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P.9.13 Congenital myopathies in a pediatric referral center
Abstracts / Neuromuscular Disorders 23 (2013) 738–852 P.9.11 Transgenic zebrafish expressing mutant skeletal muscle actin, acta1a, model human nemaline myopathy O. Ceyhan, A.H. Beggs Boston Children’s Hospital and Harvard Medical School, Division of Genetics and Program in Genomics, The Manton Center for Orphan Disease Research, Boston, United States The nemaline myopathies (NMs) are a heterogeneous group of congenital myopathies defined by moderate to severe muscle weakness and accumulation of rod-like structures (nemaline bodies) in myofibers. Heterozygous dominant mutations of the skeletal muscle a-actin gene (ACTA1) account for 25% of all NM cases. Despite our current understanding of normal actin function, the mechanisms underlying muscle pathology remain unclear and there are no curative therapies available for patients. In order to elucidate the molecular defects behind ACTA1related NM, we are generating a panel of transgenic zebrafish lines that express a series of disease-linked dominant mutations on the zebrafish acta1a transcript, the predominant actin expressed in fast myofibers. We initially tested whether three of these variants, acta1a H42Y, M134V and V165M would lead to a muscle phenotype by injecting the mutant mRNA into zebrafish embryos. The fish overexpressing mutant actins displayed myopathic phenotypes characterized by delayed hatching from the chorion and curved bodies to variable degrees that corresponded with the severity of disease in patients with these mutations. We next generated an acta1a V165M transgenic line that stably expresses mutant actin in all fast myofibers and characterized its neuromuscular phenotypes by morphological and histological analysis at different time points. The acta1a V165M fish display muscle weakness as evidenced by their thin bodies and curved tails at 2 days-post-fertilization (dpf) and reduced motility in touchevoked escape response assay at 5 dpf. Whole-mount phalloidin staining at 2 dpf revealed actin aggregates in the affected fish muscle, while electron microscopy demonstrated Z-line thickening and severe myofibrillar disorganization. These results provide proof of concept that zebrafish models of actin mutations recapitulate the human disease and have robust myopathic phenotypes that may be amenable for high-throughput chemical screening. http://dx.doi:10.1016/j.nmd.2013.06.524
P.9.12 Exploring the pathological mechanism of Actin myopathies in zebrafish T. Sztal 1, M. Zhao 1, P. Currie 2, N. Laing 3, K. Nowak 3, R. Bryson-Richardson 1 1 Monash University, School of Biological Sciences, Melbourne, Australia; 2 Monash University, Australian Regenerative Medicine Institute, Melbourne, Australia; 3 University of Western Australia, Western Australian Institute for Medical Research, Centre for Medical Research, Nedlands, Australia Congenital myopathies are characterized by progressive muscle weakness and low muscle tone. Mutations in ACTA1 are responsible for up to 20% of all congenital myopathies ranging in severity from long-term survival with minor muscle weakness to lethality prior to or shortly after birth. To date over 200 different ACTA1 mutations have been described. At the cellular level ACTA1 mutations result in multiple pathologies including; nemaline bodies, intranuclear rods, actin aggregates, congenital fibre type disproportion, or cores, however, the exact pathophysiology of muscle weakness remains unknown. To investigate the mechanism by which mutations results in the distinct pathologies we have generated stable transgenic zebrafish strains expressing human ACTA1 tagged with GFP. We show, that expression of ACTA1 variants in zebrafish recapitulates the histo-pathological hallmarks of their respective diseases. Using the advantages of the zebrafish model system we followed the progression
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of disease in vivo and identified distinct mechanisms of muscle weakness. We show that in ACTA1D286G-GFP fish, nemaline bodies initiate as punctate actin aggregations at the muscle attachment sites before elongating to form the characteristic rod shape. In addition to its inclusion in rods, ACTA1D286G-GFP is incorporated into sarcomeres, which is the likely cause of muscle weakness. By contrast the formation of actin aggregates in ACTA1D154N myopathy occurs in the cytoplasm without a clearly defined site of origin and aggregates remain punctate, never elongating to form rods. The accumulation of aggregates triggers cell death leading to muscle weakness. The generation of zebrafish models and identification of distinct mechanisms of disease provides an experimental platform for the development, testing and specific targeting of potential therapies to combat Actin myopathies. http://dx.doi:10.1016/j.nmd.2013.06.525
P.9.13 Congenital myopathies in a pediatric referral center M. Karakaya 1, B. Talim 2, G. Kale 2, H. Topaloglu 1 1 Hacettepe University Department of Pediatrics, Neurology Unit, Ankara, Turkey; 2 Hacettepe University Department of Pediatrics, Pathology Unit, Ankara, Turkey Congenital myopathies are a group of inherited neuromuscular disorders presenting in childhood and mainly characterised by muscle biopsy features. Among them, nemaline myopathy, centronuclear/myotubular myopathy and core myopathy are the most common forms. Diagnosis is sometimes challenging since there is clinical and genetic overlap between different forms of congenital myopathies and some genetically confirmed patients may not show typical histopathological findings, at least in early stages. We aimed to document congenital myopathy cases diagnosed in our pediatric center in the last 10 years. We reviewed clinical and histopathological data of cases with a clinicopathological diagnosis of congenital myopathy. Of the 91 cases with a clinical diagnosis of congenital myopathy and compatible muscle biopsy features, mean age of diagnosis was 5 years, ranging from 6 days to 16 years. Classification according to histopathological features yielded 29 cases with nemaline myopathy, 12 myotubular/centronuclear myopathy, 9 core myopathy and 6 congenital fiber type disproportion. In 19 cases, muscle biopsy showed various combination of features compatible with congenital myopathy (increase in central or internal nuclei, uneven staining or core like areas in oxidative enzyme stains, type I fiber predominance) but not sufficient to classify into a typical subgroup. In 16 cases, histological findings were more subtle or showed non-specific myopathic findings. Our results show that nemaline myopathy is the most frequent congenital myopathy in our cohort (31%), while some cases could not be classified solely on histopathological grounds. Molecular analysis, especially in this group of un-classified patients, is important for definite diagnosis and better delineation of milder or earlier histopathological changes. http://dx.doi:10.1016/j.nmd.2013.06.526
