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Zebrafish models of inherited skeletal muscle disorders
S258
Abstracts / Neuromuscular Disorders 25 (2015) S184–S316 11
London, UK; Evelina Children’s Hospital, Guy’s & St. Thomas’ NHS Foundation Trust, Department of Paediatric Neurology, London, UK; 12 Rhabdomyolysis, Study Group, International Collaboration, UK The skeletal muscle ryanodine receptor (RYR1) gene encodes the principal sarcoplasmic reticulum calcium release channel (RyR1) playing a crucial role in excitation–contraction coupling, the process whereby a neuronal electrical impulse is translated into intracellular calcium release and, ultimately, muscle contraction. Dominant and recessive RYR1 mutations have been associated with a wide range of congenital myopathies, including Central Core Disease, Multiminicore Disease, Centronuclear Myopathy and Congenital Fibre Type Disproportion. In addition, dominant RYR1 mutations may cause “induced myopathies” in previously healthy individuals, as part of the Malignant Hyperthermia Susceptibility (MHS) spectrum, a pharmacogenetic response to halogenated anaesthetics and muscle relaxants, or manifesting as rhabdomyolysis in response to exercise and other triggers. Here we describe a large group of 51 individuals with RYR1-related induced myopathies, identified through next generation sequencing of 224 patients presenting with exerciseinduced myalgia and rhabdomyolysis and applying a panel of n genes known to be associated with these features. We identified a total of 38 probably pathogenic RYR1 variants, 11 of them previously reported and 27 novel. We report clinico-pathological features from RYR1 mutated patients, in particular exercise profiles and triggers of rhabdomyolysis, and present a structured approach to pathogenicity assessment of novel RYR1 variants, applying in silico analysis, functional studies and insights derived from recent crystallographic data of the RyR1 receptor. We conclude that RYR1 mutations are a common and increasingly recognized cause of exercise-induced myalgia and/or rhabdomyolysis, but exact pathogenicity assignment of the large number of novelRYR1 variants remains challenging and requires a multilevel approach. The precise relationship to the MHS trait in this cohort will require further clarification. http://dx.doi.org/10.1016/j.nmd.2015.06.263
G.P.239 Zebrafish models of inherited skeletal muscle disorders J. Patrick *, N. Wali, I. Sealy, J. Collins, E. Busch-Nentwich, D. Stemple Wellcome Trust Sanger Institute, Cambridge, UK Inherited skeletal muscle disorders comprise a clinically and genetically heterogeneous set of neuromuscular diseases associated with muscle weakness or degeneration. Recently, whole exome sequencing of human patients with muscular disorders has accelerated the discovery of potential causative mutations. We are using the zebrafish as a model to define the molecular phenotypes of these inherited muscle disorders, which have a potential to reveal new pathways for therapy. Orthologues of most human muscular disease genes can be identified in the zebrafish genome and the zebrafish has already proved an effective model for both muscular dystrophy and myopathy. Disruption to muscle development and integrity can be observed easily both in live embryos due to the translucency of the embryo and by whole mount immunohistochemistry as changes to the distinct structural features. One gene whose loss of function causes core-related myopathy is the ryanodine receptor, RYR1. We have studied a ryr1b zebrafish mutant generated within the Zebrafish Mutation Project and found a previously undescribed morphological phenotype by immunohistochemistry, with disruption to myofibre structure and protein mislocalisation. To explore the phenotype at a molecular level we have used a high throughput sequencing strategy, DeTCT (Differential expression Transcript Counting Technique), to quantitatively analyse changes in the mRNA expression levels between mutant embryos and their wild-type siblings. Surprisingly we found a vast number of differentially abundant transcripts, suggesting a dramatic change in the transcriptional profile as a result of the ryr1b mutation. Gene ontology and anatomical analysis shows enrichment of transcription factors in both muscle and neuronal tissues within our differentially abundant gene set. This suggests a compensatory response to the ryr1b mutation and may help
us to elucidate molecular mechanisms involved in the manifestation of the human condition. http://dx.doi.org/10.1016/j.nmd.2015.06.264
G.P.240 Clinical, pathology and imaging heterogeneity in autosomal recessive RYR1-related myopathy G. Tasca *,1, F. Fattori 1, D. Cassandrini 2, M. Catteruccia 1, M. Verardo 1, G. Vasco 1, M. Monforte 3, E. Ricci 3, E. Bertini 1, A. D’Amico 1 1 Bambino Gesù Children’s Research Hospital, Unit of Neuromuscular Disorders, Rome, Italy; 2 IRCCS Stella Maris, Pisa, Italy; 3 Catholic University School of Medicine, Rome, Italy RYR1-related myopathies are increasingly recognized with the availability of next-generation sequencing techniques, which often return a complexity of mutations. Together with the classic autosomal dominant forms generally causing central core disease or susceptibility to malignant hyperthermia, different other phenotypes have been identified including multiminicore disease, centronuclear myopathies, congenital fibre type disproportion, rhabdomyolysis, and severe neonatal forms, mostly caused by