- Email: [email protected]
Dystrophin expression in the non-DMD population: What is normal?
S160
Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212
treatments, while the majority of methods currently in place rely on the use of standard constitutive genes not always validated for the muscle or tissue culture experimental conditions. Thus, it is crucial to identify a set of genes whose expression remains constant across patients with and without treatment in our particular model system. We have chosen a candidate reference gene panel (ACTB, B2M, GAPDH, GUSB, HPRT1, PPIA, RPLP, RRN18S, TBP, TUBB, UBC, YWHAZ) to test their stability under our experimental conditions and treatments. In parallel, we have evaluated the feasibility of using an upstream region of dystrophin mRNA as a normaliser.
dystrophinopathy baseline nor ‘normal’ dystrophin levels have been defined, and anecdotal reports suggest substantial variation within the healthy population. We report that immunoblot on muscle extracts from forty different non-Duchenne muscular dystrophy individuals (same muscle) reveals wide variation in dystrophin levels, associated with normal muscle architecture and equivalent expression of sarcomeric proteins. Since no universal dystrophin standard is available, each study must stand-alone and be accompanied by appropriate reference control samples, meaning that dystrophin expression in one study cannot be compared with data from another that uses different reference standards and methodologies.
http://dx.doi.org/10.1016/j.nmd.2016.06.268 http://dx.doi.org/10.1016/j.nmd.2016.06.270 P.239 Development of a validated western blot method for quantification of human dystrophin protein F. Schnell 1, C. Donoghue 1, J. Dworzak 1, J. Charleston 1, D. Frank 1, B. Wentworth 1, S. Wilton 2, S. Lewis 3, J. Mendell 3, L. Rodino-Klapac 3, Z. Sahenk 3 1 Sarepta Therapeutics, Cambridge, USA; 2 Murdoch University, Perth, Australia; 3 Nationwide Children’s Hospital, Columbus, USA Duchenne muscular dystrophy (DMD) is primarily caused by whole-exon deletions resulting in a shift of the mRNA reading frame that prevents production of functional dystrophin protein. Therapeutic agents aimed at restoring dystrophin protein, including oligonucleotides such as the phosphorodiamidate morpholino oligomer (PMO) eteplirsen, are currently under investigation in late-stage clinical trials. Currently, there are no available validated methods to quantify dystrophin protein in muscle biopsies. Western blotting has been traditionally used as the confirmatory assay for clinical diagnosis of DMD or Becker muscular dystrophy (BMD), but the methods are not standardized and lack true quantitative capacity. A sensitive and quantitative Western blot method has been developed to detect dystrophin in human muscle biopsies utilizing a standard curve composed of non-DMD/BMD (normal control) muscle lysate spiked into DMD muscle lysate to maintain equivalent protein load. Running a 5-point standard curve ranging from 0.25% to 4% normal control on every gel allows for normalization of slight fluctuations in dystrophin signal intensity between gels. Optimized film exposure time ensures standards and samples are not oversaturated and remain within the dynamic range of the assay. Dystrophin can be reproducibly quantified down to 0.25% of normal control muscle across multiple users and runs. This method was validated according to FDA Draft Guidance for Industry, Bioanalytical Method Validation, and provides confirmation of the mechanism of action for any dystrophin protein restoring therapy. http://dx.doi.org/10.1016/j.nmd.2016.06.269
P.240 Dystrophin expression in the non-DMD population: What is normal? R. Johnsen, S. Wilton, S. Fletcher Murdoch University, Perth, Australia Dystrophin is a low abundance, large protein interacting with intracellular and sarcolemmal components to stabilize the muscle membrane during contraction. Mutations in the DMD gene leading to loss of dystrophin cause Duchenne muscular dystrophy, a fatal, X-linked disease characterized by progressive muscle wasting. Molecular therapies designed to restore dystrophin expression are reported to induce dystrophin expression and delayed disease progression in subsets of Duchenne muscular dystrophy patients. However, despite concerted efforts to standardize methods for precise dystrophin quantitation, analysis of the low dystrophin expression levels in clinical studies remains controversial. Immunohistochemistry and western blot have been used routinely to assess dystrophin for diagnosing Duchenne and Becker muscular dystrophy for over two decades, and it is common practice to analyze patient material alongside a reference sample from healthy muscle. However, neither
