G.P.6.06 Systematic screening for genomic deletions and duplications in the dysferlin gene using multiplex ligation-dependant probe amplification and CGH microarrays

Abstracts / Neuromuscular Disorders 19 (2009) 543–660

tative assessment of muscle proteins. Results: Eight patients were found with deficient dysferlin protein in immunohistochemistry and western blot analysis. Clinical study showed that weakness was more proximal in 5 patients, proximal and distal in 3 patients. It affected lower limbs more than upper limbs in all patients. It was asymmetrical in both sides of body in 5 patients, symmetrical in 3 patients. Some muscles were uniformly affected in most of the patients: glutei especially Gluteus maximus, Hamstrings, Gastrocnemius and Deltoids. Pathological study showed dystrophic changes of all patients, mitochondrial changes in 2 patients and inflammatory changes in another 2 patients. Conclusions: Dysferlinopathy is a common condition in Egypt. Many clinical and pathological characteristics are found and helps in its diagnosis. doi:10.1016/j.nmd.2009.06.132

G.P.6.04 Nitric oxide signaling in selective muscle wasting of Miyoshi myopathy R. Dhanarajan1, M. Alexander 2, A. Oommen 1 1

Neurochemistry Laboratory, Christian Medical College, Vellore, India, Department of Neurological Sciences, Christian Medical College, Vellore, India 2

Selective muscle wasting of muscular dystrophies is an enigma. Muscular dystrophies, irrespective of their primary protein defect, exhibit common features of progressive muscle wasting suggesting common secondary signaling pathways maybe responsible for disease progression and selectivity. Understanding these mechanisms is necessary for therapy. The aim of this study was to elucidate secondary biochemical pathways that occur in selective muscle wasting of Miyoshi myopathy. Biopsies of severely affected gastroncnemius and relatively spared hamstring muscles from a Miyoshi myopathy patient, obtained with informed consent, were compared to each other and to 5 normal muscles for: (i) total protein content (ii) actin and myosin levels determined by SDS–PAGE (iii) lipid peroxidation (iv) protein tyrosine nitration and cysteine nitrosylation on western blots and (v) the ubiquitin proteasomal degradation pathway determined on western blots. Results: Total protein of the gastroncnemius muscle was 160 mg/gm tissue, of the hamstring muscle 145 mg/gm tissue and 254 ± 56 mg/gm tissue in normal muscle. Gastrocnemius myosin heavy chain (5-fold) and actin (3.1-fold) were lower than normal. Myofibrillar proteins in hamstring muscle were normal. Lipid peroxidation was higher in myopathic muscle (gastrocnemius750 TBARS nmols/g protein; hamstring-1724 TBARS nmols/g protein) compared to normal (498 ± 99 TBARS nmols/g protein). Nitrotyrosyinylated proteins were elevated in gastroncnemius muscle (5-fold), in hamstring muscle (2.3-fold) compared to normal. Nitrosothiolyated proteins were 1.5-fold higher in hamstring muscle, 0.65fold lower in gastroncnemius muscle than normal. Ubiquitinylated proteins were elevated in myopathic muscle (gastrocnemius – 1.6fold; hamstring – 2.6-fold) compared to normal. Conclusion: Degradation of myofibrillar proteins is associated with selective and pronounced muscle wasting in Miyoshi myopathy. Nitrotyrosinylated proteins maybe involved in this selectivity. doi:10.1016/j.nmd.2009.06.133

G.P.6.05 Attenuated muscle regeneration is a key factor in dysferlinopathy Y.H. Chiu, S.H. Laval, L.H. Jorgensen, M.A. Hornsey, L. Klinge, R. Charlton, R. Barresi, V. Straub, H. Lochmuller, K. Bushby

