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P58 Mitochondrial respiratory chain enzyme deficiency expressed during muscle development
Posters, UK Neuromuscular Translational Research Conference 2011 / Neuromuscular Disorders 21S1 (2011) S7–S30
P58 Mitochondrial respiratory chain enzyme deficiency expressed during muscle development J.-W. Taanman1 , S. Rahman2 , P. Clayton2 , J.V. Leonard2 , R. King1 , M. Orth1 . 1 Department of Clinical Neurosciences, Institute of Neurology, University College London, Rowland Hill Street, London NW3 2PF, 2 Biochemistry, Endocrinology and Metabolism Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK We studied myoblast cell cultures from four unrelated paediatric patients with marked mitochondrial respiratory chain enzyme deficiencies in skeletal muscle, but with no apparent mitochondrial DNA abnormalities. Despite the clear enzyme defects in muscle tissue, biochemical assays revealed only mildly affected respiratory chain enzyme activities in a myoblast culture from one patient, while the activities were normal in myoblast cultures from the other three patients. Immunoblot analysis of blue native gels and cytochemical staining of the myoblast cultures corroborated the results of the biochemical assays. Myotubes, however, were clearly affected in all four patient cultures. Myotubes derived from the patient myoblast culture with mild respiratory chain enzyme deficiency showed no cytochrome-c oxidase staining and did not fully develop. Myotubes derived from the three patient myoblast cultures with normal respiratory chain enzyme activity did fully develop but showed uneven cytochrome-c oxidase staining. In both patient and control myotubes, steady-state levels of respiratory chain enzyme subunits were higher than in myoblasts; however, in contrast to control myotubes, patient myotubes contained swollen and unevenly distributed mitochondria. The life span of patient myotubes was dramatically shorter than that of control myotubes. Our results suggest that respiratory chain enzyme defects seen in skeletal muscle biopsies from these patients may be due to, or enhanced by, a failure of normal mitochondrial biogenesis during muscle development. P59 Respiratory chain complex I deficiency caused by mitochondrial DNA mutations R.W. Taylor1 , H. Swalwell1 , D.M. Kirby2 , E.L. Blakely1 , A. Mitchell1 , R. Salemi2 , C. Sugiana2,3 , A.G. Compton2 , E.J. Tucker2,3 , B.-X. Ke2 , P.J. Lamont4 , D.M. Turnbull1 , R. McFarland1 , D.R. Thorburn2,3,5 . 1 Mitochondrial Research Group, Newcastle University, UK; 2 Murdoch Childrens Research Institute, Melbourne, 3 Department of Paediatrics, University of Melbourne, 4 Neurogenetic Unit, Royal Perth Hospital, Australia, 5 Genetic Health Services Victoria, Royal Children’s Hospital, Melbourne, Australia Mitochondrial respiratory chain defects are associated with a wide range of clinical presentations and may be caused by mutations in either the nuclear or mitochondrial genome (mtDNA). Isolated complex I deficiency is the most frequently observed enzyme defect in mitochondrial disease, particularly in the paediatric population where family history is often consistent with sporadic or autosomal recessive inheritance, implicating an underlying nuclear genetic cause. Although a number of recurrent, pathogenic mtDNA mutations have been described, historically these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency referred to a single national reference laboratory for diagnostic testing. Pathogenic mtDNA mutations were found in 29/101 probands (29%), 21 in MTND genes encoding structural complex I subunits and 8 in mt-tRNA genes. Nuclear gene defects were inferred in 38/101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. Median age of onset was higher in mtDNA patients (12 months)
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than patients with a nuclear gene defect (3 months), although considerable overlap existed with onset ranging from 0m to >60m in both groups. Our data confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children and as such sequencing the entire mitochondrial genome is a key step in the diagnostic algorithm to elucidate the underlying molecular genetic abnormality. P60 Dominant and recessive RRM2B mutations cause familial PEO and multiple mtDNA deletions in muscle C. Fratter1 , P. Raman2 , C. Alston2 , E.L. Blakely2 , K. Craig2 , C. Smith1 , J. Evans1 , A. Seller1 , B. Czermin3 , M.G. Hanna4 , J. Poulton5 , C. Brierley6 , T.G. Staunton7 , P.D. Turnpenny8 , A.M. Schaefer2 , P.F. Chinnery2 , R. Horvath2 , D.M. Turnbull2 , G.S. Gorman2 , R.W. Taylor2 . 