- Email: [email protected]
P17 Whole exome sequencing in patients with congenital myopathies
Abstracts, 7th Annual UK Neuromuscular Translational Research Conference, 2014 / Neuromuscular Disorders 24S1 (2014) S7–S27 P16 Towards a consensus on biochemical outcome measures for Duchenne muscular dystrophy clinical trials S. Torelli1 , K. Anthony1 , V. Arechavala-Gomeza1 , L.E. Taylor2 , A. Vulin2 , Y. Kaminoh2 , L. Feng1 , N. Janghra1 , G. Bonne3 , M. Beuvin3 , R. Barresi4 , M. Henderson4 , S. Laval4 , A. Lourbakos5 , G. Campion5 , V. Straub4 , T. Voit3 , C. Sewry1 , J. Ellis1 , J. Morgan1 , K.M. Flanigan2 , F. Muntoni1 . 1 Dubowitz Neuromuscular Unit, UCL, London, UK; 2 Nationwide Children’s Hospital, Columbus, OH, USA; 3 Institut de Myologie, INSERM, Paris, France; 4 Institute of Genetic Medicine, Newcastle upon Tyne, UK; 5 Prosensa Therapeutics, Leiden, Netherlands Background: Experimental therapies aimed at restoring dystrophin expression are progressing through clinical trials, but their efficacy is difficult to reliably assess and compare due to a lack of standardised biochemical outcome measures to quantify dystrophin. Aims: To compare dystrophin quantification methods and reach a consensus on the most reliable method. Methods: Five laboratories performed a comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using previously agreed standardised quantitative immunohistochemistry and western blotting protocols. Results: Results were highly concordant with minimal inter- and intra-laboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by two alternative quantitative immunohistochemistry methods were essentially identical. Conclusion: The combined use of quantitative immunohistochemistry and western blotting are reliable biochemical outcome measures for DMD clinical trials, and standardised protocols can be comparable between competent laboratories. The methodology validated here will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval. P17 Whole exome sequencing in patients with congenital myopathies I. Zaharieva1 , I. Colombo1,2 , M. Sframeli1 , J.H. Sigurdsson3 , L. Feng1 , R. Phadke1 , C.A. Sewry1 , J.E. Morgan1 , F. Muntoni1 . 1 Dubowitz Neuromuscular Centre, ICH, UCL, UK; 2 Neuromuscular and Rare Disorders Unit, Milan University, Italy; 3 deCODE genetics, Reykjavik, Iceland During the last decade a growing number of genes causing Congenital myopathies (CMs) have been discovered, however, additional novel genes are yet to be identified as genetic diagnosis has not been established in many patients with CMs. We carried out whole exome sequencing (WES) in eighteen patients with CM, where mutations in suspected genes had been excluded. The WES was performed by deCODE genetics and the data analysed using deCODE Clinical Sequence Miner Tool. Recently, a homozygous missense mutation in exon 10 of STAC3 gene was identified in patients with Native American myopathy. Analysis of WES data in our cases identified a homozygous STAC3 mutation in a patient with King-Denborough syndrome and corelike changes on muscle biopsy. A second patient with a severe phenotype and muscle biopsy changes suggestive of nemaline myopathy, carried a homozygous missense mutation in KLHL40. Mutations in KLHL40 have been very recently identified as a frequent cause of severe autosomalrecessive nemaline myopathy. Two heterozygous truncating TTN mutations were detected in a patient with severe cardiomyopathy and muscle biopsy suggestive of a centronuclear myopathy (CNM), supporting the emerging data that TTN mutations should be investigated as causative in cases with unresolved CNM.
