, G.E.; Ellis, J.M. ); Fairbrother, U.; Edwards, Y.H. ); Slater, C.P. ); Parry, D.J. )

    1991-04-15

    The authors have previously reported a dystrophin-related locus (DMDL for Duchenne muscular dystrophy-like) on human chromosome 6 that maps close to the dy mutation on mouse chromosome 10. Here they show that this gene is expressed in a wide range of tissues at varying levels. The transcript is particularly abundant in several human fetal tissues, including heart, placenta, and intestine. Studies with antisera raised against a DMDL fusion protein identify a 400,000 M{sub r} protein in all mouse tissues tested, including those of mdx and dy mice. Unlike the dystrophin gene, the DMDL gene transcript is not differentially spliced at the 3{prime} end in either fetal muscle or brain.

  18. Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs.

    PubMed

    Yokota, Toshifumi; Nakamura, Akinori; Nagata, Tetsuya; Saito, Takashi; Kobayashi, Masanori; Aoki, Yoshitsugu; Echigoya, Yusuke; Partridge, Terence; Hoffman, Eric P; Takeda, Shin'ichi

    2012-10-01

    Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD. PMID:22888777

  19. Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

    PubMed

    Gilbert, R; Nalbantoglu, J; Howell, J M; Davies, L; Fletcher, S; Amalfitano, A; Petrof, B J; Kamen, A; Massie, B; Karpati, G

    2001-09-20

    Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer. PMID:11560768

  20. Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy

    PubMed Central

    Kayali, Refik; Ku, Jin-Mo; Khitrov, Gregory; Jung, Michael E.; Prikhodko, Olga; Bertoni, Carmen

    2012-01-01

    Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD. PMID:22692682

  1. Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy.

    PubMed

    Kayali, Refik; Ku, Jin-Mo; Khitrov, Gregory; Jung, Michael E; Prikhodko, Olga; Bertoni, Carmen

    2012-09-15

    Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD. PMID:22692682

  2. Delayed Cardiomyopathy in Dystrophin Deficient mdx Mice Relies on Intrinsic Glutathione Resource

    PubMed Central

    Khouzami, Lara; Bourin, Marie-Claude; Christov, Christo; Damy, Thibaud; Escoubet, Brigitte; Caramelle, Philippe; Perier, Magali; Wahbi, Karim; Meune, Christophe; Pavoine, Catherine; Pecker, Françoise

    2010-01-01

    Oxidative stress contributes to the pathogenesis of Duchenne muscular dystrophy (DMD). Although they have been a model for DMD, mdx mice exhibit slowly developing cardiomyopathy. We hypothesized that disease process was delayed owing to the development of an adaptive mechanism against oxidative stress, involving glutathione synthesis. At 15 to 20 weeks of age, mdx mice displayed a 33% increase in blood glutathione levels compared with age-matched C57BL/6 mice. In contrast, cardiac glutathione content was similar in mdx and C57BL/6 mice as a result of the balanced increased expression of glutamate cysteine ligase catalytic and regulatory subunits ensuring glutathione synthesis in the mdx mouse heart, as well as increased glutathione peroxidase-1 using glutathione. Oral administration from 10 weeks of age of the glutamate cysteine ligase inhibitor, l-buthionine(S,R)-sulfoximine (BSO, 5 mmol/L), led to a 33% and 50% drop in blood and cardiac glutathione, respectively, in 15- to 20-week-old mdx mice. Moreover, 20-week-old BSO-treated mdx mice displayed left ventricular hypertrophy associated with diastolic dysfunction, discontinuities in β-dystroglycan expression, micronecrosis and microangiopathic injuries. Examination of the glutathione status in four DMD patients showed that three displayed systemic glutathione deficiency as well. In conclusion, low glutathione resource hastens the onset of cardiomyopathy linked to a defect in dystrophin in mdx mice. This is relevant to the glutathione deficiency that DMD patients may suffer. PMID:20696779

  3. Accurate quantification of dystrophin mRNA and exon skipping levels in duchenne muscular dystrophy.

    PubMed

    Spitali, Pietro; Heemskerk, Hans; Vossen, Rolf H A M; Ferlini, Alessandra; den Dunnen, Johan T; 't Hoen, Peter A C; Aartsma-Rus, Annemieke

    2010-09-01

    Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains approximately 1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model. PMID:20458276

  4. Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma.

    PubMed

    Chamberlain, Jeffrey S; Metzger, Joseph; Reyes, Morayma; Townsend, DeWayne; Faulkner, John A

    2007-07-01

    Duchenne muscular dystrophy (DMD) is the most common, lethal genetic disorder of children. A number of animal models of muscular dystrophy exist, but the most effective model for characterizing the structural and functional properties of dystrophin and therapeutic interventions has been the mdx mouse. Despite the approximately 20 years of investigations of the mdx mouse, the impact of the disease on the life span of mdx mice and the cause of death remain unresolved. Consequently, a life span study of the mdx mouse was designed that included cohorts of male and female mdx and wild-type C57BL/10 mice housed under specific pathogen-free conditions with deaths restricted to natural causes and with examination of the carcasses for pathology. Compared with wild-type mice, both mdx male and female mice had reduced life spans and displayed a progressively dystrophic muscle histopathology. Surprisingly, old mdx mice were prone to develop muscle tumors that resembled the human form of alveolar rhabdomyosarcoma, a cancer associated with poor prognosis. Rhabdomyosarcomas have not been observed previously in nontransgenic mice. The results substantiate the mdx mouse as an important model system for studies of the pathogenesis of and potential remedies for DMD. PMID:17360850

  5. MLC1 is associated with the Dystrophin-Glycoprotein Complex at astrocytic endfeet

    PubMed Central

    Boor, Ilja; Nagtegaal, Machiel; Kamphorst, Wouter; van der Valk, Paul; Pronk, Jan C.; van Horssen, Jack; Dinopoulos, Argirios; Bove, Kevin E.; Pascual-Castroviejo, Ignacio; Muntoni, Francesco; Estévez, Raúl; Scheper, Gert C.

    2007-01-01

    Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive cerebral white matter disease with onset in childhood, caused by mutations in the MLC1 gene. MLC1 is a protein with unknown function that is mainly expressed in the brain in astrocytic endfeet at the blood–brain and cerebrospinal fluid–brain barriers. It shares its localization at astrocytic endfeet with the dystrophin-associated glycoprotein complex (DGC). The objective of the present study was to investigate the possible association of MLC1 with the DGC. To test this hypothesis, (co)-localization of DGC-proteins and MLC1 was analyzed by immunohistochemical stainings in gliotic brain tissue from a patient with multiple sclerosis, in glioblastoma tissue and in brain tissue from an MLC patient. In control tissue, a direct protein interaction was tested by immunoprecipitation. Results revealed that MLC1 is co-localized with DGC-proteins in gliotic brain tissue. We demonstrated that both MLC1 and aquaporin-4, a member of the DGC, were redistributed in glioblastoma cells. In MLC brain tissue, we showed absence of MLC1 and altered expression of several DGC-proteins. We demonstrated a direct protein interaction between MLC1 and Kir4.1. From these results we conclude that MLC1 is associated with the DGC at astrocytic endfeet. PMID:17628813

  6. Structural and Functional Alterations of Skeletal Muscle Microvasculature in Dystrophin-Deficient mdx Mice.

    PubMed

    Latroche, Claire; Matot, Béatrice; Martins-Bach, Aurea; Briand, David; Chazaud, Bénédicte; Wary, Claire; Carlier, Pierre G; Chrétien, Fabrice; Jouvion, Grégory

    2015-09-01

    Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease, caused by an absence of dystrophin, inevitably leading to death. Although muscle lesions are well characterized, blood vessel alterations that may have a major impact on muscle regeneration remain poorly understood. Our aim was to elucidate alterations of the vascular network organization, taking advantage of Flk1(GFP/+) crossed with mdx mice (model for human DMD where all blood vessels express green fluorescent protein) and functional repercussions using in vivo nuclear magnetic resonance, combining arterial spin-labeling imaging of perfusion, and (31)P-spectroscopy of phosphocreatine kinetics. For the first time, our study focused on old (12-month-old) mdx mice, displaying marked chronic muscle lesions, similar to the lesions observed in human DMD, in comparison to young-adult (3-month-old) mdx mice displaying only mild muscle lesions with no fibrosis. By using an original approach combining a specific animal model, state-of-the-art histology/morphometry techniques, and functional nuclear magnetic resonance, we demonstrated that the microvascular system is almost normal in young-adult in contrast to old mdx mice, displaying marked microvessel alterations, and the functional repercussions on muscle perfusion and bioenergetics after a hypoxic stress vary depending on stage of pathology. This original approach clarifies disease evolution and paves the way for setting up new diagnostic markers or therapeutic strategies. PMID:26193666

  7. Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration

    PubMed Central

    Roberts, Thomas C.; Johansson, Henrik J.; McClorey, Graham; Godfrey, Caroline; Blomberg, K. Emelie M.; Coursindel, Thibault; Gait, Michael J.; Smith, C.I. Edvard; Lehtiö, Janne; EL Andaloussi, Samir; Wood, Matthew J.A.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA–target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle. PMID:26385637

  8. Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors.