P.241 Optimizing dystrophin quantification in DMD and BMD patients: A new semi-automated acquisition and analysis method V. Sardone 1, M. Ellis 2, S. Torelli 1, L. Feng 2, D. Chambers 2, R. Phadke 2, C. Sewry 2, J. Morgan 1, F. Muntoni 1 1 Institute of Child Health UCL, London, UK; 2 Institute of Neurology UCL, London, UK Different experimental therapies are currently under investigation in order to restore dystrophin expression in Duchenne muscular dystrophy (DMD) patients. Antisense oligonucleotide (AON) therapies are novel pharmacological approaches aiming to induce exon skipping in the DMD transcripts and to produce a truncated dystrophin protein mimicking what occurs in Becker muscular dystrophy (BMD) patients. The primary biological endpoint of these AON clinical trials is therefore to restore functional dystrophin in muscles of DMD patients. A reliable, unbiased and reproducible method for quantification of dystrophin protein is crucial to monitor the biochemical outcome of such treatments. We have developed a new semi-automated and semi-quantitative immunohistochemistry method, based on the acquisition of an entire muscle section, which is capable of collecting data from all of the muscle fibres in the section (from 103 to 104 fibres). We have used a panel of antibodies against sarcolemma components to verify sarcolemma integrity (anti spectrin and anti laminin) as well as dystrophin antibodies recognizing different protein epitopes (anti dystrophin abcam-exon 77- and mandys106-exon 43). Each image was analysed by a script generated in house using the Definiens software. This new method allows the simultaneous quantification of many parameters such as dystrophin sarcolemmal and cytoplasmic intensity expression, spectrin or laminin cytoplasmic and sarcolemmal intensity expression, number of fibres, thickness of the sarcolemma membrane and area of each sarcolemmal and cytoplasmic compartments. We have analysed muscle sections from different DMD and BMD patients expressing variable levels of dystrophin and we present data showing that this method is highly sensitive, reliable and reproducible. The accurate and unbiased quantification of dystrophin will provide a robust pharmacodynamics endpoint in support of clinical trials aimed at dystrophin restoration. http://dx.doi.org/10.1016/j.nmd.2016.06.271
NEXT GENERATION SEQUENCING P.242 RD-Connect: Data sharing and analysis for rare disease research within the integrated platform and through GA4GH beacon and matchmaker exchange A. Roos 1, S. Beltran 2, D. Piscia 2, S. Laurie 2, J. Protasio 2, A. Cañada 3, J. Fernández 3, R. Kaliyaperumal 4, S. Lair 5, P. Sernadela 6, M. Girdea 7, R. Thompson 1, V. Straub 1, M. Roos 4, P. T’Hoen 4, A. Valencia 3, D. Salgado 8, C. Béroud 8, I. Gut 2, H. Lochmüller 1 1 Institute of Genetic Medicine, Newcastle upon Tyne, UK; 2 Center for Genomic Regulation, Barcelona, Spain; 3 Centro Nacional de Investigaciones
Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212
treatments, while the majority of methods currently in place rely on the use of standard constitutive genes not always validated for the muscle or tissue culture experimental conditions. Thus, it is crucial to identify a set of genes whose expression remains constant across patients with and without treatment in our particular model system. We have chosen a candidate reference gene panel (ACTB, B2M, GAPDH, GUSB, HPRT1, PPIA, RPLP, RRN18S, TBP, TUBB, UBC, YWHAZ) to test their stability under our experimental conditions and treatments. In parallel, we have evaluated the feasibility of using an upstream region of dystrophin mRNA as a normaliser.
dystrophinopathy baseline nor ‘normal’ dystrophin levels have been defined, and anecdotal reports suggest substantial variation within the healthy population. We report that immunoblot on muscle extracts from forty different non-Duchenne muscular dystrophy individuals (same muscle) reveals wide variation in dystrophin levels, associated with normal muscle architecture and equivalent expression of sarcomeric proteins. Since no universal dystrophin standard is available, each study must stand-alone and be accompanied by appropriate reference control samples, meaning that dystrophin expression in one study cannot be compared with data from another that uses different reference standards and methodologies.
