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P77 Collagen XII; novel disease-causing candidate gene for Bethlem-like patients
Abstracts, 7th Annual UK Neuromuscular Translational Research Conference, 2014 / Neuromuscular Disorders 24S1 (2014) S7–S27 Results: The revised scales together with the North Star Ambulatory Assessment and 6 minute walk test are being tested prospectively across the participating sites. Conclusion: It is anticipated SMA REACH UK will merge with the SMA Registry facilitating recruitment into clinical trials. Once the SMA REACH database, assessment tools and functional measures are finalised, national UK sites with a history of successful SMA enrolment will be invited to participate and collect high quality longitudinal data.
Other P75 Exome sequencing identifies EPG5 mutations in two siblings with a childhood onset vacuolar myopathy T. Whyte1 , T. Cullup2 , S. Robb1 , C. Sewry1 , H. Jungbluth3 , F. Muntoni1 . 1 Dubowitz Neuromuscular Centre, UCL Institute of Child Health/Great Ormond Street Hospital for Children, London, United Kingdom; 2 DNA Laboratory, GSTS Pathology, Guy’s Hospital, London, UK; 3 Department of Paediatric Neurology, Evelina Children’s Hospital, Guy’s and St Thomas’ National Health Service (NHS) Foundation Trust, London, UK Vici syndrome is a severe, recessively inherited multisystem disorder characterized by vacuolar myopathy, callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. Ectopic P-Granules Autophagy Protein 5 Homolog (EPG5) has been identified as the primary cause of Vici syndrome. EPG5 encodes a protein that plays a crucial role in the later stages of the autophagy pathway during the clearance of autophagosomal cargo, as evidenced by defects in phagolysosome formation in the absence of Epg-5. Null mutations in this gene have consistently presented with a similar range of severe phenotypic features. We present here a UK Caucasian sib pair presenting in the early teens with severely progressive proximal, axial and distal muscle weakness, elevated creatine kinase (5–10 times normal values), who developed respiratory insufficiency in the late teens. Muscle pathology showed a vacuolar myopathy. In collaboration with the Sanger Centre UK10K project whole exome sequencing was performed on the sisters. A heterozygous frame shift mutation was discovered in EPG5 which is also present in their unaffected father (g.43534647G>A, c.721C>T, p.R241*). Further investigation into the EPG5 transcript by RT-PCR revealed a rearrangement in her other transcribed allele, in which non-coding material from the X chromosome was inserted into exon 23 of the EPG5 gene. We speculate that this inserted sequence partially disrupted the EPG5 protein function, hence the milder phenotype compared to typical Vici syndrome observed in these siblings. P76 Clinical research activity in the Newcastle MRC centre for neuromuscular disease J. Maddison2 , B. Davis1 , J. Worley1 , G. Kenyon1 , V. Straub1 , H. Lochmuller1 , D. Turnbull2 , P. Chinnery1 , M. Trenell3 , R. Horvath1 , R. McFarland2 , G. Gorman2 , J. Miller4 , K. Bushby1 . 1 Institute of Genetic Medicine, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 2 Institute of Ageing and Health, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 3 Institute of Cellular Medicine, University of Newcastle upon Tyne, UK; 4 Newcastle Upon Tyne NHS Hospitals Foundation Trust, UK The Newcastle MRC Neuromuscular team encompasses a number of specialists involved in the diagnosis, treatment, management and research into a broad spectrum of neuromuscular and mitochondrial diseases, ranging from Duchenne muscular dystrophy, congenital myopathies and limb girdle muscular dystrophies to
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mitochondrial cytopathies and inherited neuropathies. The team aims to use information gathered from translational research to offer patients suffering from genetic and acquired neuromuscular diseases the opportunity to take part in studies and clinical trials, which may lead to new treatments and improve the quality of life for all patients and their families. The clinical research team includes clinicians, clinical research associates, physiotherapists, research nurses, psychologists, physiologists, clinical trial coordinators/ project managers and PhD students working together on a number of studies in adult and paediatric patients. Current and pending projects include drug (17) and exercise intervention studies (7), translational research (6), natural history studies (9), registries (6), BioBanks (3), behavioral intervention (3) and clinical outcome studies (3). The team is active in the conception and design of local, national and international commercial and academic studies. The coordination team is responsible for obtaining Ethical and Research and Development approval, National Institute for Health Research support and adoption and study management throughout the whole process. Every member of the clinical research team is instrumental in conducting research in line with Good Clinical Practice (GCP) which is facilitated by the coordination team to produce the highest professional level of neuromuscular clinical research in Newcastle and the North East. P77 Collagen XII; novel disease-causing candidate gene for Bethlem-like patients G.T. Farsani1 , D. Hicks1 , S. Laval1 , J. Collins2 , A. Sarkozy1 , E. Martoni3 , A. Shah1 , Y. Zou4 , M. Koch5 , C.G. Bonnemann4 , M. Roberts6 , V. Straub1 , K. Bushby1 , H. Lochmuller1 . 