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Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom Dysferlinopathy patients are characterized by adult onset, slow progression, high serum CK and a prominent inflammatory infiltrate. To date, the pathomechanism of dysferlinopathy is still unclear. Our previous studies have demonstrated that the dysferlin protein translocates from the cytoplasmic space to the sarcolemma during muscle regeneration and biopsies of dysferlinopathy patients show an excess of immature fibers compared to other types of muscular dystrophy. This indicates that dysferlin might be involved in the process of muscle regeneration. To test this hypothesis, muscle regeneration was induced by injecting notexin into the right tibialis anterior (TA) muscles of wild-type C57BL/10 and dysferlin deficient C57BL/10.SJLDysf mice. Two days after notexin injection, the muscle had been severely damaged in both strains. In wild-type animals, the damaged fibers were completely removed and replaced by regenerating fibers at day 7 after notexin injection. In contrast, abundant necrotic fibers were still observed even at 28 days after injury in dysferlin deficient mice. Satellite cell activation and myoblast fusion seemed to be unaffected; however, the number of neutrophils recruited to the site of injury was significantly reduced in dysferlin deficient animals. Since dysferlin is expressed in the pro-macrophage monocyte lineage, but neither in neutrophils nor inflammatory monocytes, these early changes in inflammatory cell recruitment are likely to be driven by defective myokine secretion. Cytokine assays showed reduced MCP-1 secretion from C57BL/10.SJL-Dysf primary myotubes after stimulating with IFN-gamma or wounding with saponin. Altogether, we hypothesised that dysferlin might play a role in muscle regeneration by controlling cytokine secretion. Without dysferlin, the inflammatory response including neutrophil recruitment was attenuated, resulted in incomplete of muscle regeneration and cumulatively acted to accelerate the development of muscular dystrophy. doi:10.1016/j.nmd.2009.06.134

G.P.6.06 Systematic screening for genomic deletions and duplications in the dysferlin gene using multiplex ligation-dependant probe amplification and CGH microarrays M. Krahn, N. Wein, A. Borges, P. Bourgeois, V. Labelle, P. Negre, C. Pecheux, M. Bartoli, N. Lévy Department of Medical Genetics and INSERM UMR 910, APHM and Université de la Méditerranée, Marseille, France Mutations in DYSF cause primary dysferlinopathies, autosomal recessive diseases which mainly present clinically as Limb Girdle Muscular Dystrophy type 2B (LGMD2B; MIM#253601) and Miyoshi myopathy (MM; MIM#254130). The dysferlin coding sequence encompasses 6243bp divided into 55 exons. DYSF has a large mutational spectrum with more than 350 different sequence variants reported to date (Leiden Muscular Dystrophy pagesÓ, www.dmd.nl). Both genomic and transcriptional mutation screening have proven efficient for routine DYSF analysis. However, none of these approaches is exhaustive. In particular, possible large genomic deletions/duplications are not readily detectable using these strategies. To determine the existence of DYSF exonic deletions and/or duplications, we used a new kit for Multiplex Ligation-dependant Probe Amplification (MLPAÒ) (MRC- Holland BV, Amsterdam, The Netherlands). In addition, we are developing CGH-microarrays based on the NimblegenÒ technology for the detection of such mutational events, and characterisation of corresponding genomic breakpoints, for both the dysferlin gene, and related candidate genes.

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Among a series of 12 samples from patients with diagnostic suspicion of primary dysferlinopathy, MLPAÒ analysis evidenced exonic rearrangements in five patients, including four different exonic deletions and one duplication. Using 385K-probe CGH microarrays, several mutational events could be retrieved, thus validating this technology in this indication. We are currently developing 2.1Mprobe microarrays for higher density coverage and refinement of detectable deletions/duplications. Altogether, our findings confirm the existence of exonic rearrangements as disease-causing mutations in primary dysferlinopathies, and validate both MLPAÒ and CGH microarrays for the systematic screening of such mutational events. doi:10.1016/j.nmd.2009.06.135

G.P.6.07 The association of dysferlin and affixin is regulated by calcium concentration C. Matsuda1, K. Kameyama 1, I. Nishino 2, Y.K. Hayashi 2 1 AIST, Neuroscience Research Institute, Tsukuba, Japan, 2 NCNP, Department of Neuromuscular Research, Tokyo, Japan

Background: Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). Affixin (b-parvin) is an integrin-linked kinase- binding protein that is involved in the linkage between integrin and actin cytoskeleton. We previously reported that affixin is a dysferlin binding protein that colocalizes with dysferlin at the sarcolemma of normal human skeletal muscle. The immunoreactivity of affixin was reduced in sarcolemma of MM and LGMD2B muscles. The interaction of dysferlin and affixin was confirmed by immunoprecipitation study using normal human skeletal muscles. We also reported that affixin activates Rac1 via GDP/GTP nucleotide exchange factor (GEF) and regulates the reorganization of cytoskeletal actin. Objective: To examine the possibility that the association of dysferlin and affixin is regulated by calcium. Methods: Dysferlin and affixin or caveolin-3 were transiently co-expressed in COS-7 cells. Transfectants were lysed with the 1% NP-40 buffer containing 5 mM EGTA or 5 mM EGTA and 5 mM CaCl2 ([Ca2+] = 5 lM) and immunoprecipitated. The immune complex was separated on SDS–PAGE followed by immunoblotting. Results: Dysferlin was co-immunoprecipitated with affixin and vice versa under calcium free condition. The association of dysferlin and affixin was abolished in the presence of calcium. However, the association of dysferlin and caveolin-3 was not affected by calcium concentration. We are examining the molecular behavior of affixin during membrane repair using differentiated C2C12 myotube and mouse single muscle fiber. doi:10.1016/j.nmd.2009.06.136