homozygous or compound heterozygous mutations. The aim of our study is to review the features of a cohort of patients with recessive mutations in RYR1. Our patients were studied by means of clinical examination, muscle histology, muscle imaging and genetic testing through targeted sequencing of RYR1 completed by Sanger sequencing. In our cohort, variability in clinical phenotype was notable and included patients with or without ophthalmoparesis, proximal contractures resembling Emery– Dreifuss phenotype without cardiomyopathy, muscle fatigability with decremental response on repetitive nerve stimulation, severe early-onset forms, and phenotypes characterized by muscle hypertrophy. Pathology showed classic central core disease, multiminicore disease, centronuclear myopathy with or without core-like features, or only unspecific myopathic changes. Muscle imaging which is very useful in identifying dominant forms appears less specific in recessive RYR1-myopathies and overlapping with other diseases, with scans often showing peculiar or even misleading features. Our findings further expand the clinicopathological and imaging spectrum of RYR1-related myopathies. The thorough characterization of recessive families may provide insights to clarify the pathogenic significance of new mutations and the effect of different allelic combinations on the phenotype. http://dx.doi.org/10.1016/j.nmd.2015.06.265
G.P.241 Literature synthesis of RyR1 and its interacting proteins J. Witherspoon 1, M. Jain *,2, K. Meilleur 1 1 National Institutes of Health, National Institute of Nursing Research, Bethesda, USA; 2 National Institutes of Health, Rehabilitation Medicine Department, Bethesda, USA RYR1-related myopathies (RYR1-RM) comprise a group of congenital myopathies which affect 1/90,000 people in the United States. Most patients present in childhood with delayed motor milestones, extremity muscle weakness, impaired ambulation, joint contractures, progressive scoliosis, and in some cases eye movement paralysis, respiratory failure, or susceptibility to malignant hyperthermia. No treatments exist to date. Delivery of effective treatment requires knowledge of RyR1 pathomechanisms. This literature synthesis will summarize the work published to date on the RyR1 pathway and implicate potential regions for targeting treatment. Using PubMed and other resources, synthesis matrices were created for each regulatory complex to organize the data from several studies related to different parts of the pathway. Search terms included RYR1 + mutations, RyR1 protein interaction + skeletal muscle, myopathy, post-translational modification, oxidation, and more. Additionally, we linked the previously discovered mutations with the affected areas of the pathways. RyR1 interactions can be divided into six regulatory
Abstracts / Neuromuscular Disorders 25 (2015) S184–S316 11
London, UK; Evelina Children’s Hospital, Guy’s & St. Thomas’ NHS Foundation Trust, Department of Paediatric Neurology, London, UK; 12 Rhabdomyolysis, Study Group, International Collaboration, UK The skeletal muscle ryanodine receptor (RYR1) gene encodes the principal sarcoplasmic reticulum calcium release channel (RyR1) playing a crucial role in excitation–contraction coupling, the process whereby a neuronal electrical impulse is translated into intracellular calcium release and, ultimately, muscle contraction. Dominant and recessive RYR1 mutations have been associated with a wide range of congenital myopathies, including Central Core Disease, Multiminicore Disease, Centronuclear Myopathy and Congenital Fibre Type Disproportion. In addition, dominant RYR1 mutations may cause “induced myopathies” in previously healthy individuals, as part of the Malignant Hyperthermia Susceptibility (MHS) spectrum, a pharmacogenetic response to halogenated anaesthetics and muscle relaxants, or manifesting as rhabdomyolysis in response to exercise and other triggers. Here we describe a large group of 51 individuals with RYR1-related induced myopathies, identified through next generation sequencing of 224 patients presenting with exerciseinduced myalgia and rhabdomyolysis and applying a panel of n genes known to be associated with these features. We identified a total of 38 probably pathogenic RYR1 variants, 11 of them previously reported and 27 novel. We report clinico-pathological features from RYR1 mutated patients, in particular exercise profiles and triggers of rhabdomyolysis, and present a structured approach to pathogenicity assessment of novel RYR1 variants, applying in silico analysis, functional studies and insights derived from recent crystallographic data of the RyR1 receptor. We conclude that RYR1 mutations are a common and increasingly recognized cause of exercise-induced myalgia and/or rhabdomyolysis, but exact pathogenicity assignment of the large number of novelRYR1 variants remains challenging and requires a multilevel approach. The precise relationship to the MHS trait in this cohort will require further clarification. http://dx.doi.org/10.1016/j.nmd.2015.06.263