    PubMed

    Todaro, Marian; Quigley, Anita; Kita, Magdalena; Chin, Judy; Lowes, Kym; Kornberg, Andrew J; Cook, Mark J; Kapsa, Robert

    2007-08-01

    Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction. PMID:17394239

  9. Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

    PubMed

    Zhao, Chunli; Farruggio, Alfonso P; Bjornson, Christopher R R; Chavez, Christopher L; Geisinger, Jonathan M; Neal, Tawny L; Karow, Marisa; Calos, Michele P

    2014-01-01

    A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies. PMID:24781921

  10. Sequence characterisation of deletion breakpoints in the dystrophin gene by PCR

    SciTech Connect

    Abbs, S.; Sandhu, S.; Bobrow, M.

    1994-09-01

    Partial deletions of the dystrophin gene account for 65% of cases of Duchenne muscular dystrophy. A high proportion of these structural changes are generated by new mutational events, and lie predominantly within two `hotspot` regions, yet the underlying reasons for this are not known. We are characterizing and sequencing the regions surrounding deletion breakpoints in order to: (i) investigate the mechanisms of deletion mutation, and (ii) enable the design of PCR assays to specifically amplify mutant and normal sequences, allowing us to search for the presence of somatic mosaicism in appropriate family members. Using this approach we have been able to demonstrate the presence of somatic mosaicism in a maternal grandfather of a DMD-affected male, deleted for exons 49-50. Three deletions, namely of exons 48-49, 49-50, and 50, have been characterized using a PCR approach that avoids any cloning procedures. Breakpoints were initially localized to within regions of a few kilobases using Southern blot restriction analyses with exon-specific probes and PCR amplification of exonic and intronic loci. Sequencing was performed directly on PCR products: (i) mutant sequences were obtained from long-range or inverse-PCR across the deletion junction fragments, and (ii) normal sequences were obtained from the products of standard PCR, vectorette PCR, or inverse-PCR performed on YACs. Further characterization of intronic sequences will allow us to amplify and sequence across other deletion breakpoints and increase our knowledge of the mechanisms of mutation in the dystophin gene.

  11. RT-PCR analysis of dystrophin mRNA in DND/BMD patients

    SciTech Connect

    Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D.

    1994-09-01

    Duchenne and Becker muscular dystrophies (DMD, BMD) are X-linked recessive disorders caused by mutations in the dystrophin (dys) gene. The majority of these mutations are intragenic deletions of duplications routinely detected by Southern biots and multiplex PCR. The remainder are very likely, smaller mutations, mostly point-mutations. Detection of these mutations is very difficult due to the size and complexity of the dys gene. We applied RT-PCR to analyse the entire dys mRNA of three DMD patients with no detectable genomic defect. In two unrelated patients, a duplication of the 62 bp exon 2 was identified. This causes a frameshift sufficient to explain the DMD phenotype. In the third patient, who had congenital DMD and severe mental retardation, a complex pattern of aberrant splicing at the 3-prime exons 67-79 was observed. Sural nerve biopsy in this patient showed the complete absence of Dp116. PCR-SSCP studies are presently in progress to identify the mutations responsible for the aberrant splicing patterns.

  12. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons.

    PubMed

    Fujimoto, Takahiro; Itoh, Kyoko; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions. PMID:25152393

  13. Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.

    PubMed

    Roberts, Thomas C; Johansson, Henrik J; McClorey, Graham; Godfrey, Caroline; Blomberg, K Emelie M; Coursindel, Thibault; Gait, Michael J; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle. PMID:26385637

  14. Defective T-lymphocyte migration to muscles in dystrophin-deficient mice.

    PubMed

    Cascabulho, Cynthia M; Bani Corrêa, Cristiane; Cotta-de-Almeida, Vinícius; Henriques-Pons, Andrea

    2012-08-01

    Duchenne muscular dystrophy (DMD), an X-linked recessive disorder affecting 1 in 3500 males, is caused by mutations in the dystrophin gene. DMD leads to degeneration of skeletal and cardiac muscles and to chronic inflammation. The mdx/mdx mouse has been widely used to study DMD; this model mimics most characteristics of the disease, including low numbers of T cells in damaged muscles. In this study, we aimed to assess migration of T cells to the heart and to identify any alterations in adhesion molecules that could possibly modulate this process. In 6-week-old mdx/mdx mice, blood leukocytes, including T cells, were CD62L(+), but by 12 weeks of age down-modulation was evident, with only approximately 40% of T cells retaining this molecule. Our in vitro and in vivo results point to a P2X7-dependent shedding of CD62L (with high levels in the serum), which in 12-week-old mdx/mdx mice reduces blood T cell competence to adhere to cardiac vessels in vitro and to reach cardiac tissue in vivo, even after Trypanosoma cruzi infection, a known inducer of lymphoid myocarditis. In mdx/mdx mice treated with Brilliant Blue G, a P2X7 blocker, these blood lymphocytes retained CD62L and were capable of migrating to the heart. These results provide new insights into the mechanisms of inflammatory infiltration and immune regulation in DMD. PMID:22733008

  15. Haplotypes in the Dystrophin DNA Segment Point to a Mosaic Origin of Modern Human Diversity

    PubMed Central

    Ziętkiewicz, Ewa; Yotova, Vania; Gehl, Dominik; Wambach, Tina; Arrieta, Isabel; Batzer, Mark; Cole, David E. C.; Hechtman, Peter; Kaplan, Feige; Modiano, David; Moisan, Jean-Paul; Michalski, Roman; Labuda, Damian

    2003-01-01

    Although Africa has played a central role in human evolutionary history, certain studies have suggested that not all contemporary human genetic diversity is of recent African origin. We investigated 35 simple polymorphic sites and one Tn microsatellite in an 8-kb segment of the dystrophin gene. We found 86 haplotypes in 1,343 chromosomes from around the world. Although a classical out-of-Africa topology was observed in trees based on the variant frequencies, the tree of haplotype sequences reveals three lineages accounting for present-day diversity. The proportion of new recombinants and the diversity of the Tn microsatellite were used to estimate the age of haplotype lineages and the time of colonization events. The lineage that underwent the great expansion originated in Africa prior to the Upper Paleolithic (27,000–56,000 years ago). A second group, of structurally distinct haplotypes that occupy a central position on the tree, has never left Africa. The third lineage is represented by the haplotype that lies closest to the root, is virtually absent in Africa, and appears older than the recent out-of-Africa expansion. We propose that this lineage could have left Africa before the expansion (as early as 160,000 years ago) and admixed, outside of Africa, with the expanding lineage. Contemporary human diversity, although dominated by the recently expanded African lineage, thus represents a mosaic of different contributions. PMID:14513410

  16. Morphological abnormalities in elasmobranchs.

    PubMed

    Moore, A B M

    2015-08-01

    A total of 10 abnormal free-swimming (i.e., post-birth) elasmobranchs are reported from The (Persian-Arabian) Gulf, encompassing five species and including deformed heads, snouts, caudal fins and claspers. The complete absence of pelvic fins in a milk shark Rhizoprionodon acutus may be the first record in any elasmobranch. Possible causes, including the extreme environmental conditions and the high level of anthropogenic pollution particular to The Gulf, are briefly discussed. PMID:25903257

  17. Chromosome abnormalities in glioma

    SciTech Connect

    Li, Y.S.; Ramsay, D.A.; Fan, Y.S.

    1994-09-01

    Cytogenetic studies were performed in 25 patients with gliomas. An interesting finding was a seemingly identical abnormality, an extra band on the tip of the short arm of chromosome 1, add(1)(p36), in two cases. The abnormality was present in all cells from a patient with a glioblastoma and in 27% of the tumor cells from a patient with a recurrent irradiated anaplastic astrocytoma; in the latter case, 7 unrelated abnormal clones were identified except 4 of those clones shared a common change, -Y. Three similar cases have been described previously. In a patient with pleomorphic astrocytoma, the band 1q42 in both homologues of chromosome 1 was involved in two different rearrangements. A review of the literature revealed that deletion of the long arm of chromosome 1 including 1q42 often occurs in glioma. This may indicate a possible tumor suppressor gene in this region. Cytogenetic follow-up studies were carried out in two patients and emergence of unrelated clones were noted in both. A total of 124 clonal breakpoints were identified in the 25 patients. The breakpoints which occurred three times or more were: 1p36, 1p22, 1q21, 1q25, 3q21, 7q32, 8q22, 9q22, 16q22, and 22q13.

