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Poster Abstracts

POSTER SESSION 1 (ODD NUMBERED POSTERS)

POSTER SESSION 2 (EVEN NUMBERED POSTERS)

POSTER SESSION 1 (ODD NUMBERED POSTERS)

#1 - Quantification of Oxidative Stress, Specially Protein Thiol Oxidation, in Muscles of Dysferlin deficient A/J mice

Hannah G Radley-Crabb1, Jessica R Terrill 1,2, Peter G Arthur2, Miranda D Grounds1

1School of Anatomy and Human Biology, the University of Western Australia, Crawley, WA, Australia; 2School of Biomedical, Biomolecular & Chemical Sciences, the University of Western Australia, Crawley, WA, Australia; 3

Background: Dysferlin is a Ca2+ binding protein and plays an important role in membrane repair in skeletal muscle. Lack of dysferlin can lead to disregulation of cellular calcium. Elevated calcium concentrations lead to an increase in mitochondrial calcium, which drives an increase in reactive oxygen species (ROS) production by mitochondria. ROS can cause irreversible damage to macromolecules including proteins, with extensive damage ultimately leading to cell death. An alternate mode of action of ROS involves altering protein function through reversible thiol modifications of protein cysteine residues. Alterations in the redox status of protein thiols can profoundly affect cell function. We hypothesize that interactions between elevated oxidative stress and calcium are a major pathogenic mechanism leading to myofiber necrosis in dysferlinopathies. Methods: We are studying intracellular protein oxidation in dysferlindeficiency using the protein carbonylation technique that measures by-products of irreversible protein oxidation, and aquantitative method (2tag) to measure protein thiol oxidation in tissues that was developed in our laboratory. Serum CK levels and myofibre necrosis (measured histologically) from various muscles will be presented to test for correlation between disease pathology and protein oxidation. Results: We will present the levels of both irreversible and reversible protein oxidation in the A/J mouse in numerous different muscles.

#3 - Detection of Dysferlin Gene Mutations in Patients with Limb-Girdle Muscular Dystrophy in the Indian Population.

Rashna S. Dastur1, Pradnya S. Gaitonde1, Satish V. Khadilkar2, A. Nalini3, A. K. Meena4, Madhuri R. Hegde5

1Institute for Advanced Training & Research in Interdisciplinary Sciences (TDM-labs), Mumbai, India; 2Department of Neurology, Bombay Hospital, Mumbai, India; 3Department of Neurology, National Institute of Mental Health and Neurosciences, Bangalore, India; 4Department of Neurology, Nizam's Institute of Medical Sciences, Hyderabad, India;5Department of Human Genetics, Emory School of Medicine, Atlanta USA.

Background: Limb- Girdle Muscular Dystrophy (LGMD) is the most common adult onset muscular dystrophy encountered in India.A large number of cases with robust clinical data have been generated in India. Among LGMDs in India, sarcoglycanopathy has been most extensively studied followed by dysferlinopathy. The exact prevalence is ofdyferlinopathy in India is not known.The diagnostic workup of suspected LGMD in India is based on biochemistry and electrophysiology tests followed by histology and immunohistochemistry which helps to the establish the nature of the dystrophic process. As immunocytochemistry has its own inherent limitations, genetic studies are essential, to specify the type of LGMD.This Jain Foundation-funded project includes Phase I - blood-based screening for defectivedysferlin protein expression by immunoblot analysis using whole peripheral blood mononuclear cells (PBMCs). Phase II - Detection of dysferlin gene mutations by sequencing in the cases confirmed as dysferlinopathies based on the monocyte assay. Methods: (i) Collection of blood samples of the patients diagnosed as LGMD2B on clinical,histopathological and immunohistochemical evaluation. (ii) The method of monocyte assay /Western blot will be followed as developed by Emory University to ascertain the status of dysferlin protein expression.Results: Cases ofdysferlinopathies are expected to be identified on monocyte assay. These will be further processed for DNA sequencing to determine the dysferlin gene mutations in Indian population. Conclusions: With the availability of this test, more information about this group of myopathies (LGMD2B) confirming the diagnosis will be discernible. Given the diversity of ethnicity and genetic background of the Indian population, this test followed by sequencing studies will be of immense relevance in identifying population specific and founder mutations in Indian patients.

#5 - New Methods of Protein Engineering to Facilitate Crystallization of the C2 Domains of Dysferlin

Kerry Fuson1, R. Bryan Sutton1

1Dept of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX, USA

Background:Our goal is to understand the complete 3D folding structure of dysferlin and how this structure relates to disease. Dysferlin is an exceptionally large and presumably flexible molecule; this would make crystallization of the entire protein intractable. However, due to the domain structure of dysferlin, we will pursue a "divide and conquer" strategy; that is, isolate, purify, and crystallize the individual domains of dysferlin. Once this is done, we can re-assemble these high-resolution structures onto a lower resolution EM reconstruction of the complete molecule. To date, we have successfully solved the structure of the C2A domain of human dysferlin. We have also crystallized the C2A domain of dysferlin variant 1 (C2A_V1). For reasons we do not yet understand, the remaining C2 domains have proven to be difficult to isolate. Therefore, we have decided to try approaches that have been successful in the Protein Structure Initiative. Methods: There are several ways to deal with protein aggregation. First, we had problems with inappropriate disulfide bonding of some of the domains. This was addressed by re-engineering the domains to minimize the number of free cysteine residues that could mediate disulfide-linked domain aggregation. Second, we started to use surface entropy optimized maltose binding protein fusions to serve as crystallization scaffolds for C2D and C2F. Third, we will use nanobodies to improve the solubility of C2E. Results: We have new crystals for the isolated C2A_V1 domain. For the C2D and C2F domains, the MBP system markedly improves the solubility of the domain from very low to >40mg/ml. This is an important consideration for crystallization. Conclusion:The biophysical properties of the non-C2A domains are very different, as they tend to be sticky and difficult to work with. These new techniques at crystallization show great progress in overcoming these difficulties.

#7 - Unmasking Potential Intracellular Roles for Dysferlin Through Improved Immunolabeling Methods

Jaclyn P. Kerr1 ,Robert J Bloch1, Lisa W Ru1, Andrea M O'Neill1, Wendy G Resneck1, Richard M Lovering2,1, Joseph A Roche1

1Physiology, University of Maryland School of Medicine, Baltimore, MD, U.S.A.; 2Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, U.S.A.

Background: Mutations in the DYSF gene that severely reduce the levels of the dysferlin protein are implicated in muscle wasting syndromes known as dysferlinopathies.Although studies of its function in skeletal muscle have focused on its potential role in repairing the plasma membrane, dysferlin has also been found, albeit inconsistently, in the sarcoplasm of muscle fibers.Aim: To study the localization of dysferlin in skeletal muscle through optimizedimmunolabeling methods.Methods: We studied the localization of dysferlin in wild-type rat skeletal muscle, using several different methods of tissue collection and subsequent immunolabeling. We then applied our optimizedimmunolabeling methods on human cadaveric muscle, control and dystrophic human muscle biopsies, and control anddysferlin-deficient mouse muscle.Results: Our data suggest that dysferlin is present in a reticulum within the sarcoplasm, similar but not identical to that containing the dihydropyridine receptors and distinct from the distribution of the sarcolemmal protein, dystrophin.Conclusions: Our data illustrate the importance of tissue fixation and antigen unmasking for proper immunolocalization of dysferlin.They suggest that dysferlin has an important function in the internal membrane systems of skeletal muscle, involved in calcium homeostasis and excitation-contraction coupling.Funded by the Jain Foundation Inc.