http://dx.doi.org/10.1016/j.nmd.2016.06.268 http://dx.doi.org/10.1016/j.nmd.2016.06.270 P.239 Development of a validated western blot method for quantification of human dystrophin protein F. Schnell 1, C. Donoghue 1, J. Dworzak 1, J. Charleston 1, D. Frank 1, B. Wentworth 1, S. Wilton 2, S. Lewis 3, J. Mendell 3, L. Rodino-Klapac 3, Z. Sahenk 3 1 Sarepta Therapeutics, Cambridge, USA; 2 Murdoch University, Perth, Australia; 3 Nationwide Children’s Hospital, Columbus, USA Duchenne muscular dystrophy (DMD) is primarily caused by whole-exon deletions resulting in a shift of the mRNA reading frame that prevents production of functional dystrophin protein. Therapeutic agents aimed at restoring dystrophin protein, including oligonucleotides such as the phosphorodiamidate morpholino oligomer (PMO) eteplirsen, are currently under investigation in late-stage clinical trials. Currently, there are no available validated methods to quantify dystrophin protein in muscle biopsies. Western blotting has been traditionally used as the confirmatory assay for clinical diagnosis of DMD or Becker muscular dystrophy (BMD), but the methods are not standardized and lack true quantitative capacity. A sensitive and quantitative Western blot method has been developed to detect dystrophin in human muscle biopsies utilizing a standard curve composed of non-DMD/BMD (normal control) muscle lysate spiked into DMD muscle lysate to maintain equivalent protein load. Running a 5-point standard curve ranging from 0.25% to 4% normal control on every gel allows for normalization of slight fluctuations in dystrophin signal intensity between gels. Optimized film exposure time ensures standards and samples are not oversaturated and remain within the dynamic range of the assay. Dystrophin can be reproducibly quantified down to 0.25% of normal control muscle across multiple users and runs. This method was validated according to FDA Draft Guidance for Industry, Bioanalytical Method Validation, and provides confirmation of the mechanism of action for any dystrophin protein restoring therapy. http://dx.doi.org/10.1016/j.nmd.2016.06.269
P.240 Dystrophin expression in the non-DMD population: What is normal? R. Johnsen, S. Wilton, S. Fletcher Murdoch University, Perth, Australia Dystrophin is a low abundance, large protein interacting with intracellular and sarcolemmal components to stabilize the muscle membrane during contraction. Mutations in the DMD gene leading to loss of dystrophin cause Duchenne muscular dystrophy, a fatal, X-linked disease characterized by progressive muscle wasting. Molecular therapies designed to restore dystrophin expression are reported to induce dystrophin expression and delayed disease progression in subsets of Duchenne muscular dystrophy patients. However, despite concerted efforts to standardize methods for precise dystrophin quantitation, analysis of the low dystrophin expression levels in clinical studies remains controversial. Immunohistochemistry and western blot have been used routinely to assess dystrophin for diagnosing Duchenne and Becker muscular dystrophy for over two decades, and it is common practice to analyze patient material alongside a reference sample from healthy muscle. However, neither
P.241 Optimizing dystrophin quantification in DMD and BMD patients: A new semi-automated acquisition and analysis method V. Sardone 1, M. Ellis 2, S. Torelli 1, L. Feng 2, D. Chambers 2, R. Phadke 2, C. Sewry 2, J. Morgan 1, F. Muntoni 1 1 Institute of Child Health UCL, London, UK; 2 Institute of Neurology UCL, London, UK Different experimental therapies are currently under investigation in order to restore dystrophin expression in Duchenne muscular dystrophy (DMD) patients. Antisense oligonucleotide (AON) therapies are novel pharmacological approaches aiming to induce exon skipping in the DMD transcripts and to produce a truncated dystrophin protein mimicking what occurs in Becker muscular dystrophy (BMD) patients. The primary biological endpoint of these AON clinical trials is therefore to restore functional dystrophin in muscles of DMD patients. A reliable, unbiased and reproducible method for quantification of dystrophin protein is crucial to monitor the biochemical outcome of such treatments. We have developed a new semi-automated and semi-quantitative immunohistochemistry method, based on the acquisition of an entire muscle section, which is capable of collecting data from all of the muscle fibres in the section (from 103 to 104 fibres). We have used a panel of antibodies against sarcolemma components to verify sarcolemma integrity (anti spectrin and anti laminin) as well as dystrophin antibodies recognizing different protein epitopes (anti dystrophin abcam-exon 77- and mandys106-exon 43). Each image was analysed by a script generated in house using the Definiens software. This new method allows the simultaneous quantification of many parameters such as dystrophin sarcolemmal and cytoplasmic intensity expression, spectrin or laminin cytoplasmic and sarcolemmal intensity expression, number of fibres, thickness of the sarcolemma membrane and area of each sarcolemmal and cytoplasmic compartments. We have analysed muscle sections from different DMD and BMD patients expressing variable levels of dystrophin and we present data showing that this method is highly sensitive, reliable and reproducible. The accurate and unbiased quantification of dystrophin will provide a robust pharmacodynamics endpoint in support of clinical trials aimed at dystrophin restoration. http://dx.doi.org/10.1016/j.nmd.2016.06.271
NEXT GENERATION SEQUENCING P.242 RD-Connect: Data sharing and analysis for rare disease research within the integrated platform and through GA4GH beacon and matchmaker exchange A. Roos 1, S. Beltran 2, D. Piscia 2, S. Laurie 2, J. Protasio 2, A. Cañada 3, J. Fernández 3, R. Kaliyaperumal 4, S. Lair 5, P. Sernadela 6, M. Girdea 7, R. Thompson 1, V. Straub 1, M. Roos 4, P. T’Hoen 4, A. Valencia 3, D. Salgado 8, C. Béroud 8, I. Gut 2, H. Lochmüller 1 1 Institute of Genetic Medicine, Newcastle upon Tyne, UK; 2 Center for Genomic Regulation, Barcelona, Spain; 3 Centro Nacional de Investigaciones