  18. [Congenital foot abnormalities].

    PubMed

    Delpont, M; Lafosse, T; Bachy, M; Mary, P; Alves, A; Vialle, R

    2015-03-01

    The foot may be the site of birth defects. These abnormalities are sometimes suspected prenatally. Final diagnosis depends on clinical examination at birth. These deformations can be simple malpositions: metatarsus adductus, talipes calcaneovalgus and pes supinatus. The prognosis is excellent spontaneously or with a simple orthopedic treatment. Surgery remains outstanding. The use of a pediatric orthopedist will be considered if malposition does not relax after several weeks. Malformations (clubfoot, vertical talus and skew foot) require specialized care early. Clubfoot is characterized by an equine and varus hindfoot, an adducted and supine forefoot, not reducible. Vertical talus combines equine hindfoot and dorsiflexion of the forefoot, which is performed in the midfoot instead of the ankle. Skew foot is suspected when a metatarsus adductus is resistant to conservative treatment. Early treatment is primarily orthopedic at birth. Surgical treatment begins to be considered after walking age. Keep in mind that an abnormality of the foot may be associated with other conditions: malposition with congenital hip, malformations with syndromes, neurological and genetic abnormalities. PMID:25524290

  19. Abnormal pressures as hydrodynamic phenomena

    USGS Publications Warehouse

    Neuzil, C.E.

    1995-01-01

    So-called abnormal pressures, subsurface fluid pressures significantly higher or lower than hydrostatic, have excited speculation about their origin since subsurface exploration first encountered them. Two distinct conceptual models for abnormal pressures have gained currency among earth scientists. The static model sees abnormal pressures generally as relict features preserved by a virtual absence of fluid flow over geologic time. The hydrodynamic model instead envisions abnormal pressures as phenomena in which flow usually plays an important role. This paper develops the theoretical framework for abnormal pressures as hydrodynamic phenomena, shows that it explains the manifold occurrences of abnormal pressures, and examines the implications of this approach. -from Author

  20. Preservation of Muscle Force in Mdx3cv Mice Correlates with Low-Level Expression of a Near Full-Length Dystrophin Protein

    PubMed Central

    Li, Dejia; Yue, Yongping; Duan, Dongsheng

    2008-01-01

    The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mouse muscle but was expressed in mdx3cv mice as a near full-length protein at ∼5% of normal levels. Consistent with previous reports, we found dystrophic muscle pathology in both mouse strains. Surprisingly, mdx3cv extensor digitorium longus muscle showed significantly higher tetanic force and was also more resistant to eccentric contraction-induced injury than mdx4cv extensor digitorium longus muscle. Furthermore, mdx3cv mice had stronger forelimb grip strength than mdx4cv mice. Immunostaining revealed utrophin up-regulation in both mouse strains. The dystrophin-associated glycoprotein complex was also restored in the sarcolemma in both strains although at levels lower than those in normal mice. Our results suggest that subtherapeutic expression levels of near full-length, membrane-bound dystrophin, possibly in conjunction with up-regulated utrophin levels, may help maintain minimal muscle force but not arrest muscle degeneration or necrosis. Our findings provide valuable insight toward understanding delayed clinical onset and/or slow disease progression in certain Becker muscular dystrophy patients. PMID:18385524

  1. Feeling Abnormal: Simulation of Deviancy in Abnormal and Exceptionality Courses.

    ERIC Educational Resources Information Center

    Fernald, Charles D.

    1980-01-01

    Describes activity in which student in abnormal psychology and psychology of exceptional children classes personally experience being judged abnormal. The experience allows the students to remember relevant research, become sensitized to the feelings of individuals classified as deviant, and use caution in classifying individuals as abnormal.…

  2. Distribution of dystrophin- and utrophin-associated protein complexes (DAPC/UAPC) in human hematopoietic stem/progenitor cells.

    PubMed

    Teniente-De Alba, Carmen; Martínez-Vieyra, Ivette; Vivanco-Calixto, Raúl; Galván, Iván J; Cisneros, Bulmaro; Cerecedo, Doris

    2011-10-01

    Hematopoietic stem cells (HSC) are defined by their cardinal properties, such as sustained proliferation, multilineage differentiation, and self-renewal, which give rise to a hierarchy of progenitor populations with more restricted potential lineage, ultimately leading to the production of all types of mature blood cells. HSC are anchored by cell adhesion molecules to their specific microenvironment, thus regulating their cell cycle, while cell migration is essentially required for seeding the HSC of the fetal bone marrow (BM) during development as well as in adult BM homeostasis. The dystrophin-associated protein complex (DAPC) is a large group of membrane-associated proteins linking the cytoskeleton to the extracellular matrix and exhibiting scaffolding, adhesion, and signaling roles in muscle and non-muscle cells including mature blood cells. Because adhesion and migration are mechanisms that influence the fate of the HSC, we explored the presence and the feasible role of DAPC. In this study, we characterized the pattern expression by immunoblot technique and, by confocal microscopy analysis, the cellular distribution of dystrophin and utrophin gene products, and the dystrophin-associated proteins (α-, β-dystroglycan, α-syntrophin, α-dystrobrevin) in relation to actin filaments in freshly isolated CD34+ cells from umbilical cord blood. Immunoprecipitation assays demonstrated the presence of Dp71d/Dp71Δ110m ∼DAPC and Up400/Up140∼DAPC. The subcellular distribution of the two DAPC in actin-based structures suggests their dynamic participation in adhesion and cell migration. In addition, the particular protein pattern expression found in hematopoietic stem/progenitor cells might be indicative of their feasible participation during differentiation. PMID:21623922

  3. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

    SciTech Connect

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N.

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  4. Abnormal human sex chromosome constitutions

    SciTech Connect

    1993-12-31

    Chapter 22, discusses abnormal human sex chromosome constitution. Aneuploidy of X chromosomes with a female phenotype, sex chromosome aneuploidy with a male phenotype, and various abnormalities in X chromosome behavior are described. 31 refs., 2 figs.

  5. Exercises to Improve Gait Abnormalities

    MedlinePlus

    ... Home About iChip Articles Directories Videos Resources Contact Exercises to Improve Gait Abnormalities Home » Article Categories » Exercise and Fitness Font Size: A A A A Exercises to Improve Gait Abnormalities Next Page The manner ...

  6. Altered astrocyte morphology and vascular development in dystrophin-Dp71-null mice.

    PubMed

    Giocanti-Auregan, Audrey; Vacca, Ophélie; Bénard, Romain; Cao, Sijia; Siqueiros, Lourdes; Montañez, Cecilia; Paques, Michel; Sahel, José-Alain; Sennlaub, Florian; Guillonneau, Xavier; Rendon, Alvaro; Tadayoni, Ramin

    2016-05-01

    Understanding retinal vascular development is crucial because many retinal vascular diseases such as diabetic retinopathy (in adults) or retinopathy of prematurity (in children) are among the leading causes of blindness. Given the localization of the protein Dp71 around the retinal vessels in adult mice and its role in maintaining retinal homeostasis, the aim of this study was to determine if Dp71 was involved in astrocyte and vascular development regulation. An experimental study in mouse retinas was conducted. Using a dual immunolabeling with antibodies to Dp71 and anti-GFAP for astrocytes on retinal sections and isolated astrocytes, it was found that Dp71 was expressed in wild-type (WT) mouse astrocytes from early developmental stages to adult stage. In Dp71-null mice, a reduction in GFAP-immunopositive astrocytes was observed as early as postnatal day 6 (P6) compared with WT mice. Using real-time PCR, it was showed that Dp71 mRNA was stable between P1 and P6, in parallel with post-natal vascular development. Regarding morphology in Dp71-null and WT mice, a significant decrease in overall astrocyte process number in Dp71-null retinas at P6 to adult age was found. Using fluorescence-conjugated isolectin Griffonia simplicifolia on whole mount retinas, subsequent delay of developing vascular network at the same age in Dp71-null mice was found. An evidence that the Dystrophin Dp71, a membrane-associated cytoskeletal protein and one of the smaller Duchenne muscular dystrophy gene products, regulates astrocyte morphology and density and is associated with subsequent normal blood vessel development was provided. GLIA 2016;64:716-729. PMID:26711882

  7. Disruption of action potential and calcium signaling properties in malformed myofibers from dystrophin-deficient mice

    PubMed Central

    Hernández-Ochoa, Erick O; Pratt, Stephen J P; Garcia-Pelagio, Karla P; Schneider, Martin F; Lovering, Richard M