1 Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, 2 Mitochondrial Research Group, Newcastle University, UK; 3 Medical Genetic Centre, Munich, Germany; 4 UCL MRC Centre for Neuromuscular Diseases, Institute of Neurology, Queen Square, London, 5 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, UK, 6 Department of Neurology, The West Suffolk Hospital, Bury St Edmunds, Suffolk, 7 Department of Neurology, Norfolk and Norwich University Hospital, Norwich, 8 Department of Clinical Genetics, Royal Devon and Exeter Hospital, Exeter, UK Progressive External Ophthalmoplegia (PEO) is a common manifestation of patients with mitochondrial myopathy, characterised by a progressive paralysis of the extraocular muscles leading to ptosis and ophthalmoparesis. The molecular genetic defect involves either primary mitochondrial DNA (mtDNA) mutations or mutations in nuclear-encoded mtDNA maintenance proteins leading to secondary mtDNA changes including multiple deletions. Mendelian forms of PEO are caused by mutations in five genes – POLG, POLG2, SLC25A4, PEO1 and OPA1. A dominantly-inherited truncating mutation in a sixth gene – RRM2B – which encodes a subunit of the p53-inducible ribonucleotide reductase protein has recently been described in two families. We have determined the frequency of RRM2B mutations in a cohort of 75 adult patients with PEO and multiple mtDNA deletions in muscle in whom mutations in known candidate genes had been excluded. Novel RRM2B variants were detected in 12 subjects (16% of patients). Ten patients with ptosis and ophthalmoparesis as their predominant clinical features harboured single, heterozygous changes; seven of these had dominant family histories and nonsense mutations in exon 9. Two patients with childhood-onset of symptoms harboured compound heterozygous, missense variants thus providing the first description of recessive RRM2B mutations associated with multiple mtDNA deletions. These cases displayed a variable spectrum of the clinical features of RRM2B mutations, which can show multisystem involvement when associated with infantile mtDNA depletion syndrome. Our data confirm RRM2B mutations to be a frequent cause of PEO and multiple mtDNA deletions, and that sequencing of this gene should be considered early in the diagnostic algorithm for multiple mtDNA deletion disorders.
P58 Mitochondrial respiratory chain enzyme deficiency expressed during muscle development J.-W. Taanman1 , S. Rahman2 , P. Clayton2 , J.V. Leonard2 , R. King1 , M. Orth1 . 1 Department of Clinical Neurosciences, Institute of Neurology, University College London, Rowland Hill Street, London NW3 2PF, 2 Biochemistry, Endocrinology and Metabolism Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK We studied myoblast cell cultures from four unrelated paediatric patients with marked mitochondrial respiratory chain enzyme deficiencies in skeletal muscle, but with no apparent mitochondrial DNA abnormalities. Despite the clear enzyme defects in muscle tissue, biochemical assays revealed only mildly affected respiratory chain enzyme activities in a myoblast culture from one patient, while the activities were normal in myoblast cultures from the other three patients. Immunoblot analysis of blue native gels and cytochemical staining of the myoblast cultures corroborated the results of the biochemical assays. Myotubes, however, were clearly affected in all four patient cultures. Myotubes derived from the patient myoblast culture with mild respiratory chain enzyme deficiency showed no cytochrome-c oxidase staining and did not fully develop. Myotubes derived from the three patient myoblast cultures with normal respiratory chain enzyme activity did fully develop but showed uneven cytochrome-c oxidase staining. In both patient and control myotubes, steady-state levels of respiratory chain enzyme subunits were higher than in myoblasts; however, in contrast to control myotubes, patient myotubes contained swollen and unevenly distributed mitochondria. The life span of patient myotubes was dramatically shorter than that of control myotubes. Our results suggest that respiratory chain enzyme defects seen in skeletal muscle biopsies from these patients may be due to, or enhanced by, a failure of normal mitochondrial biogenesis during muscle development. P59 Respiratory chain complex I deficiency caused by mitochondrial DNA mutations R.W. Taylor1 , H. Swalwell1 , D.M. Kirby2 , E.L. Blakely1 , A. Mitchell1 , R. Salemi2 , C. Sugiana2,3 , A.G. Compton2 , E.J. Tucker2,3 , B.