#9 - Dysferlin Deficiency Impairs Functional Recovery fromIschaemia-Reperfusion Injury.

Frances Lemckert1, Joanne Hawkes2, Tanya Butler2, David Winlaw2, Kathryn North1, Jonathan Egan2, Sandra Cooper1

1Institute for Neuroscience and Muscle Research, The Children's Hospital at Westmead; 2Kid's Heart Research, The Children's Hospital at Westmead

Background: Dysferlin is proposed to play the role of “fusogen” in calcium-activated membrane repair, sealing “endomembrane repair patches” into plasma-membrane lesions.While dysferlinopathy patients typically display a late-onset dystrophy, it has recently been reported that some patients also display a mild cardiomyopathy.As dysferlin is highly expressed in cardiac muscle, we established a model of cardiac membrane challenge (ischaemia-reperfusion injury) to examine a role for dysferlin in this setting.Methods: Using the Langendorff apparatus, we ran both A/Jdysferlin-null, and A/J dysferlin-WT hearts through a protocol of 10 mins baseline perfusion, 15 mins globalischaemia, and 30 mins reperfusion.Hearts were externally paced at 480 beats-per-minute.Functional cardiac measurement of Diastolic Left Ventricular Developed Pressure (DLVDP), Systolic LVDP and heart-rate were recorded throughout the experiment, and samples of cardiac effluent were analysed for the membrane permeability biomarker lactate dehydrogenase (LDH).Results:During reperfusion, A/J null hearts had higher DLVDP and lower SLVDP functional measurements than WT hearts. Ventricular-fibrillation post-ischaemia was more common and more prolonged in null hearts compared to WT hearts.LDH release during reperfusion was significantly higher in null hearts than WT hearts.Conclusions: Our data demonstrate that dysferlin deficiency is associated with impaired recovery fromischaemia-reperfusion injury to the heart.LVDP values are physiological indicators of cardiac muscle function, and in the null hearts reflect an inability of the ventricle to fully relax in diastole (increased DLVDP), reducing its ability to fill, and contract forcefully in systole (reduced SLVDP). Higher LDH release from the A/J null hearts indicates greatercardiomyocyte membrane permeability post-ischaemia, consistent with previous laser-wounding studies demonstrating impaired myocyte membrane repair in dysferlin-deficiency (Han et al, J.Clin.Invest. 2007).Our findings lend extra caution to real-life settings such as myocardial infarction or to bypass surgery, where the planned ischaemia of surgery may be poorly tolerated by dysferlinopathy patients.

#11 - Proteomic Analysis of Dysferlin-Interacting Proteins in Human Vascular Endothelial Cells

Cleo Leung1,2, Soraya Utokaparch1,2, Arpeeta Sharma1,2, Carol Yu1,2, Christoph Borchers3, Pascal N Bernatchez1,2

1Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia, Vancouver, BC, Canada; 2UBC James Hogg Research Centre, Heart + Lung Health, St. Paul's Hospital; 3BC Proteomic Centre, University of Victoria, Victoria, BC, Canada

Background: We previously found that dysferlin is expressed in vascular endothelium, an innermost layer of the blood vessels. Down-regulation of dysferlin in vascular endothelial cells (VEC) causes a deficiency in cell adhesion ofsubconfluent VEC, but not in confluent cells. This phenomenon is due to decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1), indicating that dysferlin has a functional role in maintaining vascular homeostasis in blood vessels that perfused skeletal muscles. However, the mechanistic pathway of dysferlin and membrane protein trafficking remains unknown. Goal: In this study, we aim to identify dysferlin-interacting proteins that may unravel the underlying linkage between dysferlin and protein trafficking. Methods: To identify dysferlin-interacting protein complexes, we generated glutathione-S-transferase (GST)-dysferlin recombinant proteins as a baitfor pulldown assays. Briefly, truncated dysferlin sequences containing the first 3 C2 domains (C2A to C2C), last 4 C2 domains (C2D to C2G) and highly conserved domains (DysF N and C domains) were amplified by polymerase chain reaction and inserted into glutathione-S-transferase (GST) vector. Resins bound with GST-dysferlin were incubated with human umbilical vein endothelial cell (HUVEC) lysates to pull down dysferlin-interacting protein complexes. Dysferlin-interacting proteins were identified by mass spectrometry. Results: With the use of proteomics, we identified several transport, signaling, contractile and motor proteins that interact with dysferlin directly or indirectly. These results further suggest that dysferlin mediates the trafficking of vesicles that contain protein cargos. Conclusion:Collectively, our study identifies dysferlin-interacting proteins in human VEC and may initiate subsequent hypothesis-driven research to further elucidate the mechanistic pathway of dysferlin function in human vasculature and also intact skeletal muscles.

#13 - The Ferlin and EHD proteins in Muscle Development and Repair

Alexis R. Demonbreun1, Avery D. Posey Jr.2, Peter Pytel3, Panayota Rigas1, Judy U. Earley1, Michele Hadhazy1, Mark Rainey4, Hamid Band4, Elizabeth M. McNally1,2,5

1Department of Medicine, University of Chicago, Chicago, IL, USA; 2Committee on Genetics, Genomics and Systems Biology, University of Chicago, Chicago, IL, USA; 3Department of Pathology, University of Chicago, Chicago, IL, USA; 4Eppley Institute for Cancer and Allied Diseases, University of Nebraska Medical Center, NE, USA; 5Department of Human Genetics, University of Chicago, Chicago, IL, USA

Background: The ferlin family of proteins binds to negatively charged phospholipids in a calcium dependent manner.This binding is thought to be critical for mediating vesicle fusion and trafficking important for regulating muscle membrane repair and growth.EHD proteins are a family of four cytoskeletal proteins that mediate intracellular trafficking, especially endocytic recycling.Methods: We hypothesize that the EHD interaction with ferlin proteins helps mediate the cytoskeletal rearrangements necessary to accommodate vesicle fusion.We showed that the NPF motif within the C2B domains of myoferlin or Fer1L5 can interact with both EHD1 and EHD2, and that these proteins can be co-immunoprecipitated.Results: Mutation of this binding motif to SPL, that contained in dysferlin, reduced binding of both EHD1 and EHD2 but did not abolish binding completely, consistent with dysferlin having a reduced interaction with EHD proteins.Using siRNA, we found that Fer1L5, EHD1, and EHD2 are important for propermyotube growth.We now show in vivo that loss of EHD1 leads to a decrease in the number of largest muscle fibers and a reduction in muscle mass similar to what is seen in myoferlin null muscle.Interestingly, we discovered EHD1-null myoblasts also accumulate aggregates of Fer1l5 protein near the nucleus consistent with vesicle trafficking defects.

#15 - What can we Learn about Dysferlin from its Relationship with Syntaxin-4?