    2015-01-01

    Duchenne muscular dystrophy (DMD), the most common and severe muscular dystrophy, is caused by the absence of dystrophin. Muscle weakness and fragility (i.e., increased susceptibility to damage) are presumably due to structural instability of the myofiber cytoskeleton, but recent studies suggest that the increased presence of malformed/branched myofibers in dystrophic muscle may also play a role. We have previously studied myofiber morphology in healthy wild-type (WT) and dystrophic (MDX) skeletal muscle. Here, we examined myofiber excitability using high-speed confocal microscopy and the voltage-sensitive indicator di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS) to assess the action potential (AP) properties. We also examined AP-induced Ca2+ transients using high-speed confocal microscopy with rhod-2, and assessed sarcolemma fragility using elastimetry. AP recordings showed an increased width and time to peak in malformed MDX myofibers compared to normal myofibers from both WT and MDX, but no significant change in AP amplitude. Malformed MDX myofibers also exhibited reduced AP-induced Ca2+ transients, with a further Ca2+ transient reduction in the branches of malformed MDX myofibers. Mechanical studies indicated an increased sarcolemma deformability and instability in malformed MDX myofibers. The data suggest that malformed myofibers are functionally different from myofibers with normal morphology. The differences seen in AP properties and Ca2+ signals suggest changes in excitability and remodeling of the global Ca2+ signal, both of which could underlie reported weakness in dystrophic muscle. The biomechanical changes in the sarcolemma support the notion that malformed myofibers are more susceptible to damage. The high prevalence of malformed myofibers in dystrophic muscle may contribute to the progressive strength loss and fragility seen in dystrophic muscles. PMID:25907787

  8. Effects of Dantrolene Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice.

    PubMed

    Quinn, James L; Huynh, Tony; Uaesoontrachoon, Kitipong; Tatem, Kathleen; Phadke, Aditi; Van der Meulen, Jack H; Yu, Qing; Nagaraju, Kannaboyina

    2013-01-01

    Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice (4 weeks old) with daily intraperitoneal injections of 40mg/kg of Dantrolene for 6 weeks and measured functional (grip strength, in vitro force contractions), behavioral (open field digiscan), imagining (optical imaging for inflammation), histological (H&E), and molecular (protein and RNA) endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice. PMID:24270550

  9. Effects of Dantrolene Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice

    PubMed Central

    Quinn, James L; Huynh, Tony; Uaesoontrachoon, Kitipong; Tatem, Kathleen; Phadke, Aditi; Van der Meulen, Jack H; Yu, Qing; Nagaraju, Kannaboyina

    2013-01-01

    Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice (4 weeks old) with daily intraperitoneal injections of 40mg/kg of Dantrolene for 6 weeks and measured functional (grip strength, in vitro force contractions), behavioral (open field digiscan), imagining (optical imaging for inflammation), histological (H&E), and molecular (protein and RNA) endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice. PMID:24270550

  10. C-Terminal-Truncated Microdystrophin Recruits Dystrobrevin and Syntrophin to the Dystrophin-Associated Glycoprotein Complex and Reduces Muscular Dystrophy in Symptomatic Utrophin/Dystrophin Double-Knockout Mice

    PubMed Central

    Yue, Yongping; Liu, Mingju; Duan, Dongsheng

    2007-01-01

    C-terminal-truncated (ΔC) microdystrophin is being developed for Duchenne muscular dystrophy gene therapy. Encouraging results have been achieved in the mdx mouse model. Unfortunately, mdx mice do not display the same phenotype as human patients. Evaluating ΔC microdystrophin in a symptomatic model will be of significant relevance to human trials. Utrophin/dystrophin double-knockout (u-dko) mice were developed to model severe dystrophic changes in human patients. In this study we evaluated the therapeutic effect of the ΔR4-R23/ΔC microdystrophin gene (ΔR4/ΔC) after serotype-6 adeno-associated virus-mediated gene transfer in neonatal u-dko muscle. At 2 months after gene transfer, the percentage of centrally nucleated myofiber was reduced from 89.2 to 3.4% and muscle weight was normalized. Furthermore, we have demonstrated for the first time that ΔC microdystrophin can eliminate interstitial fibrosis and macrophage infiltration and restore dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex. Interestingly neuronal nitric oxide synthase was not restored. The most impressive results were achieved in muscle force measurement. Neonatal gene therapy increased twitch- and tetanic-specific force. It also brought the response to eccentric contraction-induced injury to the normal range. In summary, our results suggest that the ΔR4/ΔC microgene holds great promise in preventing muscular dystrophy. PMID:16563874

  11. Spirometric abnormalities among welders

    SciTech Connect

    Rastogi, S.K.; Gupta, B.N.; Husain, T.; Mathur, N.; Srivastava, S. )

    1991-10-01

    A group of manual welders age group 13-60 years having a mean exposure period of 12.4 {plus minus} 1.12 years were subjected to spirometry to evaluate the prevalence of spirometric abnormalities. The welders showed a significantly higher prevalence of respiratory impairment than that observed among the unexposed controls as a result of exposure to welding gases which comprised fine particles of lead, zinc, chromium, and manganese. This occurred despite the lower concentration of the pollutants at the work place. In the expose group, the smoking welders showed a prevalence of respiratory impairment significantly higher than that observed in the nonsmoking welders. The results of the pulmonary function tests showed a predominantly restrictive type of pulmonary impairment followed by a mixed ventilatory defect among the welders. The effect of age on pulmonary impairment was not discernible. Welders exposed for over 10 years showed a prevalence of respiratory abnormalities significantly higher than those exposed for less than 10 years. Smoking also had a contributory role.

  12. Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method.

    PubMed

    Iyombe-Engembe, Jean-Paul; Ouellet, Dominique L; Barbeau, Xavier; Rousseau, Joël; Chapdelaine, Pierre; Lagüe, Patrick; Tremblay, Jacques P

    2016-01-01

    The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides. PMID:26812655

  13. In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans

    PubMed Central

    Zhan, Hong; Stanciauskas, Ramunas; Stigloher, Christian; Dizon, Kevin K.; Jospin, Maelle; Bessereau, Jean-Louis; Pinaud, Fabien

    2014-01-01

    Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca2+ channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals. PMID:25232639

  14. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy

    PubMed Central

    Li, Mei; Arner, Anders

    2015-01-01

    Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf) of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf) was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM). This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively. PMID:26536238

  15. Proteomic Profiling of the Dystrophin-Deficient MDX Heart Reveals Drastically Altered Levels of Key Metabolic and Contractile Proteins

    PubMed Central

    Lewis, Caroline; Jockusch, Harald; Ohlendieck, Kay

    2010-01-01

    Although Duchenne muscular dystrophy is primarily classified as a neuromuscular disease, cardiac complications play an important role in the course of this X-linked inherited disorder. The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood. We therefore carried out a fluorescence difference in-gel electrophoretic analysis of 9-month-old dystrophin-deficient versus age-matched normal heart, using the established MDX mouse model of muscular dystrophy-related cardiomyopathy. Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains. Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins. PMID:20508850

  16. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy.

    PubMed

    Li, Mei; Arner, Anders

    2015-01-01

    Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf) of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf) was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM). This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively. PMID:26536238

  17. Three-Dimensional Regulation of Radial Glial Functions by Lis1-Nde1 and Dystrophin Glycoprotein Complexes

    PubMed Central

    Pawlisz, Ashley S.; Feng, Yuanyi

    2011-01-01

    Radial glial cells (RGCs) are distinctive neural stem cells with an extraordinary slender bipolar morphology and dual functions as precursors and migration scaffolds for cortical neurons. Here we show a novel mechanism by which the Lis1-Nde1 complex maintains RGC functions through stabilizing the dystrophin/dystroglycan glycoprotein complex (DGC). A direct interaction between Nde1 and utrophin/dystrophin allows for the assembly of a multi-protein complex that links the cytoskeleton to the extracellular matrix of RGCs to stabilize their lateral membrane, cell-cell adhesion, and radial morphology. Lis1-Nde1 mutations destabilized the DGC and resulted in deformed, disjointed RGCs and disrupted basal lamina. Besides impaired RGC self-renewal and neuronal migration arrests, Lis1-Nde1 deficiencies also led to neuronal over-migration. Additional to phenotypic resemblances of Lis1-Nde1 with DGC, strong synergistic interactions were found between Nde1 and dystroglycan in RGCs. As functional insufficiencies of LIS1, NDE1, and dystroglycan all cause lissencephaly syndromes, our data demonstrated that a three-dimensional regulation of RGC's cytoarchitecture by the Lis1-Nde1-DGC complex determines the number and spatial organization of cortical neurons as well as the size and shape of the cerebral cortex. PMID:22028625