-X. Ke2 , P.J. Lamont4 , D.M. Turnbull1 , R. McFarland1 , D.R. Thorburn2,3,5 . 1 Mitochondrial Research Group, Newcastle University, UK; 2 Murdoch Childrens Research Institute, Melbourne, 3 Department of Paediatrics, University of Melbourne, 4 Neurogenetic Unit, Royal Perth Hospital, Australia, 5 Genetic Health Services Victoria, Royal Children’s Hospital, Melbourne, Australia Mitochondrial respiratory chain defects are associated with a wide range of clinical presentations and may be caused by mutations in either the nuclear or mitochondrial genome (mtDNA). Isolated complex I deficiency is the most frequently observed enzyme defect in mitochondrial disease, particularly in the paediatric population where family history is often consistent with sporadic or autosomal recessive inheritance, implicating an underlying nuclear genetic cause. Although a number of recurrent, pathogenic mtDNA mutations have been described, historically these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency referred to a single national reference laboratory for diagnostic testing. Pathogenic mtDNA mutations were found in 29/101 probands (29%), 21 in MTND genes encoding structural complex I subunits and 8 in mt-tRNA genes. Nuclear gene defects were inferred in 38/101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. Median age of onset was higher in mtDNA patients (12 months)
S23
than patients with a nuclear gene defect (3 months), although considerable overlap existed with onset ranging from 0m to >60m in both groups. Our data confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children and as such sequencing the entire mitochondrial genome is a key step in the diagnostic algorithm to elucidate the underlying molecular genetic abnormality. P60 Dominant and recessive RRM2B mutations cause familial PEO and multiple mtDNA deletions in muscle C. Fratter1 , P. Raman2 , C. Alston2 , E.L. Blakely2 , K. Craig2 , C. Smith1 , J. Evans1 , A. Seller1 , B. Czermin3 , M.G. Hanna4 , J. Poulton5 , C. Brierley6 , T.G. Staunton7 , P.D. Turnpenny8 , A.M. Schaefer2 , P.F. Chinnery2 , R. Horvath2 , D.M. Turnbull2 , G.S. Gorman2 , R.W. Taylor2 . 1 Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford, 2 Mitochondrial Research Group, Newcastle University, UK; 3 Medical Genetic Centre, Munich, Germany; 4 UCL MRC Centre for Neuromuscular Diseases, Institute of Neurology, Queen Square, London, 5 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, UK, 6 Department of Neurology, The West Suffolk Hospital, Bury St Edmunds, Suffolk, 7 Department of Neurology, Norfolk and Norwich University Hospital, Norwich, 8 Department of Clinical Genetics, Royal Devon and Exeter Hospital, Exeter, UK Progressive External Ophthalmoplegia (PEO) is a common manifestation of patients with mitochondrial myopathy, characterised by a progressive paralysis of the extraocular muscles leading to ptosis and ophthalmoparesis. The molecular genetic defect involves either primary mitochondrial DNA (mtDNA) mutations or mutations in nuclear-encoded mtDNA maintenance proteins leading to secondary mtDNA changes including multiple deletions. Mendelian forms of PEO are caused by mutations in five genes – POLG, POLG2, SLC25A4, PEO1 and OPA1. A dominantly-inherited truncating mutation in a sixth gene – RRM2B – which encodes a subunit of the p53-inducible ribonucleotide reductase protein has recently been described in two families. We have determined the frequency of RRM2B mutations in a cohort of 75 adult patients with PEO and multiple mtDNA deletions in muscle in whom mutations in known candidate genes had been excluded. Novel RRM2B variants were detected in 12 subjects (16% of patients). Ten patients with ptosis and ophthalmoparesis as their predominant clinical features harboured single, heterozygous changes; seven of these had dominant family histories and nonsense mutations in exon 9. Two patients with childhood-onset of symptoms harboured compound heterozygous, missense variants thus providing the first description of recessive RRM2B mutations associated with multiple mtDNA deletions. These cases displayed a variable spectrum of the clinical features of RRM2B mutations, which can show multisystem involvement when associated with infantile mtDNA depletion syndrome. Our data confirm RRM2B mutations to be a frequent cause of PEO and multiple mtDNA deletions, and that sequencing of this gene should be considered early in the diagnostic algorithm for multiple mtDNA deletion disorders.