Frances J. Evesson1,2, Angela Lek1,2, Frances A. Lemckert1,2, David E. James3, Kathryn N. North1,2, Sandra T. Cooper1,2

1Institute of Neuroscience and Muscle Research, The Children's Hospital at Westmead, Sydney, NSW, Australia;2Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, NSW, Australia; 3Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney, NSW, Australia

Background: We have identified a novel relationship between dysferlin and syntaxin-4, a known SNARE protein important for calcium mediated intracellular transport and vesicle fusion. We have sought to further define this relationship as a way to understand the function of dysferlin in skeletal muscle, and its role in cellular trafficking.Methods: We have used a panel of dysferlin and syntaxin-4 deletion constructs to study their behaviour in transfected C2C12 mouse muscle cells. We have used staining techniques to determine protein localisation, flow cytometry assays to study protein expression, immunoprecipitation to study protein interactions, and western blot to quantify protein levels. Results: We show that over- expression of dysferlin up-regulates levels of endogenous syntaxin-4.Dysferlin over-expression also affects levels of other plasma membrane syntaxins but has no effect on endosomal syntaxins, noron known syntaxin binding partners. We demonstrate that dysferlin mutants with altered cellular localisation can change the localisation of syntaxin-4. We show that dysferlin and syntaxin-4 can co-immunoprecipitate, and form a protein complex in transfected cells. Conclusion: Our studies establish dysferlin as a syntaxin-binding protein and we will now use our dysferlin and syntaxin-4 deletion constructs to define their interactive domains. Our ongoing studies will focus on determining whether dysferlin plays a functional role in syntaxin-4 mediated fusion, and whether this interaction underlies dysferlin’s role in cellular trafficking and membrane repair.

#17 - Dysferlin-Deficient Muscle Increases in Size and Strength after Local IGF-I Overexpression.

Becky K. Brisson1,2, SooHyun Park1, Elisabeth R. Barton1,2

1Anatomy and Cell Biology, Univ. of Pennsylvania School of Dental Medicine, Philadelphia, PA, USA; 2Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA

Background: Patients with mutations in dysferlin have either limb-girdle muscular dystrophy type 2B or Miyoshi myopathy.Both diseases involve muscle wasting, presumably due to the lack of muscle membrane healing withoutdysferlin.These diseases do not have a satisfactory treatment for increasing muscle strength, or for preventing further muscle damage.A potential treatment for dysferlinopathies is Insulin-like growth factor-1 (IGF-I).IGF-I is a circulatingautocrine/paracrine factor which regulates many aspects of muscle development, repair, and growth.Increasing IGF-I in muscle causes hypertrophy, enhances regeneration after injury, and can improve many diseased muscle phenotypes and function.However, a recent study concluded that IGF-I infusion was ineffective at promoting these benefits indysferlin-null mice due to the absence of correctly localized IGF-I Receptors.Methods: To test if boosting local overexpression of IGF-I could benefit dysferlin-null muscle, we injected A/J and control mouse hind limb muscles with AAVsc vectors encoding IGF-I.The injected muscles were evaluated for function and mass.Sections of the muscles were stained for laminin and various myosin heavy chain isoforms, and muscle fiber-size and -type were analyzed.Muscles were also assessed for IGF-I Receptor activation and downstream signaling.In addition, diaphragms of A/J mice crossed with mice which overexpress IGF-I were evaluated for muscle morphology.Results: Overall, viral-mediated local overexpression of IGF-I caused equivalent hypertrophy and maintained muscle function in A/J and control mice.Conclusions: Dysferlin-deficient muscle does respond to IGF-I, and IGF-I should be considered as a potential treatment and therapy for patients with limb-girdle muscular dystrophy type 2B and Miyoshi myopathy.

#19 - Increasing Muscle Mass by Follistatin and Acvr2B/Fc in Dysferlin Mutant Mice

Yun-Sil Lee1, Se-Jin Lee1

1Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Background: Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Blocking the myostatin signaling could have important applications for treating patients with muscle degenerative diseases. Both Follistatin, expressed as a transgene in muscle, and a soluble form of the Acvr2B receptor (Acvr2B/Fc), administered in purified form, have been shown to increase muscle mass in mice. Methods: We examined the effects of the follistatin transgene expression and Acvr2B/Fc administration in dysferlin mutant (Dysf-/-) mice. Results: The follistatin transgene expression indysferlin mutant mice (F66;Dysf-/-) showed dramatic increases in muscle mass compared to control mice (Dysf-/-) until age 4 months. However, the muscle weights decreased sharply after age 6 months and F66;Dysf-/- mice showed even lower muscle weights than Dysf-/- mice at age 8 months. Beginning at age 7 months, Dysf-/- and C57BL/6 were given four i.p. injections of either ACVR2B/Fc or PBS over a span of 4 weeks. At age 8 months, Dysf-/- mice injected with ACVR2B/Fc showed dramatic increases in muscle mass comparable to C57BL/6 mice injected with ACVR2B/Fc.Conclusion: Our data suggest that the follistatin transgene expression may accelerate degeneration in Dysf-/- mice over the time period and that Acvr2B/Fc administration in Dysf-/- mice even at age 8 months promotes muscle growth.

#21 - Genetic Suppression of C. elegans fer-1

Mohan Viswanathan1

1Department of Biology, MIT, Cambridge, MA, USA

Background: Mutations in the dysferlin analog gene fer-1 in C. elegans lead to defects in spermatid maturation, which ultimately affect fertility. One such mutation fer-1(hc1), is a temperature sensitive conditional allele, in which worms are fertile at the permissive temperature and infertile at the non-permissive temperature. Methods: Using a strain harboring fer-1(hc1), we have performed a forward mutagenic screen for suppressor mutations that restore fertility.Results: We have isolated a number of suppressor lines and characterized these lines both phenotypically and genetically. We are taking a whole genome sequencing approach to identify the nature of the suppressing mutations.Conclusion: We hope that the identification of fer-1 suppressing mutations will further our understanding of the dysferlin pathway, membrane fusion biology, and perhaps lead to conserved players that can be targeted for therapeutic discovery efforts. We will present the results of our screen thus far.

#23 - Inhibition of p38a Reduces Pathology in Multiple Mouse Models of Muscular Dystrophy

Erin R. Wissing1,2, Kinya Otsu3, Jeffery D. Molkentin1

1Department of Molecular Cardiovascular Biology, Cincinnati Children's Hospital, Cincinnati, OH, USA; 2Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH, USA; 3Department of Cardiovascular Medicine, Osaka University, Osaka, Japan

Background: The muscular dystrophies are a group of inherited diseases that are characterized by progressive muscle weakness and wasting, with cycles of degeneration and regeneration of muscle fibers. Identification of the molecular effectors underlying myofiber degeneration and death, as well as the compensatory influx of inflammatory cells and fibrotic replacement, might suggest novel treatment targets. p38a mitogen-activated protein kinase (MAPK), which is a highly potent inflammatory reactive signaling factor, has been implicated in skeletal muscle development, although very little is understood about its potential role in muscular dystrophy. Methods: Here we specifically deleted p38a in the skeletal muscle of d-sarcoglycan (sgcd) null mice using a floxed p38a allele and the MLC1f-cre knock-in allele to examine its role in MD. Results: At three and six months of age we observed a significant reduction in pathologic indices and an increase in muscle endurance during forced running. A similar reduction in pathology in skeletal muscle of sgcd-/- and mdx mice was observed when treated with a p38a/ß pharmacologic inhibitor over 9 weeks.A/Jdysferlin null mice present with a large influx of inflammatory cells and fibrosis in muscle with aging. To determine if this disease pathway was also functioning in A/J mice we treated 4 month old mice with p38a/ß inhibitor, and these data will be presented. Finally, in order to determine if over activation of p38 is sufficient to cause a dystrophy-like pathology in skeletal muscle, we used two genetic approaches in mice. Skeletal muscles from constitutively active MKK6-transgenic mice, which had constitutive p38 activation, exhibited severe muscle wasting phenotype with the hallmarks of dystrophic disease. Consistent with this observation, mice lacking the p38 inactivating phosphatases DUSP1,4,10 showed a subtle, albeit significant induction of dystrophic-like pathology in skeletal muscle with aging.Conclusion: Taken together, these data suggest a detrimental role of p38a in the progression of muscular dystrophy and suggest a novel therapeutic approach that could be employed quickly in humans.