  18. Cholesterol favors the anchorage of human dystrophin repeats 16 to 21 in membrane at physiological surface pressure.

    PubMed

    Ameziane-Le Hir, Sarah; Raguénès-Nicol, Céline; Paboeuf, Gilles; Nicolas, Aurélie; Le Rumeur, Elisabeth; Vié, Véronique

    2014-05-01

    Dystrophin (DYS) is a filamentous protein that connects the cytoskeleton and the extracellular matrix via the sarcolemma, conferring resistance to muscular cells. In this study, interactions between the DYS R16-21 fragment and lipids were examined using Langmuir films made of anionic and zwitterionic lipids. The film fluidity was modified by the addition of 15% cholesterol. Whatever the lipid mixture examined, at low surface pressure (20 mN/m) few differences appeared on the protein insertion and the presence of cholesterol did not affect the protein/lipid interactions. At high surface pressure (30 mN/m), the protein insertion was very low and occurred only in zwitterionic films in the liquid-expanded phase. In anionic films, electrostatic interactions prevented the protein insertion outright, and caused accumulation of the protein on the hydrophilic part of the monolayer. Addition of cholesterol to both lipid mixtures drastically modified the protein-lipid interactions: the DYS R16-21 insertion increased and its organization in the monolayer appeared to be more homogeneous. The presence of accessible cholesterol recognition amino-acid consensus sequences in this fragment may enhance the protein/membrane binding at physiological lateral pressure. These results suggest that the anchorage of dystrophin to the membrane in vivo may be stabilized by cholesterol-rich nano-domains in the inner leaflet of sarcolemma. PMID:24440661

  19. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

    NASA Technical Reports Server (NTRS)

    Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

    2000-01-01

    Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

  20. In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans.

    PubMed

    Zhan, Hong; Stanciauskas, Ramunas; Stigloher, Christian; Dizon, Kevin K; Jospin, Maelle; Bessereau, Jean-Louis; Pinaud, Fabien

    2014-01-01

    Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals. PMID:25232639

  1. Eye movement abnormalities.

    PubMed

    Moncayo, Jorge; Bogousslavsky, Julien

    2012-01-01

    Generation and control of eye movements requires the participation of the cortex, basal ganglia, cerebellum and brainstem. The signals of this complex neural network finally converge on the ocular motoneurons of the brainstem. Infarct or hemorrhage at any level of the oculomotor system (though more frequent in the brain-stem) may give rise to a broad spectrum of eye movement abnormalities (EMAs). Consequently, neurologists and particularly stroke neurologists are routinely confronted with EMAs, some of which may be overlooked in the acute stroke setting and others that, when recognized, may have a high localizing value. The most complex EMAs are due to midbrain stroke. Horizontal gaze disorders, some of them manifesting unusual patterns, may occur in pontine stroke. Distinct varieties of nystagmus occur in cerebellar and medullary stroke. This review summarizes the most representative EMAs from the supratentorial level to the brainstem. PMID:22377853

  2. Disassembly of the cholinergic postsynaptic apparatus induced by axotomy in mouse sympathetic neurons: the loss of dystrophin and beta-dystroglycan immunoreactivity precedes that of the acetylcholine receptor.

    PubMed

    Zaccaria, M L; De Stefano, M E; Properzi, F; Gotti, C; Petrucci, T C; Paggi, P

    1998-08-01

    In mouse sympathetic superior cervical ganglion (SCG), cortical cytoskeletal proteins such as dystrophin (Dys) and beta1sigma2 spectrin colocalize with beta-dystroglycan (beta-DG), a transmembrane dystrophin-associated protein, and the acetylcholine receptor (AChR) at the postsynaptic specialization. The function of the dystrophin-dystroglycan complex in the organization of the neuronal cholinergic postsynaptic apparatus was studied following changes in the immunoreactivity of these proteins during the disassembly and subsequent reassembly of the postsynaptic specializations induced by axotomy of the ganglionic neurons. After axotomy, a decrease in the number of intraganglionic synapses was observed (t1/2 8 h 45'), preceded by a rapid decline of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha3 AChR subunit (alpha3AChR) (t1/2 3 h 45', 4 h 30' and 6 h, respectively). In contrast, the percentage of postsynaptic densities immunopositive for beta1sigma2 spectrin remained unaltered. When the axotomized neurons began to regenerate their axons, the number of intraganglionic synapses increased, as did that of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha3AChR. The latter number increased more slowly than that of Dys and beta-DG. These observations suggest that in SCG neurons, the dystrophin-dystroglycan complex might play a role in the assembly-disassembly of the postsynaptic apparatus, and is probably involved in the stabilization of AChR clusters. PMID:9720492

  3. More deletions in the 5{prime} region than in the central region of the dystrophin gene were identified among Filipino Duchenne and Becker muscular dystrophy patients

    SciTech Connect

    1995-11-06

    This report describes mutations in the dystrophin gene and the frequency of these mutations in Filipino pedigrees with Duchenne and Becker muscular dystrophy (DMD/BMD). The findings suggest the presence of genetic variability among DMD/BMD patients in different populations. 13 refs., 1 tab.

  4. Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy.

    PubMed

    Allen, David G; Whitehead, Nicholas P; Froehner, Stanley C

    2016-01-01

    Dystrophin is a long rod-shaped protein that connects the subsarcolemmal cytoskeleton to a complex of proteins in the surface membrane (dystrophin protein complex, DPC), with further connections via laminin to other extracellular matrix proteins. Initially considered a structural complex that protected the sarcolemma from mechanical damage, the DPC is now known to serve as a scaffold for numerous signaling proteins. Absence or reduced expression of dystrophin or many of the DPC components cause the muscular dystrophies, a group of inherited diseases in which repeated bouts of muscle damage lead to atrophy and fibrosis, and eventually muscle degeneration. The normal function of dystrophin is poorly defined. In its absence a complex series of changes occur with multiple muscle proteins showing reduced or increased expression or being modified in various ways. In this review, we will consider the various proteins whose expression and function is changed in muscular dystrophies, focusing on Ca(2+)-permeable channels, nitric oxide synthase, NADPH oxidase, and caveolins. Excessive Ca(2+) entry, increased membrane permeability, disordered caveolar function, and increased levels of reactive oxygen species are early changes in the disease, and the hypotheses for these phenomena will be critically considered. The aim of the review is to define the early damage pathways in muscular dystrophy which might be appropriate targets for therapy designed to minimize the muscle degeneration and slow the progression of the disease. PMID:26676145

  5. Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter

    SciTech Connect

    Yanagawa, H.; Nishio, H.; Takeshima, Y.

    1994-09-01

    The dystrophin gene, which is muted in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously-unidentified alternative promoter. The case study presented is that of patient with Duchenne muscular dystrophy who had a deletion extending from 5{prime} end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5{prime} part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5{prime} end of the new transcript indicated that the 5{prime} end of exon 3 was extended by 9 codons, only the last (most 3{prime}) of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5{prime} of the previously-identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

  6. Ictal Cardiac Ryhthym Abnormalities

    PubMed Central

    Ali, Rushna

    2016-01-01

    Cardiac rhythm abnormalities in the context of epilepsy are a well-known phenomenon. However, they are under-recognized and often missed. The pathophysiology of these events is unclear. Bradycardia and asystole are preceded by seizure onset suggesting ictal propagation into the cortex impacting cardiac autonomic function, and the insula and amygdala being possible culprits. Sudden unexpected death in epilepsy (SUDEP) refers to the unanticipated death of a patient with epilepsy not related to status epilepticus, trauma, drowning, or suicide. Frequent refractory generalized tonic-clonic seizures, anti-epileptic polytherapy, and prolonged duration of epilepsy are some of the commonly identified risk factors for SUDEP. However, the most consistent risk factor out of these is an increased frequency of generalized tonic–clonic seizures (GTC). Prevention of SUDEP is extremely important in patients with chronic, generalized epilepsy. Since increased frequency of GTCS is the most consistently reported risk factor for SUDEP, effective seizure control is the most important preventive strategy. PMID:27347227

