- Email: [email protected]
A comparative phenotype study of the key forms of Periodic Paralysis in the UK
Abstracts of the 10th Neuromuscular Translational Research Conference / Neuromuscular Disorder 27S1 (2017) S5–S44
MN14 Binding to a novel RNA-protein complex creates specificity for small molecule splicing modifiers to treat SMA M. Sivaramakrishnan1,2, K.D. McCarthy1, S. Huber1, S. Campagne3, S. Meier1,2, A. Augustin1, T. Heckel1, H. Meistermann1, M. Hug1, P. Birrer1, A. Moursy3, S. Khawaja3, R. Schmucki1, N. Berntenis1, N. Giroud1, S. Golling1, M. Tzouros1, B. Banfai1, G. Duran-Pacheco1, J. Lamerz1, Y.H. Liu2, T. Luebbers1, H. Ratni1 , A. Clery3, M. Ebeling1, S. Paushkin4, A.R. Krainer2, F. Allain3, F. Metzger1 1 F. Hoffmann-La Roche Ltd., Pharma Research & Early Development, Roche Innovation Center Basel, Basel, Switzerland; 2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, USA; 3ETH Zurich, Department of Biology, Hönggerbergring, Zürich, Switzerland;4SMA Foundation, New York, NY, USA Background: Mutations affecting splicing are thought to be the cause of over half of all disease-causing mutations. The splicing defect in spinal muscular atrophy (SMA) is well-described, resulting in truncated messenger ribonucleic acid transcripts, and small molecules targeting the gene splicing mechanism are in clinical development. Small-molecule splicing modifiers have been described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival motor neuron 2) gene have been identified and are moving towards a potential therapy for SMA. Aims and Methods: In this study, we aimed to understand the mechanism of action of small molecule splicing modifiers and the underlying cause of their high specificity by using several molecular and biochemical analysis technologies as well as NMR. Results: Here we show that these small molecule splicing modifiers directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical for the high specificity of these small molecules for SMN2 over other genes. Conclusions: The data provide evidence for structural components on the small molecules and on RNA that are required for achieving very high specificity of the compounds. In addition to the therapeutic potential of these molecules for the treatment of people with SMA, our work has broad implications for further research to identify highly specific small molecules, able to target splicing correction of specific genes through interaction with gene specific protein-RNA structures.
Neuromuscular Junction Disorders and Channelopathies NMJ+C01 A comparative phenotype study of the key forms of Periodic Paralysis in the UK D.L. Raja Rayan, M.G. Hanna MRC Centre for Neuromuscular Disease, Institute of Neurology, University College London, London, UK Background: In the UK there are three main types of Periodic Paralysis: Hypokalaemic periodic paralysis (HypoPP), Hyperkalaemic periodic paralysis (HyperPP) and Andersen Tawil Syndrome (ATS). There have been no published comparative studies of these three conditions. Aims: To investigate key phenotypic characteristics of patients with HypoPP, HyperPP and ATS and identify key differentiating features. Methods: Data was collected from a comprehensive database of over 1500 channelopathy patients seen or assessed at the MRC Centre for Neuromuscular disease. Only genetically confirmed cases were included. Results: In total 119 patients were identified and significant differences were noted between diseases. Significant differences were seen in age of onset of disease, with HyperPP patients having the youngest onset, HypoPP having the oldest onset and ATS associated with an intermediate age of onset. HyperPP patients tended to have less severe, more focal attacks of weakness and HypoPP more generalised episodes. The duration of attacks were markedly longer in ATS patients. Ictal potassium, as expected, was high in HyperPP, low in HypoPP and intermediate in ATS. Patients with HypoPP were much
S33
more likely to be hospitalised during attacks. Exercise was a common trigger in all three diseases but carbohydrates were more likely to trigger HypoPP and prolonged rest more likely to trigger HyperPP. The efficacy of acetazolamide and dichlorphenamide were similar in all groups. The prevalence of proximal weakness in each group was similar but patients with ATS had a younger age of onset and poorer functional status. Conclusions: This study illustrates some of the key differences between the three types of periodic paralysis in the UK. Overall the disease appears to be a spectrum, with HyperPP at one end and HypoPP at the other and patients with ATS having a more intermediate phenotype which correlates with the observation that ATS patients have a combination of hyper, hypo and normokalaemic attacks. These findings may help guide clinical management and genetic testing in this group of patients. NMJ+C02 Hypokalaemic periodic paralysis due to a novel ATP1A2 mutation: a new periodic paralysis gene? E. Matthews1, E. Zanoteli2, R.S. Scalco1, B. O’Callaghan1, R. Sud3, S. McCall3, M.G. Hanna1, M. Sampedro Castenada1, R. Männikkö1, H. Poulson4 