#25 - The Universal Mutation Database for Dysferlin

Gaëlle Blandin1, Dalil Hamroun2, Marc Bartoli1, Nicolas Lévy1, Christophe Béroud2, Martin Krahn1

1Department of medical genetics, APHM-CHU Timone and INSERM UMR910, Université de la MéditerranéeMarseille, France; 2Laboratoire de Génétique Moléculaire, INSERM U 827 and CHU de Montpellier IURC - InstitutUniversitaire de Recherche Clinique, Montpellier, France

Background: The compilation of mutational and clinical data from patients affected with dysferlinopathy is an essential step towards a better understanding of the natural course of this disease, and possible inclusion in future therapeutic clinical trials. As the French national reference centre for molecular diagnosis of primary dysferlinopathies, we developed the Universal Mutation Database for Dysferlin (UMD-DYSF, in collaboration with Christophe Beroud, Montpellier, France). Methods: The main objective of this Locus-Specific Database is the compilation and interactive analysis of mutational data. The database includes bioinformatics tools which allow for the statistical analysis of mutational data. Being confronted to the recurrent difficulty of interpreting newly identified intronic, missense- orisosemantic-exonic sequence variants, we integrated in UMD-DYSF specific algorithms for the prediction of a pathogenicity score, which makes this database a valuable bioinformatics support for genetic diagnosis in primarydysferlinopathies. Results: UMD-DYSF v1.0 (April, 2011) includes 742 entries corresponding to 266 different disease-causing mutations identified in 558 patients worldwide, with direct links to the corresponding publications. Online availability is planned for summer 2011 following current finalization of UMD-DYSF, supported by the Association Française contre les Myopathies and the Jain Foundation. Conclusion: Being integrated into the Natural History of Dysferlinopathies project, supported by the Jain Foundation, current developments aim to further expand UMD-DYSF to reach the status of an international patient registry according to the guidelines of TREAT-NMD, including regular updates by patients/physicians on the clinical status of patients with genetically confirmeddysferlinopathy.

#27 - Non-Dysferlin non-Anoctamin 5 Miyoshi Myopathy

Francois-Jerome Authier1,2, Bruno Eymard3, Norma B Romero3, France Leturqc4

1INSERM U955-Team 10, Paris Est-Creteil Univ., Creteil, France; 2Reference Center for Neuromuscular Diseases, Henri Mondor Hospital, Creteil, France; 3Institute of Myology, Paris 6 Univ., Paris, France; 4Cochin Institute, Paris 5 Univ. Paris, France

Background: Miyoshi myopathy (MM) is an autosomal recessive distal muscular dystrophy characterized by early calf muscle involvement and high serum CK levels. Mutations in the dysferlin gene cause MM, limb-girdle muscular dystrophy 2B (LGMD2B), distal anterior compartment myopathy, proximodistal phenotype, late-onset forms and asymptomatic hyperCKemia. Genetic heterogeneity was recognized in MM with the identification of families displaying a clinical picture mimicking MM and excluded from dysferlin involvement. Among them, some were tentatively linked to chromosome 10p (MMD2) and others with chromosome 11p (MMD3). MMD3 is allelic with LGMD2L, and caused by mutations in the anoctamin 5 (ANO5) gene (Am J Hum Genet 2010; 86: 1). Here, we report a patient with MM-type distal muscle dystrophy in whom dysferlin and anoctamin 5 were excluded. Case Report: A 21-yr old Caucasian man first presented with a 2-yr history of asymmetric distal muscle weakness and aching in lower limbs, leg and calf amyotrophy, and marked hyperCKemia (~ 3000 IU/l). EMG was myopathic. Muscle biopsy showed a dystrophic process. Expression of dystrophin, a-, b-, g- and d-sarcoglycans, dysferlin, a-dystroglycan, caveolin-3, and calpain-3 were all normal. Afterwards, the patient’s condition worsened with asymmetric progression of muscle weakness, affecting left calf, then right thigh, and more recently left arm and pectoral muscles. At 28, whole body muscle MRI showed (i) hypertrophy of upper limb girdle, arm, forearm and paraspinal muscles; (ii) symmetricamyotrophy and fat infiltration of posterior thigh muscles; (iii) asymmetric involvement of quadriceps; (iv)amyotrophy and fat infiltration of both soleus and medial gastrocnemius muscles, while lateral gastrocnemius and peroneus longus muscles were spared. Sequencing of ANO5 gene displayed no significant abnormality.Conclusion:This case points out the genetic heterogeneiety of MM phenotype, and supports the view that various gene defects may have similar effects on myofibers.

#29 - Increased Sphingosine 1-Phosphate Ameliorates Muscle Degeneration in Mouse Models of Muscular Dystrophy

Morayma Reyes1, Mario Pantoja2, Nicholas Ieronimakis1, and Hannele Ruohola-Baker

1Department of Pathology, Univ. of Washington, Seattle, WA, USA; 2Department of Biochemistry, Univ. of Washington, Seattle, WA, USA.

Background: Presently, there is no effective treatment for the lethal muscle wasting disease Duchenne Muscular Dystrophy. Methods: Through an unbiased suppression screen of the dystrophic phenotype in Drosophila, we found suppressors that upregulate the levels of sphingosine 1 phosphate (S1P) and thereby ameliorate the dystrophic phenotype in the flies. Previously, S1P has been implicated in satellite cell proliferation and myoblast differentiation in vitro. These essential roles for S1P in skeletal muscleenabled us to hypothesize that S1P mechanisms are conserved in mammals. Results: In extending the S1P studies to the mdx mouse, we found that localized elevation of S1P via direct injection into muscle led to an increase in muscle satellite cell proliferation, newly regenerated fibers as well as fiber size. Additionally, we found that the systemic administration of that 2-acetyl-4(5)-tetrahydroxybutyl imidazole(THI), an S1P lyase inhibitor that strongly suppressed dystrophic muscle wasting in Drosophila, led to a significant amelioration of known hallmarks of DMD pathology, fibrosis and fat deposition and dramatic increase in muscle fiber size in mdx mice. Similar results were also observed in the dysferlinopathy mouse model, AJ/SCID mice with direct administration of S1P. This increase in muscle fiber size can be attributed to anabolic pathways as indicated by increased levels of phosphorylated ribosomal S6. Conclusion: THI holds promise as a new pharmacotherapy to treat muscular dystrophy.