  7. Abnormal uterine bleeding.

    PubMed

    Whitaker, Lucy; Critchley, Hilary O D

    2016-07-01

    Abnormal uterine bleeding (AUB) is a common and debilitating condition with high direct and indirect costs. AUB frequently co-exists with fibroids, but the relationship between the two remains incompletely understood and in many women the identification of fibroids may be incidental to a menstrual bleeding complaint. A structured approach for establishing the cause using the Fédération International de Gynécologie et d'Obstétrique (FIGO) PALM-COEIN (Polyp, Adenomyosis, Leiomyoma, Malignancy (and hyperplasia), Coagulopathy, Ovulatory disorders, Endometrial, Iatrogenic and Not otherwise classified) classification system will facilitate accurate diagnosis and inform treatment options. Office hysteroscopy and increasing sophisticated imaging will assist provision of robust evidence for the underlying cause. Increased availability of medical options has expanded the choice for women and many will no longer need to recourse to potentially complicated surgery. Treatment must remain individualised and encompass the impact of pressure symptoms, desire for retention of fertility and contraceptive needs, as well as address the management of AUB in order to achieve improved quality of life. PMID:26803558

  8. Haem degradation in abnormal haemoglobins.

    PubMed Central

    Brown, S B; Docherty, J C

    1978-01-01

    The coupled oxidation of certain abnormal haemoglobins leads to different bile-pigment isomer distributions from that of normal haemoglobin. The isomer pattern may be correlated with the structure of the abnormal haemoglobin in the neighbourhood of the haem pocket. This is support for haem degradation by an intramolecular reaction. PMID:708385

  9. Electrocardiograph abnormalities revealed during laparoscopy

    PubMed Central

    Nijjer, Sukhjinder; Dubrey, Simon William

    2010-01-01

    This brief case presents a well patient in whom an electrocardiograph abnormality consistent with an accessory pathway was found during a routine procedure. We present the electrocardiographs, explain the underlying condition, and consider why the abnormality was revealed in this manner. PMID:22419949

  10. Abnormal pressure in hydrocarbon environments

    USGS Publications Warehouse

    Law, B.E.; Spencer, C.W.

    1998-01-01

    Abnormal pressures, pressures above or below hydrostatic pressures, occur on all continents in a wide range of geological conditions. According to a survey of published literature on abnormal pressures, compaction disequilibrium and hydrocarbon generation are the two most commonly cited causes of abnormally high pressure in petroleum provinces. In young (Tertiary) deltaic sequences, compaction disequilibrium is the dominant cause of abnormal pressure. In older (pre-Tertiary) lithified rocks, hydrocarbon generation, aquathermal expansion, and tectonics are most often cited as the causes of abnormal pressure. The association of abnormal pressures with hydrocarbon accumulations is statistically significant. Within abnormally pressured reservoirs, empirical evidence indicates that the bulk of economically recoverable oil and gas occurs in reservoirs with pressure gradients less than 0.75 psi/ft (17.4 kPa/m) and there is very little production potential from reservoirs that exceed 0.85 psi/ft (19.6 kPa/m). Abnormally pressured rocks are also commonly associated with unconventional gas accumulations where the pressuring phase is gas of either a thermal or microbial origin. In underpressured, thermally mature rocks, the affected reservoirs have most often experienced a significant cooling history and probably evolved from an originally overpressured system.

  11. Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy.

    PubMed

    Kimura, En; Han, Jay J; Li, Sheng; Fall, Brent; Ra, Jennifer; Haraguchi, Miki; Tapscott, Stephen J; Chamberlain, Jeffrey S

    2008-08-15

    Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles of myofiber necrosis and regeneration leading to loss of muscle fibers and replacement with fibrotic connective and adipose tissue. The ongoing activation and recruitment of muscle satellite cells for myofiber regeneration results in loss of regenerative capacity in part due to proliferative senescence. We explored a method whereby new myoblasts could be generated in dystrophic muscles by transplantation of primary fibroblasts engineered to express a micro-dystrophin/enhanced green fluorescent protein (muDys/eGFP) fusion gene together with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)]. Fibroblasts isolated from mdx(4cv) mice, a mouse model for DMD, were efficiently transduced with lentiviral vectors expressing muDys/eGFP and MyoD-ER(T) and underwent myogenic conversion when exposed to tamoxifen. These cells could also be induced to differentiate into muDys/eGFP-expressing myocytes and myotubes. Transplantation of transduced mdx(4cv) fibroblasts into mdx(4cv) muscles enabled tamoxifen-dependent regeneration of myofibers that express muDys. This lineage control method therefore allows replenishment of myogenic stem cells using autologous fibroblasts carrying an exogenous dystrophin gene. This strategy carries several potential advantages over conventional myoblast transplantation methods including: (i) the relative simplicity of culturing fibroblasts compared with myoblasts, (ii) a readily available cell source and ease of expansion and (iii) the ability to induce MyoD gene expression in vivo via administration of a medication. Our study provides a proof of concept for a novel gene/stem cell therapy technique and opens another potential therapeutic approach for degenerative muscle disorders. PMID:18511457

  12. Dystrophin Hot-Spot Mutants Leading to Becker Muscular Dystrophy Insert More Deeply into Membrane Models than the Native Protein.

    PubMed

    Ameziane-Le Hir, Sarah; Paboeuf, Gilles; Tascon, Christophe; Hubert, Jean-François; Le Rumeur, Elisabeth; Vié, Véronique; Raguénès-Nicol, Céline

    2016-07-26

    Dystrophin (DYS) is a membrane skeleton protein whose mutations lead to lethal Duchenne muscular dystrophy or to the milder Becker muscular dystrophy (BMD). One third of BMD "in-frame" exon deletions are located in the region that codes for spectrin-like repeats R16 to R21. We focused on four prevalent mutated proteins deleted in this area (called RΔ45-47, RΔ45-48, RΔ45-49, and RΔ45-51 according to the deleted exon numbers), analyzing protein/membrane interactions. Two of the mutants, RΔ45-48 and RΔ45-51, led to mild pathologies and displayed a similar triple coiled-coil structure as the full-length DYS R16-21, whereas the two others, RΔ45-47 and RΔ45-49, induced more severe pathologies and showed "fractional" structures unrelated to the normal one. To explore lipid packing, small unilamellar liposomes (SUVs) and planar monolayers were used at various initial surface pressures. The dissociation constants determined by microscale thermophoresis (MST) were much higher for the full-length DYS R161-21 than for the mutants; thus the wild type protein has weaker SUV binding. Comparing surface pressures after protein adsorption and analysis of atomic force microscopy images of mixed protein/lipid monolayers revealed that the mutants insert more into the lipid monolayer than the wild type does. In fact, in both models every deletion mutant showed more interactions with membranes than the full-length protein did. This means that mutations in the R16-21 part of dystrophin disturb the protein's molecular behavior as it relates to membranes, regardless of whether the accompanying pathology is mild or severe. PMID:27367833

  13. Early manifestation of alteration in cardiac function in dystrophin deficient mdx mouse using 3D CMR tagging

    PubMed Central

    Li, Wei; Liu, Wei; Zhong, Jia; Yu, Xin

    2009-01-01

    Background Duchenne muscular dystrophy (DMD) is caused by the absence of the cytoskeletal protein, dystrophin. In DMD patients, dilated cardiomyopathy leading to heart failure may occur during adolescence. However, early cardiac dysfunction is frequently undetected due to physical inactivity and generalized debilitation. The objective of this study is to determine the time course of cardiac functional alterations in mdx mouse, a mouse model of DMD, by evaluating regional ventricular function with CMR tagging. Methods In vivo myocardial function was evaluated by 3D CMR tagging in mdx mice at early (2 months), middle (7 months) and late (10 months) stages of disease development. Global cardiac function, regional myocardial wall strains, and ventricular torsion were quantified. Myocardial lesions were assessed with Masson's trichrome staining. Results Global contractile indexes were similar between mdx and C57BL/6 mice in each age group. Histology analysis showed that young mdx mice were free of myocardial lesions. Interstitial fibrosis was present in 7 month mdx mice, with further development into patches or transmural lesions at 10 months of age. As a result, 10 month mdx mice showed significantly reduced regional strain and torsion. However, young mdx mice showed an unexpected increase in regional strain and torsion, while 7 month mdx mice displayed similar regional ventricular function as the controls. Conclusion Despite normal global ventricular function, CMR tagging detected a biphasic change in myocardial wall strain and torsion, with an initial increase at young age followed by progressive decrease at older ages. These results suggest that CMR tagging can provide more sensitive measures of functional alterations than global functional indexes in dystrophin-related cardiomyopathies. PMID:19849858

  14. Chromosomal abnormalities in human sperm

    SciTech Connect

    Martin, R.H.

