G.P.281

Abstracts / Neuromuscular Disorders 24 (2014) 791–924 Mutations in the Fukutin-Related Protein gene (FKRP) account for a broad spectrum of phenotypes ranging from severe congenital muscular dystrophies to much milder limb-girdle muscular dystrophy (LGMD2I). In both, weakness and wasting of shoulder-girdle muscles, primary restrictive respiratory and cardiac involvement are classical features. To our knowledge, facial dysmorphic features have not been reported yet. The first woman developed, at age 12, a progressive pelvic weakness. Two biopsies were performed showing a dystrophic pattern with normal classical immunohistochemistry. At age 38, she was confined in a wheelchair and suffered from respiratory insufficiency. She also had a mild cardiomyopathy. As she presented macroglossia, analysis of the FKRP gene was performed and two compound mutations p.Leu276Ile and p.Glu343X were found. The second woman was born of a consanguineous union. From childhood, she had difficulties running and rising from the floor. At 12, she developed a limb-girdle weakness that confined her in a wheelchair at age 26. Vital capacity was normal, as well as US heart exam. Muscular biopsy showed classical dystrophic features with normal immunohistochemistry and Western blot. She had no macroglossia but her face looked alike our first FKRP patient. Indeed, both women presented hypertelorism, large palpebral fissures, depressed nasal bridge, prognathism and thick lips being responsible for a dramatic physical resemblance although they were unrelated. FKRP gene analysis was therefore performed and a homozygous mutation p.Leu276Ile was found. Limb-Girdle weakness associated with macroglossia and calf hypertrophy are classical clues for FKRP gene sequencing. Some mutations have more frequent macroglossia or calf hypertrophy than others. But facial dysmorphia has not been reported in this disease so far. This dysmorphia represent a real diagnosis help in our second patient and should be searched each time a LGMD patient is considered. http://dx.doi:10.1016/j.nmd.2014.06.354

G.P.279 Efficient AAV-mediated transfer of FKRP in a new mouse model of Limb Girdle Muscular Dystrophy 2I E. Gicquel, I. Richard Genethon CNRS, Evry, France Dystroglycanopathies constitute a group of genetic diseases caused by defective glycosylation of alpha-dystroglycan (aDG), a membrane glycoprotein involved in the cell/matrix anchoring of muscle fibers. The aDG glycosylation, a very complex process, requires many proteins whose functions are not fully elucidated. In particular, mutations in the FKRP gene encoding Fukutin related protein, lead to hypoglycosylation of aDG, resulting in different forms of dystroglycanopathies, among which Limb Girdle Muscular Dystrophy type 2I (LGMD2I). We generated a knock-in mouse model of LGMD2I, carrying the most frequent mutation (L276I) encountered in LGMD2I patients. Molecular characterization of this mouse model showed that the introduction of the mutation did not alter the expression of FKRP. However, the protein appears to have altered function since abnormal glycosylation of aDG and reduction of laminin binding was observed. Histologically, the muscles of this model show a dystrophic pattern starting from 6 months of age, consisting both in the presence of central nuclei and in fiber size variability. Interestingly, functional muscle impairment can be observed as early as 2 months of age by a decrease of the muscle resistance to eccentric mechanical stress. To evaluate gene transfer as a therapeutic approach, we cloned the FKRP cDNA in an AAV vector under the transcriptional control of the desmin promoter. The recombinant AAV2/9 vector was injected intramuscularly or intravenously in the mouse model. Expression of the FKRP transgene was obtained, both at RNA and protein levels. The glycosylation of aDG was restored as well as laminin binding. A histological rescue was observed by the decrease

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of fibers with centrally located nuclei. The AAV vector also improved the muscle function, since it conferred a better resistance to eccentric stress to the injected muscles. http://dx.doi:10.1016/j.nmd.2014.06.355