POSTER SESSION 2 (EVEN NUMBERED POSTERS)

#2 - Complement 5a Receptor is Expressed in Human Skeletal Muscle

Christian Herrmann1, Stephanie Adams1, Verena Schöwel1, Katrin Wenzel1, Adrian Schreiber2, Simone Spuler1

1Muscle Research Unit, Experimental and Clinical Research Center, a joint institution of the Max-Delbrück-Centrum for Molecular Medicine and the Charité Medical Faculty, Berlin, Germany; 2Department of Nephrology and Hypertension, Experimental and Clinical Research Center, a joint institution of the Max-Delbrück-Centrum for Molecular Medicine and the Charité Medical Faculty, Berlin, Germany

Background: Complement activation on the surface of non-necrotic muscle fibers plays a role in the pathogenesis and course of dysferlinopathy. Therefore, complement inhibition might be an option for therapeutical intervention. Blocking soluble complement factor C5 might be suitable. Here, we explored whether C5a receptor (C5aR) is expressed on skeletal muscle as several C5aR blocking molecules are on the market. Methods: Human skeletal muscle tissue samples, primary human myoblasts and myotubes as well as immortalized human myoblast cell lines – normal and material harboring DYSF mutations - were analyzed for expression of C5aR. Protein expression was examined by Western blot and immunofluorescence, mRNA expression was analyzed by RT-PCR. Results: Human skeletal muscle tissue, as well as primary and immortalized myogenic cell lines, expresses C5aR at the mRNA and protein level.Conclusion: Blockade of C5aR might be a therapeutical option in the treatment of dysferlinopathy.

#4 - Ultrastructural Analysis of Differentiating C2C12 Dysferlin Knockdown Cells

Rumaisa Bashir1, Usha K Ramachandran1, Helen Grindley1, Michelle M Maxwell2, Christine A Richardson1

1School of Biological and Biomedical Sciences, University of Durham, Durham, UK; 2MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA

Background: Dysferlin deficient muscle is characterized by delayed muscle regeneration, which indicates a defect in myoblast fusion. Methods: To examine the role of dysferlin in myoblast fusion we have established stable C2C12 cell lines in which dysferlin expression has been silenced using shRNA plasmids. Results: We have examined the fusion index of the dysferlin knockdown cultures which show a reduced fusion index which is associated with a significantly higher number of desmin positive myoblasts, small myotubes (2-3 nuclei) and significantly fewer large multinucleatedmyotubes (>4 nuclei), compared to control cultures. It has previously been demonstrated that dysferlin staining is enriched at sites of myoblast/myotubes fusion. We hypothesized that dysferlin silencing impairs myoblast/myotubefusion. To examine this at high resolution we are currently performing scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of dysferlin knockdown and control C2C12 cultures. Conclusion: These data will be presented and may shed light into potential mechanisms associated with the role of dysferlin in muscle regeneration.

#6 - Trans-Splicing as an Approach to Dysferlin Gene Repair

Susanne Philippi1,2, Stéphanie Lorain1, Ute Zacharias2, Andreas Marg2, Anne Bigot1, Marc Bartoli3, Martin Krahn3, Simone Spuler2, Luis Garcia1

1Institut Myologie, Groupe Hospitalier Pitié-Salpetrière, Paris, France; 2Myology Research Group, ECRC CharitéBerlin-Buch, Berlin, Germany; 3Faculté de Médecine de Marseille, Université de la Méditerranée, Marseille, France

Background: Mutations in the gene encoding dysferlin cause Limb Girdle Muscular Dystrophy Type 2B (LGMD2B), Miyoshi Myopathy (MM) and distal anterior compartment myopathy by the lack of sarcolemmal membrane repair. Several hundred mutations have been detected in the 55-exon-spanning dysferlin ORF which do not accumulate in mutational hot spots. Methods: To heal a large cohort of mutations, we designed pre-mRNA-trans-splicing molecules exchanging large regions of the dysferlin messenger RNA. By gene repair at the mRNA level we preserve natural mRNA stability and translational regulation of the gene. Results: As a test system we employ human immortalizeddysferlin-deficient myoblasts. We immortalized and characterised primary human myoblasts retrieved from patient muscle biopsies at the muscular dystrophy outpatient clinic Berlin Buch in collaboration with the cell-culture platform in Paris. We deliver the pre-mRNA-trans-splicing molecule via lentivirus and plan to employ adeno-associated virus for the delivery in dysferlin-deficient mouse models. We assess trans-splicing efficiency by quantitative PCR andimmunoblot. To prove functional recovery of the dysferlin protein we apply two cell membrane wounding assays, using atomic force and confocal laser microscopy.

#8 - Therapeutic Approach by “Genetic Compensation” forDysferlinopathies

Marc Bartoli1,2, Florian Barthélémy1,2, Nicolas Wein1,2, Virginie Kergourlay1,2, Martin Krahn1,2,3, SébastienCourrier1,2, Vincent Mouly4, Luis Garcia5, Nicolas Lévy1,2,3

1INSERM UMR S 910, "Medical genetics and functional genomics", Marseille, France; 2Faculté de Médecine de Marseille, Université de la Méditerranée, Marseille, France; 3Hopital d'enfants, departement de génétique Médicale, Marseille, France; 4Biotherapies of neuromuscular disorders, Inserm UMRS_974, Paris, France ; 5Inserm UMR_S 974, CNRS UMR 7215, Institut de Myologie, Paris, France

Background: Mutations in DYSF encoding dysferlin cause primary dysferlinopathies, autosomal recessive diseases that mainly present clinically as Limb Girdle Muscular Dystrophy type 2B and Miyoshi myopathy. Currently there is no curative treatment but some therapeutics approaches are being investigated. Dysferlin is composed of homologous C2 domains and a C-terminal transmembrane domain. Even if these C2 domains differ from each other by the interaction established with dysferlin partners, there is some evidence that they could have redundant or independent functions. Based on the clinical observation that some deletions are associated with moderate phenotypes, we are investigating two approaches of “genetic compensation” for dysferlinopathies: exon skipping and “miniproteins” transfer.Methods: Firstly, an in-frame deletion of exon 32, resulting in “quasi-dysferlin” expression, has been found in a patient presenting with a mild and late-onset phenotype. Thus, we have postulated that exon 32 of dysferlin could be a target for an exon-skipping therapeutic strategy. Results: We have developed Antisense OligoNucleotides designed to block essentials sites for the maintenance of exon 32.Similarly, a “minidysferlin”, composed only by the two last C2 domains and the transmembrane domain has been discovered in a patient with no severe symptom. Here again we used this natural proof of concept for a gene therapy. The minigene approach overcomes the limited packaging size of AAV vectors the best vector so far for muscular disorders. Conclusions: Several combinations of dysferlin domains will be tested to obtain the most functional “minidysferlin”.Finally, we think that both approaches, based on clinical data, could be the starting point for a therapy in the dysferlinopathy fields.