G.P.6.08 The evaluation of novel therapeutic strategies for the treatment of dysferlinopathy M. Hornsey, Y. Chiu, L. Jorgensen, L. Klinge, S. Laval, R. Barresi, V. Straub, H. Lochmuller, K. Bushby University of Newcastle, Institute of Human Genetics, Newcastle, United Kingdom Mutations in the dysferlin gene cause limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi Myopathy and distal anterior

compartment myopathy, collectively known as the dysferlinopathies. Dysferlin-deficient C57BL/10.SJL-Dysf mice exhibit adult onset muscular dystrophy, high serum creatine kinase (CK) levels and a prominent inflammatory infiltrate, providing an excellent model for the study of dysferlinopathy. Preliminary analyses indicate that pathology is limited to specific muscle groups even at late stages of disease, while in situ force measurement performed on pre-symptomatic tibialis anterior (TA) shows a delay in force recovery. However, dysferlin-deficient muscle does not show susceptibility to acute damage, but has an impaired ability to regenerate following muscle wounding using either the snake venom notexin or needle wounding. Histological analysis shows the delayed removal of necrotic myofibres with an initial poor recruitment of neutrophils. Our hypothesis is that the process of muscle regeneration in C57BL/10.SJL-Dysf mice can be viewed as a model for disease progression in man. We have evaluated the regenerative process in this model, especially force generation following notexin injection and frequency of necrotic fibres as markers for pathological progression and treatment efficacy. We are currently evaluating a number of agents that modify neutrophil behaviour and/or recruitment to assess their ability to enhance the muscle regenerative process in dysferlin-deficient animals. doi:10.1016/j.nmd.2009.06.137

G.P.6.09 Immunolabelling and FACS as new tools to explore dysferlinopathies W.N. Wein1, K.M. Krahn 2, C.S. Courrier 3, S.C.E. Salort-Campana 4, N.K. Nguyen 2, F.C. Fernandez 5, P.J. Pouget 4, F.C. Fossat 6, D.D. Depetris 1, L.F. Leturcq 7, L.N. Lévy 1 1

INSERM UMR_S910, Génétique Médiale et Génomique Fonctionnelle, Marseille, France, 2 AP-HM, Département de Génétique Médicale et de Biologie C, Marseille, France, 3 Faculté de Médecine, CERGM, Marseille, France, 4 AP-HM, Service de Neurologie, Pôle de Neurosciences Clini, Marseille, France, 5 AP-HM, Laboratoire d’Anatomopathologie, Marseille, France, 6 AP-HM, Laboratoire d’Hématologie, Marseille, France, 7 AP-HP, Laboratoire de Biochimie Génétique, Paris, France Dysferlinopathies are autosomal recessive muscular dystrophies caused by DYSF mutations, which lead to a reduced amount or a complete lack of Dysferlin. Diagnosis is first based on the clinical, biological (CKs) and skeletal muscle CT-scan or MRI analyses. The second diagnosis step consists in Western-blot analysis of proteins extracted from muscle biopsy, or blood monocytes, which express Dysferlin. Noteworthy, monocytes represent a cellular model easier to obtain than muscle biopsy samples, in particular for patients suffering from muscular dystrophy. In regard to the accessibility of monocytes, whole blood flow cytometry with antibodies directed against Dysferlin appeared as a promising approach. We evaluated detection of Dysferlin by FACS using three different antibodies (Hamlet-1 which recognizes amino-acids 1999–2016; Hamlet-2: aa349–366; and sc-16634: internal region of Dysferlin). Six patients were submitted to these analyzes. In three patients carrying mutations predicted to lead to truncated Dysferlin, and lack of the transmembrane domain, no signal was detected, suggesting that truncated Dysferlin is not produced or degraded. In three other patients, in whom expression of a Dysferlin molecule maintaining its transmembrane domain is predicted, a fluorescent signal was detected in monocytes. These results have been confirmed by Western-blot and immunofluorescence analyzes on monocytes. The possible usefulness of this flow cytometry analysis in routine diag-