S11
By applying WES, we were able to reveal the molecular defect in 3 out of 18 patients in whom mutations in recently described genes causing CM were identified, showing that these genes should be considered in diagnostic testing. The remaining cases carried mutations in potentially novel genes which are under investigation. P18 Developing allele-selective silencing by antisense oligonucleotide as a therapeutic strategy for autosomal dominant neuromuscular diseases H. Zhou, F. Muntoni. Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, WC1N 1EH, UK Background: Antisense oligonucleotide (AON) technology has been successfully applied as experimental therapies in neuromuscular disorders, e.g. in Duchene muscular dystrophy and spinal muscular atrophy, by manipulating pre-mRNA splicing. In addition to the strategies of exon skipping and exon inclusion, allelicspecific silencing by AON has recently been investigated in some neurodegenerative genetic diseases and has shown its therapeutic potential. This strategy is potentially applicable to dominant genetic disease in which haploinsufficiency is not pathogenic. In the paediatric neuromuscular field, typical common examples are congenital muscular dystrophy caused by dominant mutations in the 3 COL6A genes and congenital core myopathies caused by dominant mutations in RYR1 gene. Aims: We aim to develop allele-specific targeting with AON to induce out-of-frame exon skipping and deplete the mutant allele by RNA nonsense-mediated decay. This strategy is theoretically feasible to specifically silence the allele where the dominant mutation resides while keeping the wild type allele intact. Methods and Materials: Exonic single nucleotide polymorphisms (SNPs) in the RYR1 gene with high frequency of heterozygosity were identified. AONs with 2 -O-methyl phosphorothiate chemistry were designed to target the candidate SNPs. Skipping of target exons and its consequences on the expression of RNA and protein were evaluated in an immortalized skeletal muscle cell line. Results: Skipping of exon 51 in RYR1 gene was confirmed at the RNA level by reverse transcript PCR and sequencing. Monoallelic expression of SNPs in exon 51 and its flanking exons were confirmed by cDNA sequencing. Furthermore, western blot showed the reduction of RyR1 protein in differentiated myotubes after AON treatment. Conclusion: Allele-specific silencing by out-of-frame exon skipping induced by AON is a feasible approach as an experimental therapy for dominantly inherited congenital core myopathies caused by RYR1 gene mutations. P19 Low doses of antisense oligonucleotide to generate an intermediate mouse model of SMA and explore optimal timing for therapeutic intervention H. Zhou, J. Morgan, F. Muntoni. Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, WC1N 1EH, UK Background: The clinical subtypes of spinal muscular atrophy (SMA) range from the most severe form (type I), to intermediate type II, type III and the mild adult-onset (type IV). While the severe transgenic mouse models may mimic type I SMA with short lifespan and severe neuromuscular involvement, the mild mouse model is essentially free from clinical neuromuscular manifestation, therefore not recapitulating the phenotype of milder SMA patients. Aims: We aim to: (1) generate an intermediate SMA mouse model which may mimic the less severe subtypes, e.g. type II and type III SMA; and (2) explore the therapeutic window in the less severe SMA mice. Methods and Materials: Severe type I SMA mice [(SMN2)2+/− ; smn−/− ] were given a single low (sub-therapeutic) dose of a 25-mer
S11
By applying WES, we were able to reveal the molecular defect in 3 out of 18 patients in whom mutations in recently described genes causing CM were identified, showing that these genes should be considered in diagnostic testing. The remaining cases carried mutations in potentially novel genes which are under investigation. P18 Developing allele-selective silencing by antisense oligonucleotide as a therapeutic strategy for autosomal dominant neuromuscular diseases H. Zhou, F. Muntoni. Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, WC1N 1EH, UK Background: Antisense oligonucleotide (AON) technology has been successfully applied as experimental therapies in neuromuscular disorders, e.g. in Duchene muscular dystrophy and spinal muscular atrophy, by manipulating pre-mRNA splicing. In addition to the strategies of exon skipping and exon inclusion, allelicspecific silencing by AON has recently been investigated in some neurodegenerative genetic diseases and has shown its therapeutic potential. This strategy is potentially applicable to dominant genetic disease in which haploinsufficiency is not pathogenic. In the paediatric neuromuscular field, typical common examples are congenital muscular dystrophy caused by dominant mutations in the 3 COL6A genes and congenital core myopathies caused by dominant mutations in RYR1 gene. Aims: We aim to develop allele-specific targeting with AON to induce out-of-frame exon skipping and deplete the mutant allele by RNA nonsense-mediated decay. This strategy is theoretically feasible to specifically silence the allele where the dominant mutation resides while keeping the wild type allele intact. Methods and Materials: Exonic single nucleotide polymorphisms (SNPs) in the RYR1 gene with high frequency of heterozygosity were identified. AONs with 2 -O-methyl phosphorothiate chemistry were designed to target the candidate SNPs. Skipping of target exons and its consequences on the expression of RNA and protein were evaluated in an immortalized skeletal muscle cell line. Results: Skipping of exon 51 in RYR1 gene was confirmed at the RNA level by reverse transcript PCR and sequencing. Monoallelic expression of SNPs in exon 51 and its flanking exons were confirmed by cDNA sequencing. Furthermore, western blot showed the reduction of RyR1 protein in differentiated myotubes after AON treatment. Conclusion: Allele-specific silencing by out-of-frame exon skipping induced by AON is a feasible approach as an experimental therapy for dominantly inherited congenital core myopathies caused by RYR1 gene mutations. P19 Low doses of antisense oligonucleotide to generate an intermediate mouse model of SMA and explore optimal timing for therapeutic intervention H. Zhou, J. Morgan, F. Muntoni. Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, WC1N 1EH, UK Background: The clinical subtypes of spinal muscular atrophy (SMA) range from the most severe form (type I), to intermediate type II, type III and the mild adult-onset (type IV). While the severe transgenic mouse models may mimic type I SMA with short lifespan and severe neuromuscular involvement, the mild mouse model is essentially free from clinical neuromuscular manifestation, therefore not recapitulating the phenotype of milder SMA patients. Aims: We aim to: (1) generate an intermediate SMA mouse model which may mimic the less severe subtypes, e.g. type II and type III SMA; and (2) explore the therapeutic window in the less severe SMA mice. Methods and Materials: Severe type I SMA mice [(SMN2)2+/− ; smn−/− ] were given a single low (sub-therapeutic) dose of a 25-mer