G.P.239 Zebrafish models of inherited skeletal muscle disorders J. Patrick *, N. Wali, I. Sealy, J. Collins, E. Busch-Nentwich, D. Stemple Wellcome Trust Sanger Institute, Cambridge, UK Inherited skeletal muscle disorders comprise a clinically and genetically heterogeneous set of neuromuscular diseases associated with muscle weakness or degeneration. Recently, whole exome sequencing of human patients with muscular disorders has accelerated the discovery of potential causative mutations. We are using the zebrafish as a model to define the molecular phenotypes of these inherited muscle disorders, which have a potential to reveal new pathways for therapy. Orthologues of most human muscular disease genes can be identified in the zebrafish genome and the zebrafish has already proved an effective model for both muscular dystrophy and myopathy. Disruption to muscle development and integrity can be observed easily both in live embryos due to the translucency of the embryo and by whole mount immunohistochemistry as changes to the distinct structural features. One gene whose loss of function causes core-related myopathy is the ryanodine receptor, RYR1. We have studied a ryr1b zebrafish mutant generated within the Zebrafish Mutation Project and found a previously undescribed morphological phenotype by immunohistochemistry, with disruption to myofibre structure and protein mislocalisation. To explore the phenotype at a molecular level we have used a high throughput sequencing strategy, DeTCT (Differential expression Transcript Counting Technique), to quantitatively analyse changes in the mRNA expression levels between mutant embryos and their wild-type siblings. Surprisingly we found a vast number of differentially abundant transcripts, suggesting a dramatic change in the transcriptional profile as a result of the ryr1b mutation. Gene ontology and anatomical analysis shows enrichment of transcription factors in both muscle and neuronal tissues within our differentially abundant gene set. This suggests a compensatory response to the ryr1b mutation and may help
us to elucidate molecular mechanisms involved in the manifestation of the human condition. http://dx.doi.org/10.1016/j.nmd.2015.06.264
G.P.240 Clinical, pathology and imaging heterogeneity in autosomal recessive RYR1-related myopathy G. Tasca *,1, F. Fattori 1, D. Cassandrini 2, M. Catteruccia 1, M. Verardo 1, G. Vasco 1, M. Monforte 3, E. Ricci 3, E. Bertini 1, A. D’Amico 1 1 Bambino Gesù Children’s Research Hospital, Unit of Neuromuscular Disorders, Rome, Italy; 2 IRCCS Stella Maris, Pisa, Italy; 3 Catholic University School of Medicine, Rome, Italy RYR1-related myopathies are increasingly recognized with the availability of next-generation sequencing techniques, which often return a complexity of mutations. Together with the classic autosomal dominant forms generally causing central core disease or susceptibility to malignant hyperthermia, different other phenotypes have been identified including multiminicore disease, centronuclear myopathies, congenital fibre type disproportion, rhabdomyolysis, and severe neonatal forms, mostly caused by homozygous or compound heterozygous mutations. The aim of our study is to review the features of a cohort of patients with recessive mutations in RYR1. Our patients were studied by means of clinical examination, muscle histology, muscle imaging and genetic testing through targeted sequencing of RYR1 completed by Sanger sequencing. In our cohort, variability in clinical phenotype was notable and included patients with or without ophthalmoparesis, proximal contractures resembling Emery– Dreifuss phenotype without cardiomyopathy, muscle fatigability with decremental response on repetitive nerve stimulation, severe early-onset forms, and phenotypes characterized by muscle hypertrophy. Pathology showed classic central core disease, multiminicore disease, centronuclear myopathy with or without core-like features, or only unspecific myopathic changes. Muscle imaging which is very useful in identifying dominant forms appears less specific in recessive RYR1-myopathies and overlapping with other diseases, with scans often showing peculiar or even misleading features. Our findings further expand the clinicopathological and imaging spectrum of RYR1-related myopathies. The thorough characterization of recessive families may provide insights to clarify the pathogenic significance of new mutations and the effect of different allelic combinations on the phenotype. http://dx.doi.org/10.1016/j.nmd.2015.06.265
G.P.241 Literature synthesis of RyR1 and its interacting proteins J. Witherspoon 1, M. Jain *,2, K. Meilleur 1 1 National Institutes of Health, National Institute of Nursing Research, Bethesda, USA; 2 National Institutes of Health, Rehabilitation Medicine Department, Bethesda, USA RYR1-related myopathies (RYR1-RM) comprise a group of congenital myopathies which affect 1/90,000 people in the United States. Most patients present in childhood with delayed motor milestones, extremity muscle weakness, impaired ambulation, joint contractures, progressive scoliosis, and in some cases eye movement paralysis, respiratory failure, or susceptibility to malignant hyperthermia. No treatments exist to date. Delivery of effective treatment requires knowledge of RyR1 pathomechanisms. This literature synthesis will summarize the work published to date on the RyR1 pathway and implicate potential regions for targeting treatment. Using PubMed and other resources, synthesis matrices were created for each regulatory complex to organize the data from several studies related to different parts of the pathway. Search terms included RYR1 + mutations, RyR1 protein interaction + skeletal muscle, myopathy, post-translational modification, oxidation, and more. Additionally, we linked the previously discovered mutations with the affected areas of the pathways. RyR1 interactions can be divided into six regulatory