G.P.280 Preserved expression of truncated telethonin in a patient with LGMD2G V. Straub 1, N.P. Davies 2, R. Barresi 3, C. Morris 3, C. Pickthall 4, K. Bushby 5 1 Newcastle University, NE2 4AZ, UK; 2 Queen Elizabeth Hospital, Birmingham, UK; 3 Newcastle NHS Hospital, NE2 4AZ, UK; 4 Newcastle NHS Hospital, Newcastle upon Tyne, UK; 5 Newcastle University, Newcastle upon Tyne, UK Limb-girdle muscular dystrophy type 2G (LGMD2G) is a disorder caused by mutations in the TCAP gene that encodes for the striated muscle specific protein telethonin. LGMD2G is extremely rare and was initially identified in the Brazilian population. Recently, a small number of LGMD2G patients have been diagnosed in populations with diverse genetic backgrounds indicating a wider geographical distribution of this disorder. Here we describe a 49 year old male patient presenting a classical LGMD phenotype. He was born from non-consanguineous healthy parents of Indian descent. He had normal motor milestones but became noticeable slower in his early teens and presented scapular winging and Achilles tendon contractures. He eventually became wheelchair bound by age 47. There has been no cardiac involvement so far and the respiratory function is only mildly reduced with nocturnal hypoventilation. CK levels were 2541 iU/L. A muscle biopsy showed well preserved fascicular architecture with mild myopathic features. Immunohistochemistry with an antibody directed to the C-terminal portion of telethonin showed complete absence of labelling. However, an antibody directed against full-length telethonin showed some labelling on sections and a single band of 10 kDa on Western blot. Sequence analysis of the TCAP gene revealed a novel homozygous nonsense c.244C>T (p.Gln82X) mutation in exon 2, predicted to generate a truncated protein of molecular mass consistent with the Western blot findings. Double labelling for telethonin and filamin C showed overlap in a cross-striated pattern, consistent with the Z-disc localization of both proteins, indicating that the mutant telethonin is retained at the Zdisc. Our results suggest that the mutant protein is correctly incorporated in the sarcomere allowing sufficient binding to titin such that functional rather than structural defects may be the mechanism that leads to LGMD2G. http://dx.doi:10.1016/j.nmd.2014.06.356

G.P.281 Detection of homozygous and compound heterozygous deletions in TRIM32 in LGMD patients analyzed by a combined strategy of CGH-array and Massive Parallel Sequencing J. Nectoux 1, R. de Cid 2, S. Baulande 3, F. Leturcq 1, J.A. Urtizberea 4, I. Penisson-Besnier 5, A. Nadaj Pakleza 5, C. Roudaut 6, A. Criqui 3, L. Orhant 1, D. Peyroulan 7, R. Ben Yaou 8, I. Nelson 8, M.C. Arne´-Bes 9, P. Nitschke 10, M. Claustres 7, G. Bonne 8, N. Le´vy 11, J. Chelly 1, I. Richard 6, M. Cosse´e 7 1 Assistance Publique – Hoˆpitaux de Paris, Hoˆpital Cochin, Paris, France; 2 Genethon, UMR8587, Evry, France; 3 PartnerChip, Evry, France; 4 Centre de re´fe´rence neuromusculaire GNMH, Hendaye, Angers, Angers, France; 6 Genethon, Evry, France; 5 CHU 7 France; CHRU Montpellier, Montpellier, France; 8 GH Pitie´-Salpeˆtrie`re - Institut de Myologie, Paris, France; 9 CHU Toulouse, Toulouse, France; 10 Universite´ Paris Descartes, Paris, France; 11 Faculte´

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Abstracts / Neuromuscular Disorders 24 (2014) 791–924

de Me´decine de Marseille, Marseille, France Defects in TRIM32 were reported in limb-girdle muscular dystrophy type 2H (LGMD2H). Few cases have been described to date, but this gene is not systematically analysed due to the absence of specific signs and difficulties in protein analysis. By using high-throughput mutation screening techniques, we identified mutations in TRIM32 in two patients presenting nonspecific progressive LGMD. We report the first case of total inactivation by homozygous deletion of the entire TRIM32 gene. Of interest, the deletion removes part of the ASTN2 gene, a large gene in which TRIM32 is nested. Despite the total TRIM32 gene inactivation, the patient does not present a more severe phenotype. However, he developed a mild progressive cognitive impairment that may be related to the loss of function of ASTN2 since association between ASTN2 heterozygous deletions and neurobehavioral disorders was previously reported. Regarding genomic characteristics at breakpoint of the deleted regions of TRIM32, we found a high density of repeated elements, suggesting a possible hotspot. These observations illustrate the importance of high-throughput technologies for identifying molecular defects in LGMD, confirm that total loss of function of TRIM32 is not associated with a specific phenotype and that ASTN2 inactivation could be associated with cognitive impairment. http://dx.doi:10.1016/j.nmd.2014.06.357