    1985-01-01

    The ability to analyze human sperm chromosome complements after penetration of zona pellucida-free hamster eggs provides the first opportunity to study the frequency and type of chromosomal abnormalities in human gametes. Two large-scale studies have provided information on normal men. We have studied 1,426 sperm complements from 45 normal men and found an abnormality rate of 8.9%. Brandriff et al. (5) found 8.1% abnormal complements in 909 sperm from 4 men. The distribution of numerical and structural abnormalities was markedly dissimilar in the 2 studies. The frequency of aneuploidy was 5% in our sample and only 1.6% in Brandriff's, perhaps reflecting individual variability among donors. The frequency of 24,YY sperm was low: 0/1,426 and 1/909. This suggests that the estimates of nondisjunction based on fluorescent Y body data (1% to 5%) are not accurate. We have also studied men at increased risk of sperm chromosomal abnormalities. The frequency of chromosomally unbalanced sperm in 6 men heterozygous for structural abnormalities varied dramatically: 77% for t11;22, 32% for t6;14, 19% for t5;18, 13% for t14;21, and 0% for inv 3 and 7. We have also studied 13 cancer patients before and after radiotherapy and demonstrated a significant dose-dependent increase of sperm chromosome abnormalities (numerical and structural) 36 months after radiation treatment.

  15. Haematological abnormalities in mitochondrial disorders

    PubMed Central

    Finsterer, Josef; Frank, Marlies

    2015-01-01

    INTRODUCTION This study aimed to assess the kind of haematological abnormalities that are present in patients with mitochondrial disorders (MIDs) and the frequency of their occurrence. METHODS The blood cell counts of a cohort of patients with syndromic and non-syndromic MIDs were retrospectively reviewed. MIDs were classified as ‘definite’, ‘probable’ or ‘possible’ according to clinical presentation, instrumental findings, immunohistological findings on muscle biopsy, biochemical abnormalities of the respiratory chain and/or the results of genetic studies. Patients who had medical conditions other than MID that account for the haematological abnormalities were excluded. RESULTS A total of 46 patients (‘definite’ = 5; ‘probable’ = 9; ‘possible’ = 32) had haematological abnormalities attributable to MIDs. The most frequent haematological abnormality in patients with MIDs was anaemia. 27 patients had anaemia as their sole haematological problem. Anaemia was associated with thrombopenia (n = 4), thrombocytosis (n = 2), leucopenia (n = 2), and eosinophilia (n = 1). Anaemia was hypochromic and normocytic in 27 patients, hypochromic and microcytic in six patients, hyperchromic and macrocytic in two patients, and normochromic and microcytic in one patient. Among the 46 patients with a mitochondrial haematological abnormality, 78.3% had anaemia, 13.0% had thrombopenia, 8.7% had leucopenia and 8.7% had eosinophilia, alone or in combination with other haematological abnormalities. CONCLUSION MID should be considered if a patient’s abnormal blood cell counts (particularly those associated with anaemia, thrombopenia, leucopenia or eosinophilia) cannot be explained by established causes. Abnormal blood cell counts may be the sole manifestation of MID or a collateral feature of a multisystem problem. PMID:26243978

  16. Metabolism of thyroxine-binding globulin in man. Abnormal rate of synthesis in inherited thyroxine-binding globulin deficiency and excess.

    PubMed Central

    Refetoff, S; Fang, V S; Marshall, J S; Robin, N I

    1976-01-01

    It has been previously suggested that inherited thyroxine-binding globulin (TBG) abnormalities in man may be due to mutations at a single X-chromosome-linked locus controlling TBG synthesis. However, abnormalities in TBG degradation have not been excluded. The availability of purified human TBG and its successful labeling with radioiodide allowed us to examine such possibility. Human TBG was purified by affinity chromatography, labeled under sterile conditions with 131I or 125I,, and mixed with [125I]thyroxine (T4) or [131I]T4, respectively, before their intravenous injection. Blood and urine samples were collected over a 10-day period, and the turnover parameters were calculated. In eight normal volunteers mean values +/-SD for TBG and T4 respectively, were as follows: Half time (t1/2) 5.3 +/- 0.4 and 7.0 +/- 0.6 days; distribution space (DS) 7.2 +/- 1.0 and 10.8 +/- 1.2 liters; and total daily degradation (D) 0.211 +/- 0.053 and 0.088 +/- 0.011 mumol/day. In all subjects, t1/2 of TBG was shorter than that of T4; and the DS was smaller. 2.4 mol of TBG was degraded for each mole of T4. In five of six subjects from four families, comprising hemizygous and heterozygous carriers of TBG absence, decrease, and excess, the t1/2 and DS for TBG were within the normal range. The D of TBG was proportional to the serum concentration of the protein. Changes in the T4 kinetics in these patients were compatible with euthyroidism and with the known alterations in the extrathyroidal T4 pool associated with the changes in serum TBG concentration. A striking decrease in the t1/2 of TBG was found only in a patient with acquired diminution in TBG concentration and in patients with thyrotoxicosis or other conditions apparently unrelated to thyroid dysfunction. TBG t1/2 was 2.5 days in a patient with multiple myeloma and 3.6 days in two patients with thyrotoxicosis. Decreased TBG t1/2 was also observed in three of six patients with nonthyroidal pathology and was associated with an

  17. Dystrophin deficient cardiomyopathy in mouse: Expression of Nox4 and Lox are associated with fibrosis and altered functional parameters in the heart

    PubMed Central

    Spurney, Christopher F.; Knoblach, Susan; Pistilli, Emidio E.; Nagaraju, Kanneboyina; Martin, Gerard R.; Hoffman, Eric P.

    2008-01-01

    Duchenne muscular dystrophy (DMD; dystrophin-deficiency) causes dilated cardiomyopathy in the second decade of life in affected males. We studied the dystrophin-deficient mouse heart (mdx) using high frequency echocardiography, histomorphometry, and gene expression profiling. Heart dysfunction was prominent at 9-10 months of age and showed significantly increased LV internal diameter (end systole) and decreased posterior wall thickness. This cardiomyopathy was associated with a 30% decrease in shortening fraction. Histologically, there was a 10-fold increase in connective tissue volume (fibrosis). mRNA profiling with RT-PCR validation showed activation of key pro-fibrotic genes, including Nox4 and Lox. The Nox gene family expression differed in mdx heart and skeletal muscle, where Nox2 was specifically induced in skeletal muscle while Nox4 was specifically induced in heart. This is the first report of an altered profibrotic gene expression profile in cardiac tissue of dystrophic mice showing echocardiographic evidence of cardiomyopathy. PMID:18440230

  18. Distal mdx muscle groups exhibiting up-regulation of utrophin and rescue of dystrophin-associated glycoproteins exemplify a protected phenotype in muscular dystrophy

    NASA Astrophysics Data System (ADS)

    Dowling, Paul; Culligan, Kevin; Ohlendieck, Kay

    2002-02-01

    Unique unaffected skeletal muscle fibres, unlike necrotic torso and limb muscles, may pave the way for a more detailed understanding of the molecular pathogenesis of inherited neuromuscular disorders and help to develop new treatment strategies for muscular dystrophies. The sparing of extraocular muscle in Duchenne muscular dystrophy is mostly attributed to the special protective properties of extremely fast-twitching small-diameter fibres, but here we show that distal muscles also represent a particular phenotype that is more resistant to necrosis. Immunoblot analysis of membranes isolated from the well established dystrophic animal model mdx shows that, in contrast to dystrophic limb muscles, the toe musculature exhibits an up-regulation of the autosomal dystrophin homologue utrophin and a concomitant rescue of dystrophin-associated glycoproteins. Thus distal mdx muscle groups provide a cellular system that naturally avoids myofibre degeneration which might be useful in the search for naturally occurring compensatory mechanisms in inherited skeletal muscle diseases.

  19. Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

    PubMed Central

    Janghra, Narinder; Morgan, Jennifer E.; Sewry, Caroline A.; Wilson, Francis X.; Davies, Kay E.; Muntoni, Francesco; Tinsley, Jonathon

    2016-01-01

    Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify

  20. Mice Lacking Dystrophin or α Sarcoglycan Spontaneously Develop Embryonal Rhabdomyosarcoma with Cancer-Associated p53 Mutations and Alternatively Spliced or Mutant Mdm2 Transcripts

    PubMed Central

    Fernandez, Karen; Serinagaoglu, Yelda; Hammond, Sue; Martin, Laura T.; Martin, Paul T.