1 MRC Center for Neuromuscular Diseases, UCL and National Hospital for Neurology and Neurosurgery, Queen Square, London, UK; 2Department of Neurology, Medical School of the University of São Paulo, São Paulo, Brazil; 3 Neurogenetics Unit, Institute of Neurology, Queen Square, London, UK; 4 Danish Research Institute of Translational Neuroscience — DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark E-mail: [email protected] Background: Hypokalaemic periodic paralysis (hypoPP) is a rare neuromuscular disorder characterised by episodes of flaccid skeletal muscle paralysis. Symptoms typically occur in the early morning, last a minimum of hours and are accompanied by low serum potassium. Carbohydrates commonly provoke an attack. Aims: A young boy with clinical features of hypoPP was referred for genetic analysis. He lacked a mutation in any of the known periodic paralysis genes (CACNA1S, SCN4A, KCNJ2) and we sought to identify another genetic explanation. Methods: Targeted exome sequencing was performed using the Agilent Focused Exome kit. Variant of interest and family segregation was confirmed by Sanger Sequencing. Functional characterisation was performed using Two Electrode Voltage Clamp and a xenopus oocyte system. Results: Pregnancy and birth history were unremarkable but he experienced global developmental delay with learning difficulties. At age 4 months he had a generalised tonic-clonic seizure. At age 2 he woke with tetraparesis and dysphagia lasting several hours. He had a concomitant lower respiratory tract infection. He had two similar although longer (4 days) episodes at age 3 and 4. From this age he had increasingly frequent episodes, averaging 5 to 10 per month. Attacks are usually on waking and last hours to days. Triggers include carbohydrate meals and viral illness. CK is elevated 587–775 IU/L. Serum potassium low during symptoms (2.4). Muscle biopsy shortly after attack showed evidence of myopathy and fibre necrosis. MRI head and EEG were unremarkable. Symptoms responded to daily potassium supplemments but were significantly worsened by acetazolamide. Genetic analysis identified a novel variant in the ATP1A2 gene. Preliminary functional analysis demonstrates an aberrant inward leak current. Conclusions: ATP1A2 codes for an isoform of sodium-potassium ATP-ase that is expressed in both brain and skeletal muscle. The identified variant is highly conserved and an integral residue for a potassium binding site of the pump. Known genes associated with hypoPP alter sarcolemmal excitability by introducing an aberrant inward leak current that increases susceptibility to depolarisation at physiologically low potassium levels. Functional analysis demonstrates a similar pathomechanism is introduced by the ATP1A2 variant and is consistent with the clinical features.
MN14 Binding to a novel RNA-protein complex creates specificity for small molecule splicing modifiers to treat SMA M. Sivaramakrishnan1,2, K.D. McCarthy1, S. Huber1, S. Campagne3, S. Meier1,2, A. Augustin1, T. Heckel1, H. Meistermann1, M. Hug1, P. Birrer1, A. Moursy3, S. Khawaja3, R. Schmucki1, N. Berntenis1, N. Giroud1, S. Golling1, M. Tzouros1, B. Banfai1, G. Duran-Pacheco1, J. Lamerz1, Y.H. Liu2, T. Luebbers1, H. Ratni1 , A. Clery3, M. Ebeling1, S. Paushkin4, A.R. Krainer2, F. Allain3, F. Metzger1 1 F. Hoffmann-La Roche Ltd., Pharma Research & Early Development, Roche Innovation Center Basel, Basel, Switzerland; 2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, USA; 3ETH Zurich, Department of Biology, Hönggerbergring, Zürich, Switzerland;4SMA Foundation, New York, NY, USA Background: Mutations affecting splicing are thought to be the cause of over half of all disease-causing mutations. The splicing defect in spinal muscular atrophy (SMA) is well-described, resulting in truncated messenger ribonucleic acid transcripts, and small molecules targeting the gene splicing mechanism are in clinical development. Small-molecule splicing modifiers have been described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival motor neuron 2) gene have been identified and are moving towards a potential therapy for SMA. Aims and Methods: In this study, we aimed to understand the mechanism of action of small molecule splicing modifiers and the underlying cause of their high specificity by using several molecular and biochemical analysis technologies as well as NMR. Results: Here we show that these small molecule splicing modifiers directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical for the high specificity of these small molecules for SMN2 over other genes. Conclusions: The data provide evidence for structural components on the small molecules and on RNA that are required for achieving very high specificity of the compounds. In addition to the therapeutic potential of these molecules for the treatment of people with SMA, our work has broad implications for further research to identify highly specific small molecules, able to target splicing correction of specific genes through interaction with gene specific protein-RNA structures.