1 MRC Centre for Neuromuscular Disease at Newcastle, Institute of Genetic Medicine, Newcastle, UK; 2 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 3 Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy; 4 NIH, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA; 5 Institute for Dental Research and Oral Musculoskeletal Biology, Centre for Biochemistry, University of Cologne, Cologne, Germany; 6 Departments of Neurology and Neuropathology, Hope Hospital, Salford, UK Background: Bethlem Myopathy characterized by progressive muscle weakness, joint contractures and late onset of symptoms. Mutations in collagen VIA genes have been defined for only 50% of Bethlem phenotypes. Aim: a cohort of 24 Bethlem patients without mutations in the COL6A genes were analysed to identify potential disease-causing variants. Methods and Results: 12 candidate genes including collagen VI binding partners were sequenced in this cohort but no disease-causing variants were found. Whole exome sequencing (WES) revealed two novel variants (c.C5893T: p.Arg1965Cys and c.G8357A: p.Gly2786Asp) in the COL12A1 gene were identified in five patients from two families. Segregation of both variants showed an autosomal dominant pattern of inheritance. Collagen XII is a member of Fibril Associated Collagen with Interrupted Triple helices (FACIT collagens), important in stabilisation of other extracellular matrix (ECM) components [1]. In silico analysis revealed that c.G8357A: p.Gly2786Asp disrupted conserved motif of GLY-X-Y in the triple-helical domain of the protein and c.C5893T: p.Arg1965Cys which creates an unpaired cystein residue. Immunofluorescence studies revealed an intracellular retention pattern of the mutant collagens in dermal fibroblasts of patients. Conclusions: Our findings suggest a causative role for COL12A1 in Bethlem myopathy. Key words: Bethlem myopathy, COL12A1 Reference(s) [1] Koch, M., Bohrmann, B., Matthison, M., Hagios, C., Trueb, B. and Chiquet, M. (1995) Large and small splice variants of collagen XII: differential expression and ligand binding. J. Cell Biol., 130, 1005–1014.
Other P75 Exome sequencing identifies EPG5 mutations in two siblings with a childhood onset vacuolar myopathy T. Whyte1 , T. Cullup2 , S. Robb1 , C. Sewry1 , H. Jungbluth3 , F. Muntoni1 . 1 Dubowitz Neuromuscular Centre, UCL Institute of Child Health/Great Ormond Street Hospital for Children, London, United Kingdom; 2 DNA Laboratory, GSTS Pathology, Guy’s Hospital, London, UK; 3 Department of Paediatric Neurology, Evelina Children’s Hospital, Guy’s and St Thomas’ National Health Service (NHS) Foundation Trust, London, UK Vici syndrome is a severe, recessively inherited multisystem disorder characterized by vacuolar myopathy, callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. Ectopic P-Granules Autophagy Protein 5 Homolog (EPG5) has been identified as the primary cause of Vici syndrome. EPG5 encodes a protein that plays a crucial role in the later stages of the autophagy pathway during the clearance of autophagosomal cargo, as evidenced by defects in phagolysosome formation in the absence of Epg-5. Null mutations in this gene have consistently presented with a similar range of severe phenotypic features. We present here a UK Caucasian sib pair presenting in the early teens with severely progressive proximal, axial and distal muscle weakness, elevated creatine kinase (5–10 times normal values), who developed respiratory insufficiency in the late teens. Muscle pathology showed a vacuolar myopathy. In collaboration with the Sanger Centre UK10K project whole exome sequencing was performed on the sisters. A heterozygous frame shift mutation was discovered in EPG5 which is also present in their unaffected father (g.43534647G>A, c.721C>T, p.R241*). Further investigation into the EPG5 transcript by RT-PCR revealed a rearrangement in her other transcribed allele, in which non-coding material from the X chromosome was inserted into exon 23 of the EPG5 gene. We speculate that this inserted sequence partially disrupted the EPG5 protein function, hence the milder phenotype compared to typical Vici syndrome observed in these siblings. P76 Clinical research activity in the Newcastle MRC centre for neuromuscular disease J. Maddison2 , B. Davis1 , J. Worley1 , G. Kenyon1 , V. Straub1 , H. Lochmuller1 , D. Turnbull2 , P. Chinnery1 , M. Trenell3 , R. Horvath1 , R. McFarland2 , G. Gorman2 , J. Miller4 , K. Bushby1 . 