    2010-01-01

    Altered expression of proteins in the dystrophin-associated glycoprotein complex results in muscular dystrophy and has more recently been implicated in a number of forms of cancer. Here we show that loss of either of two members of this complex, dystrophin in mdx mice or α sarcoglycan in Sgca−/− mice, results in the spontaneous development of muscle-derived embryonal rhabdomyosarcoma (RMS) after 1 year of age. Many mdx and Sgca−/− tumors showed increased expression of insulin-like growth factor 2, retinoblastoma protein, and phosphorylated Akt and decreased expression of phosphatase and tensin homolog gene, much as is found in a human RMS. Further, all mdx and Sgca−/− RMS analyzed had increased expression of p53 and murine double minute (mdm)2 protein and contained missense p53 mutations previously identified in human cancers. The mdx RMS also contained missense mutations in Mdm2 or alternatively spliced Mdm2 transcripts that lacked an exon encoding a portion of the p53-binding domain. No Pax3:Fkhr or Pax7:Fkhr translocation mRNA products were evident in any tumor. Expression of natively glycosylated α dystroglycan and α sarcoglycan was reduced in mdx RMS, whereas dystrophin expression was absent in almost all human RMS, both for embryonal and alveolar RMS subtypes. These studies show that absence of members of the dystrophin-associated glycoprotein complex constitutes a permissive environment for spontaneous development of embryonal RMS associated with mutation of p53 and mutation or altered splicing of Mdm2. PMID:20019182

  1. Protein-DNA array-based identification of transcription factor activities differentially regulated in skeletal muscle of normal and dystrophin-deficient mdx mice.

    PubMed

    Dogra, Charu; Srivastava, Daya Shankar; Kumar, Ashok

    2008-05-01

    Inactivation of dystrophin gene is the primary cause of Duchenne muscular dystrophy (DMD) in humans and mdx mice. However, the underpinning mechanisms, which govern the pathogenesis of dystrophin-deficient skeletal muscle, remain poorly understood. We have previously reported activation of mitogen-activated protein kinases (MAPK), nuclear factor-kappa B (NF-kappaB), and phosphatidyl-inositol 3-kinase/Akt (PI3K/Akt) signaling pathways in diaphragm muscle of mdx mice. In this study, using a protein-DNA array-based approach, we have investigated the activation of 345 transcription factors in diaphragm muscle of 6-week old normal and dystrophin-deficient mdx mice. Our data demonstrate increased activation of a number nuclear transcription factors including AP1, HFH-3, PPARalpha, c.myb BP, ETF, Fra-1/JUN, kBF-A, N-rasBP, lactoferrin BP, Myb(2), EBP40_45, EKLF(1), p53(2), TFEB, Myc-Max; c-Rel; E2, ISRE; NF-kB; Stat1 p84/p91, Antioxidant RE, EVI-1, Stat3, AP3, p53, Stat4, AP4, HFH-1, FAST-1, Pax-5, and Beta-RE in the diaphragm muscle of mdx mice compared to corresponding normal mice. The level of activation for p53 was highest among all the transcription factors studied. Furthermore, higher activation of p53 in diaphragm muscle of mdx mice was associated with its increased phosphorylation and nuclear translocation. Collectively, our data suggest that the primary deficiency of dystrophin leads to the aberrant activation of nuclear transcription factors which might further contribute to muscle pathogenesis in mdx mice. PMID:18278580

  2. A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping

    PubMed Central

    Walmsley, Gemma L.; Arechavala-Gomeza, Virginia; Fernandez-Fuente, Marta; Burke, Margaret M.; Nagel, Nicole; Holder, Angela; Stanley, Rachael; Chandler, Kate; Marks, Stanley L.; Muntoni, Francesco; Shelton, G. Diane; Piercy, Richard J.

    2010-01-01

    Background Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion “hot spot” is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD “hot spot”. Methodology/Principal Findings Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5′ donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. Conclusions/Significance Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD. PMID:20072625

  3. Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies.

    PubMed

    Janghra, Narinder; Morgan, Jennifer E; Sewry, Caroline A; Wilson, Francis X; Davies, Kay E; Muntoni, Francesco; Tinsley, Jonathon

    2016-01-01

    Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ -sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify

  4. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    SciTech Connect

    Eom, Young Woo; Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin; Park, Won Jin; Kong, Jee Hyun; Shim, Kwang Yong; Lee, Jong In; Kim, Hyun Soo

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  5. Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9

    PubMed Central

    Li, Hongmei Lisa; Fujimoto, Naoko; Sasakawa, Noriko; Shirai, Saya; Ohkame, Tokiko; Sakuma, Tetsushi; Tanaka, Michihiro; Amano, Naoki; Watanabe, Akira; Sakurai, Hidetoshi; Yamamoto, Takashi; Yamanaka, Shinya; Hotta, Akitsu

    2014-01-01

    Summary Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases. PMID:25434822

  6. Use of dystrophin genomic and cDNA probes for solving difficulties in carrier detection and prenatal diagnosis of Duchenne muscular dystrophy.

    PubMed

    Shomrat, R; Driks, N; Legum, C; Shiloh, Y

    1992-02-01

    Duchenne muscular dystrophy (DMD) results from mutations in the X-linked gene coding for the muscular protein dystrophin. The isolation of genomic and cDNA probes for this gene has greatly facilitated the detection of DMD carriers, which previously relied mainly on measurements of serum creatine kinase (CK), and has enabled prenatal diagnosis of this disease. However, the relatively large size of the gene and the high frequency of recombination and mutation events within the dystrophin locus continue to pose difficulties in the genetic counselling and prenatal diagnosis of DMD, and render the conclusions of molecular analysis less clear cut. This communication presents examples of two such difficulties: the distinction between sporadic and inherited cases in families with a single patient and normal CK levels in all females, and the distinction between mutant and normal dystrophin alleles in families in which the patients have died. The combined use of genomic and cDNA probes allows one to make these distinctions. An additional complicating factor, gonadal mosaicism, is demonstrated. PMID:1536162

  7. Long-Term Efficacy of Systemic Multiexon Skipping Targeting Dystrophin Exons 45–55 With a Cocktail of Vivo-Morpholinos in Mdx52 Mice

    PubMed Central

    Echigoya, Yusuke; Aoki, Yoshitsugu; Miskew, Bailey; Panesar, Dharminder; Touznik, Aleksander; Nagata, Tetsuya; Tanihata, Jun; Nakamura, Akinori; Nagaraju, Kanneboyina; Yokota, Toshifumi

    2015-01-01

    Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45–55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45–55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45–55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45–55 region was induced, and the Western blot analysis exhibited the restoration of 5–27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model. PMID:25647512

  8. Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

    PubMed

    Heller, Kristin N; Montgomery, Chrystal L; Shontz, Kimberly M; Clark, K Reed; Mendell, Jerry R; Rodino-Klapac, Louise R

    2015-10-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD. PMID:26076707

  9. Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

    PubMed

    Echigoya, Yusuke; Aoki, Yoshitsugu; Miskew, Bailey; Panesar, Dharminder; Touznik, Aleksander; Nagata, Tetsuya; Tanihata, Jun; Nakamura, Akinori; Nagaraju, Kanneboyina; Yokota, Toshifumi

    2015-01-01

    Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45-55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45-55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45-55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45-55 region was induced, and the Western blot analysis exhibited the restoration of 5-27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model. PMID:25647512

  10. Activation of non-myogenic mesenchymal stem cells during the disease progression in dystrophic dystrophin/utrophin knockout mice

    PubMed Central

    Sohn, Jihee; Lu, Aiping; Tang, Ying; Wang, Bing; Huard, Johnny

    2015-01-01

    Ectopic calcification as well as fatty and fibrotic tissue accumulation occurs in skeletal muscle during the disease progression of Duchenne muscular dystrophy (DMD), a degenerative muscle disorder caused by mutations in the dystrophin gene. The cellular origin and the environmental cues responsible for this ectopic calcification, fatty and fibrotic infiltration during the disease progression, however, remain unknown. Based on a previously published preplate technique, we isolated two distinct populations of muscle-derived cells from skeletal muscle: (i) a rapidly adhering cell population, which is non-myogenic, Pax7− and express the mesenchymal stem cell (MSC) marker platelet-derived growth factor receptor alpha; hence, we termed this population of cells non-myogenic MSCs (nmMSCs); and (ii) a slowly adhering cell population which is Pax7+ and highly myogenic, termed muscle progenitor cells (MPCs). Previously, we demonstrated that the rapid progression of skeletal muscle histopathologies in dystrophin/utrophin knockout (dys−/− utro−/− dKO) mice is closely associated with a rapid depletion of the MPC population pool. In the current study, we showed that in contrast to the MPCs, the nmMSCs become activated during the disease progression in dKO mice, displaying increased proliferation and differentiation potentials (adipogenesis, osteogenesis and fibrogenesis). We also found that after co-culturing the dKO-nmMSCs with dKO-MPCs, the myogenic differentiation potential of the dKO-MPCs was reduced. This effect was found to be potentially mediated by the secretion of secreted frizzled-related protein 1 by the dKO-nmMSCs. We therefore posit that the rapid occurrence of fibrosis, ectopic calcification and fat accumulation, in dKO mice, is not only attributable to the rapid depletion of the MPC pool, but is also the consequence of nmMSC activation. Results from this study suggest that approaches to alleviate muscle weakness and wasting in DMD patients should not