#10 - Bone Marrow Transplantation in a Mouse Model of Dysferlin Myopathy

Eduard Gallardo1, Xavier Suárez-Calvet1, Jordi Diaz-Manera1, Xavier Navarro2, Renzo Mancuso2, Eva Santos-Nogueira2, Mireia Genebriera1, Jordi Barquinero3, Isabel Illa1, Barbara Flix1

1Servei de Neurologia, Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau i Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED; 2Departament de Biologia Cellular, Fisiologia i Immunologia, Institutde Neurociencies, UAB and CIBERNED; 3Departament de Terapia Cellular i Gènica Institut de Recerca Valld’Hebron.Departament de Biologia Cellular, Fisiologia i Immunologia, Institut de Neurociencies, UAB and CIBERNED

Background: Dysferlinopathies are caused by mutations in the gene DYSF. Dysferlin is a protein expressed mainly in skeletal muscle and peripheral blood monocytes (PBM). We evaluated the effect of a total bone marrow transplantation (BMT) with dysferlin+/+ cells into A/Jprmd mice. We also studied the ability of monocytic cells to fuse with skeletal muscle cells in vitro. Methods: In vitro, Green fluorescent protein-labeled HL60 cells were differentiated intomonocytic cells with vitamin D3 and co-cultured with human myotubes from dysferlin-deficient patients and healthy controls. In vivo, mice were irradiated lethally or at a lower dose to favor chimerism. Transplanted mice were studied for dysferlin expression by western blot and immunohistochemistry. Computerized analysis of locomotion (Digigait) and electrophysiological techniques were also performed to test functional improvement.Results: In vitro studies showed a low rate of fusion between monocytic cells and skeletal muscle primary cultures, that was enhanced after SDS treatment of myotubes. In vivo, we observed dysferlin expression in spleen cells from treated mice but not in its skeletal muscle after BMT. The electrophysiological studies were normal in controls and treated mice. However, locomotion analysis showed a slight improvement in transplanted mice.Conclusions: We conclude that BMT is able to restore dysferlin expression in peripheral blood but not in skeletal muscle. We propose that the mild improvement observed in the functional studies could be related with a change in the inflammatory response due to the restored expression of dysferlin in PBM.

#12 - Zinc-Finger Nuclease-Mediated Gene Correction using Single AAV Vector Transduction and Enhancement by FDA Approved Drugs

Matthew L Hirsch1, Brian L Ellis2, R J Samulski1, Matthew H Porteus2

1Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA; 2Department of Pediatrics, Stanford Medical School, Palo Alto, CA, USA

Background: A therapeutic strategy for the treatment of diseases employs genetic engineering to precisely correct the mutation(s) using a zinc-finger nuclease (ZFN) and an exogenous DNA repair substrate.This approach would be applied ex vivo and among gene delivery vectors, adeno-associated virus (AAV) has demonstrated clinical success. A drawback of these vectors is the small DNA packaging capacity (less than 5kb, which is less than the dysferlin cDNAsize).Methods: To overcome this limitation in the context of ZFN gene correction, an expression cassette was engineered wherein two ZFNs were connected by a 2A peptide and expressed from a single promoter.Given this arrangement, a 1kb repair substrate (gfpR) was included on the same vector while not exceeding the AAV packaging capacity.Results: Initially, we analyzed transduction by AAV serotypes 1-9 on 17 primary cell types, including skeletal muscle progenitor cells, and found that AAV1 and 6 were most efficient. Transduction using AAV6 packaged with the ZFN-gfpR expression cassette demonstrated efficient cellular cleavage of the ZFN polypeptide to monomers that stimulated gene correction over 1% in primary fibroblasts.Conclusion: Collectively, these experiments provide a novel reagent for gene correction using AAV single vector delivery and demonstrate FDA-approved drug enhancement of this process into values near therapeutic levels for several genetic diseases.

#14 - Site-Specific Recombinase Strategy to Create iPS cells from A/J Mice

Marisa Karow1, Christopher L. Chavez1, Alfonso P. Farruggio1, Jonathan M. Geisinger1, Michele P. Calos1

1Department of Genetics, Stanford University, Stanford, CA, USA

Background: Induced pluripotent stem cells (iPSC) have substantially opened the field of gene and stem cell therapy. An important aspect is the method applied to derive those cells from adult somatic cell sources. Methods: We have reprogrammed adipose-derived mesenchymal stem cells (ASC) from adult A/J mice by non-viral overexpression of the Yamanaka factors Oct4, Sox2, Klf4, and cMyc by applying plasmid DNA. Results: The sequence-specific phagerecombinase phiC31 was used to insert the reprogramming cassette at one preferred location in the mouse genome, producing bona fide iPSC. In a second step the reprogramming genes were removed by transient transfection of Crerecombinase, rendering the iPSC free of the ectopically expressed genes. Here we present a strategy to further target a pre-inserted phage recombination site, which was present on the reprogramming plasmid. This site remains in the genome after removal of the reprogramming genes and can be used to insert a therapeutic gene in a previously characterized genomic site. Conclusion: This strategy offers the potential to reinsert the dysferlin gene into autologous patient-derived iPSCs, to differentiate them into muscle tissue, and reimplant them into the host.

#16 - Trans-Splicing for Dysferlinopathies.

François Monjaret1, Karine Charton1, Pierre Klein1, Eduard Gallardo2, Isabelle Richard1

1Généthon, Evry, France; 2Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

Background: We are developing dysferlin RNA surgery as a new therapeutic perspective on dysferlinopathies by using spliceosome mediated RNA trans-splicing approach. This strategy has the advantage of bypassing two current main issues of genetic therapy on DYSF gene: the large size of the dysferlin cDNA, incompatible with current vector packaging capacity, and the toxicity related to dysferlin over-expression. Methods: Constructions consist of pre-mRNA trans-splicing molecules (PTM) for replacement of the last seven exons of human and murine dysferlin pre-mRNA. Co-transfection of the human constructs together with a human dysferlin minigene is performed in HER911 cells as an initial trans-splicing in vitro model. It is used to optimize the PTM by changing regulatory sequences involved in splicing. Selected PTMs are then used to drive trans-splicing on endogenous dysferlin expressed by human cells. The murine PTMs are used to target the endogenous mouse dysferlin by transfection in murine cells. Results: We showed that the human dysferlin PTM is efficient to trans-splice the human dysferlin minigene and that the trans-spliced RNA is translated. Comparison of the different PTM is on-going and will allow for selecting the most efficient PTMs to be transfected in human cells expressing dysferlin. In murine cells, transfection of a specific PTM drove the production of a trans-spliced RNA from the endogeneous dysferlin. We are now working on improving efficiency in order to obtain a detectable amount of translated protein. Conclusions: Our experiments demonstrated the feasibility of the strategy. Improvements are being pursued to obtain efficiency on human myogenic cells from dysferlin patients and to reverse the pathological signs observed in the dysferlin deficient mouse model.

#18 - Using the Isolated Myofibre to Study Muscle Growth DuringNormal and Myopathic Post-natal Murine Development.

William J. Duddy1, Stephanie Duguez1, Terence A. Partridge1

1Center for Genetic Medicine, Children's Research Institute, Children's National Medical Center, Washington, DC, USA

Background: We have recently reported on a medium-throughput method to sensitively quantify the actin content of an isolated murine myofibre (Duddy et al., Exp. Cell Res., 2011, in press).Methods: Normalized against myonuclearnumber this serves as a proxy of myonuclear domain size.In combination with counts of the numbers of myoblasts permyofibre, using myogenic markers such as Pax7 and MyoD, and counts of total myofibres per muscle cryosection, this reveals a substantially complete picture of muscle mass and changes thereof.Myopathic models such as dysferlin-deficiency and dystrophin-deficiency can be characterized at different ages or regenerative time-points to identify departure from normal muscle growth. We have begun a comparative characterization of dystrophin-deficient and wild-type mice at various ages.Results: Data are presented showing the dramatic departure of dystrophin-deficient muscle from wild-type controls from 6 weeks of age onwards and suggesting abnormal growth at earlier ages.Intriguingly, myoblast counts are severely affected over the first four weeks of age, even before initial signs of necrosis and regeneration at 3 weeks.Conclusion: We propose that the study of dysferlin-deficient models would benefit from the application of this methodology.