G.P.282 A novel mutation in DNAJB6 causes a more severe phenotype and greater loss of anti-aggregation function P.H. Jonson 1, J. Palmio 2, J. Sarparanta 1, H. Luque 1, B. Udd 1 1 Folkha¨lsan Institute of Genetics & Haartman Institute, University of Helsinki, Helsinki, Finland; 2 Dept. of Neurology, Neuromuscular Research Unit, Tampere University Hospital & University of Tampere, Tampere, Finland LGMD1D (OMIM: #603511) is caused by mutations in the co-chaperone DNAJB6. All reported disease-causing mutations (F89I, F93I, F93L, P96R, and F100I) are located in the G/F-rich domain of DNAJB6. Mutations have so far been identified in European, American and Asian populations. We have now discovered a novel mutation in the G/F-domain of DNAJB6 in a Finnish family with a more severe phenotype than previously reported for DNAJB6 mutations. The age of onset of symptoms was 10 to 12 years of age with weakness in the proximal lower limb muscles and it progressed to upper limbs and to some extent to distal lower limb muscles causing walking difficulties in early adulthood. Respiratory muscles were also involved with this mutation. Two patients had dyspnea and mild to moderate decrease in ventilation and one patient died of respiratory insufficiency at age 33. In this study we investigate the anti-aggregation effect of DNAJB6 in a filter-trap based system using transient transfection of mammalian cell lines and polyQ-huntingtin as a model for an aggregation-prone protein. We here present data for the anti-aggregation effect of all currently known mutations in DNAJB6. The new DNAJB6 mutant protein shows a greater loss of anti-aggregation activity than the other LGMD1D mutations. This loss of activity could be related to the early onset and faster progression seen in these patients. http://dx.doi:10.1016/j.nmd.2014.06.358

G.P.283 A novel mutation in DNAJB6 gene causes a very severe early-onset LGMD1D disease J.M.M. Palmio 1, A. Evila¨ 2, P.H. Jonson 2, M. Auranen 3, S. Kiuru-Enari 3, H. Pihko 4, P. Hackman 2, B. Udd 5

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Neuromuscular Research Center, Tampere University and University Hospital, Tampere, Finland; 2 Folkha¨lsan Institute of Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland; 3 Unit for Neuromuscular Diseases, Helsinki University Central Hospital, Helsinki, University Central Hospital, Helsinki, Finland; 4 Helsinki Finland; 5 Neuromuscular Research Center, Tampere University and University Hospital, Tampere, Finland DNAJB6 was recently identified as a causative gene for limb-girdle muscular dystrophy type 1D (LGMD1D). DNAJB6 belongs to a class of co-chaperones characterized by a J-domain in the N-terminus. To date, six different mutations have been identified in several families from European, North American and Asian countries. The known mutations cause mainly adult onset, slowly progressive proximal muscle weakness, although occasional patients with earlier onset have been reported. The evolution of the disease has been very mild to moderate and respiratory involvement has not been reported. A Finnish family with four affected members, three siblings and their mother, has been studied extensively in the past decade. All patients presented with marked proximal lower limb weakness at the age of 10 to13 years and many reported some weakness from early childhood. Weakness progressed to proximal upper limbs and to some extent also to distal lower limb muscles causing walking difficulties in early adulthood. The mother became wheelchair bound at age 25. Respiratory involvement was a major problem in the family; two sisters had dyspnea in their late teens and their mother died of respiratory failure at age 33. A novel mutation in DNAJB6 was identified in the family as the cause of this very severe, early onset form of LGMD1D. Functional studies of the mutation indicate a more pronounced reduction of the antiaggregation capacity compared to previously known mutations. http://dx.doi:10.1016/j.nmd.2014.06.359

G.P.284 Dysferlinopathy caused by protein misfolding: The novel murine animal model Dysf-MMex38 L. Heidt 1, M. Bader 2, S. Spuler 1, V. Schoewel 1 1 Experimental and Clinical Research Center, Charite´ University of Medicine, Berlin, Germany; 2 Max-Delbru¨ck-Center for Molecular Medicine, Berlin, Germany Limb-girdle muscular dystrophy 2B (LGMD2B) is caused by mutations in the dysferlin gene (DYSF). There are no mutational hotspots. Approximately one third of all mutations are missense mutations causing misfolding and protein aggregation, premature degradation and amyloid formation. There is no therapy. We could show that by treating patients’ primary myotubes with short dysferlin-specific peptides the endogenous produced missense mutant dysferlin can be relocated to the sarcolemma. There, it regains its proper function as a membrane repair protein. The human missense mutation DYSF p.L1341P causes all characteristics of a protein misfolding disease in dysferlinopathy. We therefore generated the first knock-in mouse model carrying the analogous murine missense mutation in exon 38 (Dysf p.L1360P, NP_001071162.1) named MMex38 (B6;129P2-Dysftm1.1Mdcb). Natural disease course was investigated at 8, 13 and 21 weeks of age (n = 36). We performed body composition, analyzed treadmill performance and voluntary running as well as protein analysis and histology of proximal and distal muscles. Evaluation of ER stress and membrane resealing assays complete the assessment. The targeted mutation leads to a dystrophic phenotype in 13 week old mice with a continuous disease progression in adulthood. Total body weight of 21 week old MMex38 was increased compared to WT controls without changes in the percentage of fat, free body fluid and lean tissue. Three weeks of treadmill exercise exposed significant endurance impairment in 19 to 21 week old MMex38 compared to controls. Histology revealed a