P.9.14 Next generation sequencing provides diagnosis for multiple foetal akinesia disorders E.J. Todd 1, R. Ong 1, C. Barnett 2, J. Slee 3, M. Ryan 4, K. Howell 4, P. Sivadorai 5, M.R. Davis 5, R.J.N. Allcock 6, N.G. Laing 1, G. Ravenscroft 1 1 Western Australian Institute for Medical Research and the Centre for Medical Research, University of Western Australia, Nedlands, Western Australia, Australia; 2 Paediatric and Reproductive Genetics Unit, South Australia Clinical Genetics Service, Women’s and Children’s Hospital,
P.9.12 Exploring the pathological mechanism of Actin myopathies in zebrafish T. Sztal 1, M. Zhao 1, P. Currie 2, N. Laing 3, K. Nowak 3, R. Bryson-Richardson 1 1 Monash University, School of Biological Sciences, Melbourne, Australia; 2 Monash University, Australian Regenerative Medicine Institute, Melbourne, Australia; 3 University of Western Australia, Western Australian Institute for Medical Research, Centre for Medical Research, Nedlands, Australia Congenital myopathies are characterized by progressive muscle weakness and low muscle tone. Mutations in ACTA1 are responsible for up to 20% of all congenital myopathies ranging in severity from long-term survival with minor muscle weakness to lethality prior to or shortly after birth. To date over 200 different ACTA1 mutations have been described. At the cellular level ACTA1 mutations result in multiple pathologies including; nemaline bodies, intranuclear rods, actin aggregates, congenital fibre type disproportion, or cores, however, the exact pathophysiology of muscle weakness remains unknown. To investigate the mechanism by which mutations results in the distinct pathologies we have generated stable transgenic zebrafish strains expressing human ACTA1 tagged with GFP. We show, that expression of ACTA1 variants in zebrafish recapitulates the histo-pathological hallmarks of their respective diseases. Using the advantages of the zebrafish model system we followed the progression
787
of disease in vivo and identified distinct mechanisms of muscle weakness. We show that in ACTA1D286G-GFP fish, nemaline bodies initiate as punctate actin aggregations at the muscle attachment sites before elongating to form the characteristic rod shape. In addition to its inclusion in rods, ACTA1D286G-GFP is incorporated into sarcomeres, which is the likely cause of muscle weakness. By contrast the formation of actin aggregates in ACTA1D154N myopathy occurs in the cytoplasm without a clearly defined site of origin and aggregates remain punctate, never elongating to form rods. The accumulation of aggregates triggers cell death leading to muscle weakness. The generation of zebrafish models and identification of distinct mechanisms of disease provides an experimental platform for the development, testing and specific targeting of potential therapies to combat Actin myopathies. http://dx.doi:10.1016/j.nmd.2013.06.525
P.9.13 Congenital myopathies in a pediatric referral center M. Karakaya 1, B. Talim 2, G. Kale 2, H. Topaloglu 1 1 Hacettepe University Department of Pediatrics, Neurology Unit, Ankara, Turkey; 2 Hacettepe University Department of Pediatrics, Pathology Unit, Ankara, Turkey Congenital myopathies are a group of inherited neuromuscular disorders presenting in childhood and mainly characterised by muscle biopsy features. Among them, nemaline myopathy, centronuclear/myotubular myopathy and core myopathy are the most common forms. Diagnosis is sometimes challenging since there is clinical and genetic overlap between different forms of congenital myopathies and some genetically confirmed patients may not show typical histopathological findings, at least in early stages. We aimed to document congenital myopathy cases diagnosed in our pediatric center in the last 10 years. We reviewed clinical and histopathological data of cases with a clinicopathological diagnosis of congenital myopathy. Of the 91 cases with a clinical diagnosis of congenital myopathy and compatible muscle biopsy features, mean age of diagnosis was 5 years, ranging from 6 days to 16 years. Classification according to histopathological features yielded 29 cases with nemaline myopathy, 12 myotubular/centronuclear myopathy, 9 core myopathy and 6 congenital fiber type disproportion. In 19 cases, muscle biopsy showed various combination of features compatible with congenital myopathy (increase in central or internal nuclei, uneven staining or core like areas in oxidative enzyme stains, type I fiber predominance) but not sufficient to classify into a typical subgroup. In 16 cases, histological findings were more subtle or showed non-specific myopathic findings. Our results show that nemaline myopathy is the most frequent congenital myopathy in our cohort (31%), while some cases could not be classified solely on histopathological grounds. Molecular analysis, especially in this group of un-classified patients, is important for definite diagnosis and better delineation of milder or earlier histopathological changes. http://dx.doi:10.1016/j.nmd.2013.06.526
P.9.14 Next generation sequencing provides diagnosis for multiple foetal akinesia disorders E.J. Todd 1, R. Ong 1, C. Barnett 2, J. Slee 3, M. Ryan 4, K. Howell 4, P. Sivadorai 5, M.R. Davis 5, R.J.N. Allcock 6, N.G. Laing 1, G. Ravenscroft 1 1 Western Australian Institute for Medical Research and the Centre for Medical Research, University of Western Australia, Nedlands, Western Australia, Australia; 2 Paediatric and Reproductive Genetics Unit, South Australia Clinical Genetics Service, Women’s and Children’s Hospital,