#20 - An Optical Assay to Measure Energy Thresholds for Membrane Wounding and Resealing Kinetics

Glen W. Humphrey1, Paul S. Blank1, Elena Mekhedov1, Joshua Zimmerberg1

1Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), Bethesda, MD USA;

Background: Mutations in the DYSF gene coding for dysferlin cause late-onset muscle wasting syndromes in humans and mice (e.g. Myoshi myopathy and LGMD2B). The muscle wasting phenotype is thought to be due to loss of adysferlin dependent, calcium-activated plasma membrane repair process. To better understand the requirement fordysferlin in muscle membrane repair, we have devised an assay to measure both the rate and extent of membrane resealing in cultured mouse myotubes. Methods: For this assay, myotubes are labeled with fluorescent calcein dye, and subjected to IR laser induced membrane wounding on a confocal microscope. An image stack is collected and analyzed to measure the rate of dye loss from the wounded cells. Results: Our results indicate that membrane resealing occurs at similar rates in dysferlin-normal C2C12 myotubes and dysferlin-negative GREG myotubes (derived from the A/J mouse), but C2C12 myotubes can reseal following wounding at higher laser energies than GREG myotubes.Conclusion: Laser wounding at a given energy level may create larger holes in the GREG myotube membrane, resulting in a larger inward flux of Ca2+, and possibly inhibiting resealing by exceeding a critical threshold for maintaining calcium homeostasis. We will use this assay to evaluate potential therapies, such as lipid modification, aimed at improving membrane repair in dysferlin-deficient muscle fibers.

#22 - Analysis of Dysferlin-Knockdown Morphant Fish

Genri Kawahara1, Peter Sarafini1, Louis M Kunkel1

1Program in Genomics, Children's Hospital Boston, Boston, MA, USA

Background: Zebrafish express a number of genes that are orthologs of muscular dystrophy causative genes, including dysferlin, a gene known to mediate vesicle fusion to the plasma membrane. Methods: To understand the role of dysferlin, expression of zebrafish dysferlin was analyzed with RT-PCR. In order to reveal dysferlin’s function and to make a fish model of dysferlinopathy, three non-overlapping antisense oligonucleotide morpholinos for translation-blocking to target zebrafish dysferlin were injected for knocking down the expression during the early development stage. These morphants were analyzed by a muscle birefringence assay, whole mount-immunohistochemistry and western blot. Results: Zebrafish dysferlin had a high identity score (68%) with the amino acid sequence of human dysferlin. In adult fish, dysferlin is expressed mainly in skeletal muscle. The expression analysis of dysferlin in developmental stages showed that the expression was detected from 1 day post fertilization (dpf) to adult. In the skeletal muscle of zebrafish, dysferlin is strongly expressed in the myosepta, in which dystrophin,laminin and other muscle components are also expressed. In three individual morphants, abnormal muscle organization was detected by birefringence assays. Immunohistochemical analysis and western blot analysis of these different morphants indicated that expression of dysferlin was reduced compared to those of wild type fish.Conclusion: In zebrafish, our results indicate that 1) zebrafish dysferlin expressed in the myosepta has high homology to human dysferlin. 2) Knocking down the expression of zebrafish dysferlin results in muscle disorganization. These initial findings suggest that it may be feasible to create a dysferlinopathy model fish. We are now making dysferlin-null fish by knocking out expression with zinc finger nuclease technology. The model fish may facilitate understand of the pathomechanism of dysferlinopathy and be useful for therapeutic drug screening.

#24 - Development of New Tools for the Identification of Disease-Causing Mutation in Dysferlinopathies

Virginie Kergourlay1,2, Marc Bartoli1,2, Gaelle Blandin1,2, Florian Barthélémy1,2, NicolasLévy1,2,3, Martin Krahn1,2,3

1INSERM UMR S 910, "Medical genetics and functional genomics", Marseille, France; 2Faculté de Médecine deMarseille, Université de la Méditerranée, Marseille, France; 3Hopital d'Enfants, Département de Génétique Médicale,Marseille, France

Background: Diagnosis of dysferlinopathies is difficult, due to the large size and the large mutational spectrum of the DYSF gene. Indeed, more than 400 different variations have been reported to date. These variants are spread along the entire coding sequence. In 20% of patients, only one disease –causing mutation can be identified. Being confronted to the recurrent problem of interpreting newly identified missense sequence variants, but also isosemantic and intronicvariants, we intend to develop assays for the validation of possible pathogenic effects at the mRNA and protein levels.Methods: This project studies sequence variants identified in patients in order to determine their impact ondysferlin. We developed several constructions: 1) we created an expression plasmid with the cDNA of the DYSF gene under the control of the dysferlin’s endogenous promoter; 2) we created a single BAC containing the entire sequence of the DYSF genomic locus. In a subsequent step, we will introduce sequence variants in the plasmid and BAC, and determine their impact on: the stability of the mRNA and protein, the splicing, the conformation, the dysferlininteraction network and the functionality of dysferlin. This strategy is potentially promising, because we are not solely looking at the impact on the protein but also at the messenger level. The information obtained for aberrant splicing will be important for several approaches such as exon-skipping and trans-splicing… Furthermore, we will try to develop these tests as easy and fast as possible to be used downstream of the molecular diagnosis of new variants. In parallel, we rely on bioinformatics tools integrated in the UMD-DYSF database for the prediction of pathogenic effects. Conclusion: The combination of bioinformatics prediction and functional validation should allow defining consensual criteria for the interpretation of newly DYSF identified sequence variants. Finally, this work could improve the knowledge of dysferlin’s physiopathology.

#26 - A Knockout Model of Dysferlinopathy in Zebrafish

Peter R. Serafini1, Genri Kawahara1, Louis M. Kunkel1, David Langenau2

1Program in Genomics, Children’s Hospital Boston, Boston, MA; 2Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA

Background: We plan to knockout dysferlin in zebrafish using zinc finger nuclease technology. Zinc finger nucleases (ZFN) are artificial restriction enzymes that make targeted double strand DNA breaks at specific genomic loci. Normal DNA repair processes often lead to the introduction of insertions or deletions at the site of these breaks. Methods and Anticipated Results: Using the “ZiFit” and “Open” programs created by Keith Joung and David Langenau, we have designed zinc finger arrays that have target specificity for zebrafish dysferlin. We will inject mRNA from two customized zinc finger constructs into one-cell-stage zebrafish embryos. The left and right zinc finger arrays willdimerize and bind dysferlin, thereby activating the fok1 nuclease for double-strand DNA cleavage (Foley, et al., 2009). Steps to creating a dysferlin-null fish line:To identify dysferlin mutants, F0 generation males and females will be outcrossed with wild-type fish to create heterozygous carriers. Two out-of-frame deletions and one in-frame deletion will be selected from F1 offspring by genotyping. The selected F1 mutants will be crossed to wild-type fish to create F2 heterozygotes that can be mated to produce homozygous dysferlin-null fish. For raising the dysferlin-null fish line, we will follow a methodology previously reported by Jeff Guyon for a splicing mutant of zebrafishdystrophin (Guyon, et al., 2009). Characterization of mutant zebrafish:We will characterize the dysferlin mutants by confirming the nature of the mutation, checking dysferlin expression using immunohistochemistry and immunoblotanalysis. We will also observe the muscle quality by immunohistochemistry of muscle cross-sections of both early embryos as well as adult fish.We will also use motility and birefringence assays to develop assays of the dysferlin-deficient phenotype. Once characterized, we will use our dysferlin-null fish to screen small molecule libraries for compounds that might alter the phenotype.