1 Institute of Genetic Medicine, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 2 Institute of Ageing and Health, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 3 Institute of Cellular Medicine, University of Newcastle upon Tyne, UK; 4 Newcastle Upon Tyne NHS Hospitals Foundation Trust, UK The Newcastle MRC Neuromuscular team encompasses a number of specialists involved in the diagnosis, treatment, management and research into a broad spectrum of neuromuscular and mitochondrial diseases, ranging from Duchenne muscular dystrophy, congenital myopathies and limb girdle muscular dystrophies to
S27
mitochondrial cytopathies and inherited neuropathies. The team aims to use information gathered from translational research to offer patients suffering from genetic and acquired neuromuscular diseases the opportunity to take part in studies and clinical trials, which may lead to new treatments and improve the quality of life for all patients and their families. The clinical research team includes clinicians, clinical research associates, physiotherapists, research nurses, psychologists, physiologists, clinical trial coordinators/ project managers and PhD students working together on a number of studies in adult and paediatric patients. Current and pending projects include drug (17) and exercise intervention studies (7), translational research (6), natural history studies (9), registries (6), BioBanks (3), behavioral intervention (3) and clinical outcome studies (3). The team is active in the conception and design of local, national and international commercial and academic studies. The coordination team is responsible for obtaining Ethical and Research and Development approval, National Institute for Health Research support and adoption and study management throughout the whole process. Every member of the clinical research team is instrumental in conducting research in line with Good Clinical Practice (GCP) which is facilitated by the coordination team to produce the highest professional level of neuromuscular clinical research in Newcastle and the North East. P77 Collagen XII; novel disease-causing candidate gene for Bethlem-like patients G.T. Farsani1 , D. Hicks1 , S. Laval1 , J. Collins2 , A. Sarkozy1 , E. Martoni3 , A. Shah1 , Y. Zou4 , M. Koch5 , C.G. Bonnemann4 , M. Roberts6 , V. Straub1 , K. Bushby1 , H. Lochmuller1 . 1 MRC Centre for Neuromuscular Disease at Newcastle, Institute of Genetic Medicine, Newcastle, UK; 2 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 3 Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy; 4 NIH, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA; 5 Institute for Dental Research and Oral Musculoskeletal Biology, Centre for Biochemistry, University of Cologne, Cologne, Germany; 6 Departments of Neurology and Neuropathology, Hope Hospital, Salford, UK Background: Bethlem Myopathy characterized by progressive muscle weakness, joint contractures and late onset of symptoms. Mutations in collagen VIA genes have been defined for only 50% of Bethlem phenotypes. Aim: a cohort of 24 Bethlem patients without mutations in the COL6A genes were analysed to identify potential disease-causing variants. Methods and Results: 12 candidate genes including collagen VI binding partners were sequenced in this cohort but no disease-causing variants were found. Whole exome sequencing (WES) revealed two novel variants (c.C5893T: p.Arg1965Cys and c.G8357A: p.Gly2786Asp) in the COL12A1 gene were identified in five patients from two families. Segregation of both variants showed an autosomal dominant pattern of inheritance. Collagen XII is a member of Fibril Associated Collagen with Interrupted Triple helices (FACIT collagens), important in stabilisation of other extracellular matrix (ECM) components [1]. In silico analysis revealed that c.G8357A: p.Gly2786Asp disrupted conserved motif of GLY-X-Y in the triple-helical domain of the protein and c.C5893T: p.Arg1965Cys which creates an unpaired cystein residue. Immunofluorescence studies revealed an intracellular retention pattern of the mutant collagens in dermal fibroblasts of patients. Conclusions: Our findings suggest a causative role for COL12A1 in Bethlem myopathy. Key words: Bethlem myopathy, COL12A1 Reference(s) [1] Koch, M., Bohrmann, B., Matthison, M., Hagios, C., Trueb, B. and Chiquet, M. (1995) Large and small splice variants of collagen XII: differential expression and ligand binding. J. Cell Biol., 130, 1005–1014.