Neuromuscular Junction Disorders and Channelopathies NMJ+C01 A comparative phenotype study of the key forms of Periodic Paralysis in the UK D.L. Raja Rayan, M.G. Hanna MRC Centre for Neuromuscular Disease, Institute of Neurology, University College London, London, UK Background: In the UK there are three main types of Periodic Paralysis: Hypokalaemic periodic paralysis (HypoPP), Hyperkalaemic periodic paralysis (HyperPP) and Andersen Tawil Syndrome (ATS). There have been no published comparative studies of these three conditions. Aims: To investigate key phenotypic characteristics of patients with HypoPP, HyperPP and ATS and identify key differentiating features. Methods: Data was collected from a comprehensive database of over 1500 channelopathy patients seen or assessed at the MRC Centre for Neuromuscular disease. Only genetically confirmed cases were included. Results: In total 119 patients were identified and significant differences were noted between diseases. Significant differences were seen in age of onset of disease, with HyperPP patients having the youngest onset, HypoPP having the oldest onset and ATS associated with an intermediate age of onset. HyperPP patients tended to have less severe, more focal attacks of weakness and HypoPP more generalised episodes. The duration of attacks were markedly longer in ATS patients. Ictal potassium, as expected, was high in HyperPP, low in HypoPP and intermediate in ATS. Patients with HypoPP were much
S33
more likely to be hospitalised during attacks. Exercise was a common trigger in all three diseases but carbohydrates were more likely to trigger HypoPP and prolonged rest more likely to trigger HyperPP. The efficacy of acetazolamide and dichlorphenamide were similar in all groups. The prevalence of proximal weakness in each group was similar but patients with ATS had a younger age of onset and poorer functional status. Conclusions: This study illustrates some of the key differences between the three types of periodic paralysis in the UK. Overall the disease appears to be a spectrum, with HyperPP at one end and HypoPP at the other and patients with ATS having a more intermediate phenotype which correlates with the observation that ATS patients have a combination of hyper, hypo and normokalaemic attacks. These findings may help guide clinical management and genetic testing in this group of patients. NMJ+C02 Hypokalaemic periodic paralysis due to a novel ATP1A2 mutation: a new periodic paralysis gene? E. Matthews1, E. Zanoteli2, R.S. Scalco1, B. O’Callaghan1, R. Sud3, S. McCall3, M.G. Hanna1, M. Sampedro Castenada1, R. Männikkö1, H. Poulson4 1 MRC Center for Neuromuscular Diseases, UCL and National Hospital for Neurology and Neurosurgery, Queen Square, London, UK; 2Department of Neurology, Medical School of the University of São Paulo, São Paulo, Brazil; 3 Neurogenetics Unit, Institute of Neurology, Queen Square, London, UK; 4 Danish Research Institute of Translational Neuroscience — DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark E-mail: [email protected] Background: Hypokalaemic periodic paralysis (hypoPP) is a rare neuromuscular disorder characterised by episodes of flaccid skeletal muscle paralysis. Symptoms typically occur in the early morning, last a minimum of hours and are accompanied by low serum potassium. Carbohydrates commonly provoke an attack. Aims: A young boy with clinical features of hypoPP was referred for genetic analysis. He lacked a mutation in any of the known periodic paralysis genes (CACNA1S, SCN4A, KCNJ2) and we sought to identify another genetic explanation. Methods: Targeted exome sequencing was performed using the Agilent Focused Exome kit. Variant of interest and family segregation was confirmed by Sanger Sequencing. Functional characterisation was performed using Two Electrode Voltage Clamp and a xenopus oocyte system. Results: Pregnancy and birth history were unremarkable but he experienced global developmental delay with learning difficulties. At age 4 months he had a generalised tonic-clonic seizure. At age 2 he woke with tetraparesis and dysphagia lasting several hours. He had a concomitant lower respiratory tract infection. He had two similar although longer (4 days) episodes at age 3 and 4. From this age he had increasingly frequent episodes, averaging 5 to 10 per month. Attacks are usually on waking and last hours to days. Triggers include carbohydrate meals and viral illness. CK is elevated 587–775 IU/L. Serum potassium low during symptoms (2.4). Muscle biopsy shortly after attack showed evidence of myopathy and fibre necrosis. MRI head and EEG were unremarkable. Symptoms responded to daily potassium supplemments but were significantly worsened by acetazolamide. Genetic analysis identified a novel variant in the ATP1A2 gene. Preliminary functional analysis demonstrates an aberrant inward leak current. Conclusions: ATP1A2 codes for an isoform of sodium-potassium ATP-ase that is expressed in both brain and skeletal muscle. The identified variant is highly conserved and an integral residue for a potassium binding site of the pump. Known genes associated with hypoPP alter sarcolemmal excitability by introducing an aberrant inward leak current that increases susceptibility to depolarisation at physiologically low potassium levels. Functional analysis demonstrates a similar pathomechanism is introduced by the ATP1A2 variant and is consistent with the clinical features.