#28 - Immortalized Human Dysferlin-Deficient Myoblasts as a Model to Study Dysferlinopathy

Ute Zacharias1, Susanne Philippi1, Anne Bigot2, Gillian Butler-Browne2, Vincent Mouly2, Simone Spuler1

1Muscle Research Unit, Experimental and Clinical Research Center, Charité University Medicine Berlin, Germany;2Thérapie des maladies du muscle strié/ Institut de Myologie, UM76, UPMC Univ. Paris 6, Paris, France

Background: Dysferlin (DYSF) gene mutations cause limb girdle muscular dystrophy 2B and Myoshi myopathy due to defects in muscle membrane repair. Because the access to primary human myoblasts from patients is limited, there is a strong interest in immortalized cell lines harbouring disease-causing mutations. Methods: Immortalized cell lines were generated from primary human myoblasts by transduction with both telomerase and cyclin-dependent kinase 4.Results: Four stable cell lines were established from primary human dysferlin-deficient myoblasts harboring different mutations in DYSF resulting in the absence or the intracellular aggregation of dysferlin (homozygous: c.4022T>C; c.2810+2T>A; compound heterozygous: c.855+1delG c.895G>A; c.1448C>A c.107T>A). We compared primary human myoblasts and myotubes with disease-causing mutations in DYSF to their immortalized counterparts and to normal controls with respect to the expression of muscle-specific differentiation markers and the time course and pattern of differentiation into myotubes. Our focus was on dysferlin protein expression, subcellular localization and function in membrane repair. Overall, the immortalized myoblast cell lines showed a high proliferative potential and displayed properties similar to their non-immortalized counterparts with respect to myogenic differentiation anddysferlin expression. Conclusion: These human myoblast cell lines represent a useful model to further investigatedysferlin function, to study the pathophysiological mechanisms involved in dysferlinopathy and to test therapeutic strategies for this disease on the cellular level.

#30 - Improvements in Monocyte Assay as Diagnostic Tool for Dysferlin

Madhuri Hegde1, A Ankala1, C Wilson1

1Department of Human Genetics, Emory University School of Medicine

Background: We are working on validating and optimizing the dysferlin detection assay from peripheral blood as an efficient clinical diagnostic assay.The monocyte assay can help reduce the cost of molecular analysis for the LGMD2B and MM patients, who may directly undergo dysferlin specific analysis, avoiding comprehensive panel-based testing if found abnormal for dysferlin expression. Patients with clinical heterogeneity and overlapping phenotype can opt for rapid screening for dysferlin deficiency without invasive biopsies prior to extensive molecular diagnosis. The study also aims to evaluate the efficacy of the test as a less expensive and quick carrier screening tool. Further, theproteotype-phenotype correlation studies included in this proposal will strongly help in better diagnosing and discriminating the disease subtype. Moreover, experimental therapies intended to restore the levels of dysferlin indysferlinopathy patients can be continuously tracked for efficacy of the treatment by monitoring blood dysferlinlevels. Methods: So far we have successfully validated and optimized the assay in our laboratory using known carriers and patient blood samples. Performing densitometry analysis on the western blots we have been able to predict relative expression of the dysferlin protein in carriers and controls. Results: While dysferlinopathy patients do not show any detectable dysferlin protein, carriers with different mutations involving missense to nonsense or frameshift deletions, show at least half the expression as controls. Conclusion: With an extensive screening of a large set of carriers, controls as well as affected we aim to set-up definitive expression thresholds for carrier screening and discern mutation type and its effect on protein expression or phenotypic severity.

#31 - Exon Skipping for Dysferlinopathies?

Annemieke Aartsma-Rus1, Isabella Houweling-Gazzoli1, Nisha Verwey1, Gert-Jan van Ommen1, Silvère M van der Maarel1

1Department of Human Genetics, Leiden Univ. Medical Center, Leiden, the Netherlands

Background: Dysferlinopathies are generally caused by small mutations within DYSF exons. There are some suggestions from the literature that internally deleted dysferlin proteins may be partially functional. Thus, antisense-mediated exon skipping of the mutation-containing exon could be a potential therapy for dysferlinopathies. However, it is not yet known which domains are essential for dysferlin function. Furthermore, most mutations would require the skipping of two exons to maintain the open reading frame. Finally, skipping of a certain exon (combination) would apply to only a small subset of patients. Our aim is therefore to assess whether dysferlins lacking exons that would be applicable to larger groups of patients are stable and functional. Methods: Our focus will be on exon 32 (shown to be dispensable), exon 30 (applicable to 8.4% of patients) and exons 20&21 (applicable to 7.1% of patients). Antisense oligonucleotides were designed for these exons, and tested in wild type cells and patient-derived fibroblasts with a point mutation in exon 20. Results: Exon 32 and 30 skipping can be achieved at high efficiency. Skipping exon 20 and exon 21 requires further optimization as exon skipping levels are too low (<5%). We are currently testing a combination of different exon 20 and exon 21 specific antisense oligonucleotides. Following optimization will test exon 20&21 skipping in patient-derived fibroblasts to allow assessment of the stability and correct localization of exon 20&21 deleted dysferlin. Furthermore, we intend to generate dysferlin deletion mouse models, using vivo-morpholinos (allowing high levels of exon skipping) to assess in vivo functionality. Conclusion: Exon skipping is feasible for DYSF, but it is not yet known whether internally-deleted dysferlin proteins are functional.

#32 - In Vivo Imaging and Modulation of Dysferlin via Intracellular Expression of VHH Antibody Fragments

Annemieke Aartsma-Rus1, Laure Grand Moursel1, Antoine de Morree1,2, Rinse Klooster1, Silvere M van der Maarel1

1Department of Human Genetics, Leiden Univ Medical Center, Leiden, the Netherlands; 2Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA

Background: Defects in the DYSF gene underlie several progressive myopathies including Limb Girdle Muscular Dystrophy 2B and Miyoshi Myopathy. The DYSF gene encodes dysferlin, a 230 kDa cyotosol faced transmembraneprotein. Dysferlin contains multiple intracellular C2 domains as well as several other domains that are conserved between ferlin proteins and anchors to the muscle membrane through a single pass transmembrane domain at its far C-terminus. Several interaction partners are described but comprehensive insight in the dysferlin protein complex and its spatiotemporal dynamics is largely missing. Spatiotemporal localization studies are typically done with fluorescently tagged recombinant proteins in living cells, which may not accurately reflect the biological behaviour of the endogenous protein. Methods: Studying the localization of endogenous dysferlin is possible with specific antibodies, but requires fixed cells. These issues could be circumvented by expression of specific affinity binders in live cells. Several affinity binders specific for dysferlin are (commercially) available but these are all classical multidomainantibodies composed of two heavy and two light chains, rendering them unsuitable for intracellular expression.Results: We therefore selected alternative recombinant affinity binders against dysferlin from phage display libraries of the minimal antigen recognition fragment (VHH) of llama heavy chain antibodies. We show that these binders can be expressed in live cells to detect recombinant dysferlin and study its spatiotemporal behavior. Conclusion: By selecting VHH antibody fragments against different domains of dysferlin we plan to modulate the behavior ofdysferlin to gain insight into its function. Moreover, this tool can be applied to study the fate and behavior of dysferlinprotein variants that are created through exon skipping of mutant exons.