D.P.3.10 Partial caveolin 3 deficiency in acquired rippling muscle disease

Abstracts / Neuromuscular Disorders 18 (2008) 724–833 ingly, dense electron dense inclusions, which are probably amyloid, are seen in areas where myofibrils are well preserved, indicating that these are seen very early in the stage of the disease. Moreover, the level of serum beta amyloid is higher in these DMRV mice compared to littermates, and is observed to be age-dependent. Conclusion: Our results suggest that amyloid deposition is an upstream event in the pathogenesis of DMRV/hIBM. This underscores the possibility of using anti-amyloid treatment as a strategy at least in delaying the progression of DMRV/hIBM. doi:10.1016/j.nmd.2008.06.148

D.P.3.07 Welander distal myopathy: The evasive gene P. Hackman 1; S. Hollo 1; H. Luque 1; M. Tokola 1; J. Kere 2; L. Edstrom 3; G. Ahlberg 3; B. Udd 1 1 Folkha¨lsan Institute of Genetics and the University of Helsinki, Department of Medical Genetics, Helsinki, Finland; 2 Karolinska Institutet, Department of Biosciences and Nutrition, Stockholm, Sweden; 3 Karolinska Hospital, Department of Clinical Neuroscience, Stockholm, Sweden

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capacity (FVC), peak expiratory flow (PEF), manual muscle test using Medical Research Council scale (MRC), 2 min walking test, Functional Reach test, Timed Stands Test (TST) and Brookes functional test and self-rated measurements, Egenklassifikation (EK scale), Barthel and Medical Outcomes Study 36-item Short Form (SF 36). The results confirm that respiratory failure is an early symptom. The pattern of weakness is early most proximal, symmetric upper limb, then deteriorating over time both regarding degree of weakness and number of muscle groups involved. The ability of independent gait is in this study dependent on, at least MRC 4 of the knee extension and ability to perform TST. There is a wide range from independence to complete dependence regarding activity of daily living (ADL). Independent gait remains long time after the debut of the need for respirator support (17 years). The quality of life, SF 36, showed that all but one person rated the physical health lower compared to mean for a Swedish population, while five of eight rated their mental health higher than mean. Either the ADL ability or the quality of life is mainly related to the degree of respiratory failure. doi:10.1016/j.nmd.2008.06.150

D.P.3.09 Welander distal myopathy (WDM) is a late onset autosomal dominant disease characterized by slow progression of distal muscle weakness. Usually hands are first affected with weakness of the finger extensor muscles. A few much more severely affected patients proved to be homozygotes. The ˚ hlberg et al. in 1999. WDM locus was mapped to chromosome 2p13 by A Further genotyping during 2004–2007, using additional new microsatellite markers narrowed down the linked region to <980 kb, with a core region of >530 kb. All known coding sequences in the linked region, including their putative promotor regions, as well as known hypothetical genes, have been sequenced. Several linked SNP polymorphisms have been identified, but the disease causing mutation is still pending. Large deletions and duplications in the area were excluded by screening with a 250 K SNP microarray linkage chip. Analyses of cDNA of all linked genes synthesized from mRNA isolated from muscle biopsies, are being finalized in search of potential splicing mutations. Studies of expression levels with RT-PCR (Taqman) of four linked genes have been performed, and the expression levels of the rest of the genes are being analyzed. No conclusive results have so far been obtained and the underlying gene is still unidentified. Total sequencing of the linked genomic area, including all introns and regions between genes, has been started using high throughput sequencing in microfabricated high-density picolitre reactors. With all these extended methods we hope to identify the underlying genetic defect causing WDM. doi:10.1016/j.nmd.2008.06.149

D.P.3.08 Physiotherapeutic description of an uncommon form of severe autosomal dominant limb girdle muscular dystrophy (LGMD) with early respiratory failure M. Ka˚na˚hols 1; K. Dahlbom 2 1 ¨ ¨ rebro, Orebro University Hospital, Department of Physiotherapy, O ¨ rebro University Hospital, Department Neurology and Clinical Sweden; 2 O ¨ rebro, Sweden Neurophysiology, O LGMD collects a great number of various inherited dystrophic muscular syndromes. Early respiratory failure is not common but characteristic for some LGMDs. This study use a physiotherapeutic perspective to describe patients with a Swedish type of autosomal dominant LGMD, Myopathy with Proximal Weakness and Early Respiratory Muscle Involvement, Type 1 (Nicolao, 1999). We examined eight of nine identified persons with the disorder, 35–73 years of age. The time from debut of symptoms defined as need for intermittent or continuous ventilator support varied between two and 30 years. Examination included: Forced vital

Caveolinopathy – New mutations and additional symptoms A. Aboumousa 1; J. Hoogendijk 2; R. Barresi 1; R. Charlton 1; R. Herrmann 3; T. Voit 4; J. Hudson 1; M. Roberts 5; D. Hilton-Jones 6; M. Eagle 1; K. Bushby 1; V. Straub 1 1 Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, UK; 2 University Medical Centre Utrecht, Department of Neurology, Utrecht, Netherlands; 3 University Hospital Essen, Department of Paediatrics, Essen, Germany; 4 Groupe Hospitalier Pitie´-Salpeˆtrie`re, Institut de Myologie, Paris, France; 5 Hope Hospital, Greater Manchester Neurosciences Centre, Salford, UK; 6 John Radcliffe Hospital, Department of Clinical Neurology, Oxford, UK Mutations in the caveolin-3 gene (CAV3) can lead to a broad spectrum of clinical phenotypes. Phenotypes that have so far been associated with primary caveolin-3-deficiency include limb girdle muscular dystrophy, rippling muscle disease, distal myopathy and hyperCKemia. This is the first report describing the clinical, pathological and genetic features of patients with caveolinopathy from the UK. Ten patients (six families) were identified via the National Commissioning Group (NCG) service for patients with limb girdle muscle dystrophy in Newcastle. A standardised clinical evaluation was conducted with each patient. Myalgia was the most prominent symptom in our cohort of patients and for 50% it was the reason for referral. Muscle weakness was only found in 60% of the patients, whereas rippling muscle movement was present in 80%. One of the patients reported episodes of myoglobinuria and another one episodes of hypoglycaemia. Five different mutations were identified, two of which were novel and three that had previously been described. Caveolinopathy needs to be considered as a differential diagnosis in a range of clinical situations, including in patients who do not have any weakness. Indeed, rippling muscles are a more frequent symptom than weakness, and can be detected in childhood. Presentation with myalgia is common and management of it as well as of myoglobinuria and hypoglycaemia may have a major impact on the patients’ quality of life. doi:10.1016/j.nmd.2008.06.151

D.P.3.10 Partial caveolin 3 deficiency in acquired rippling muscle disease M. Mirabella 1; R. Charlton 2; E.M. Valente 3; S. Petrini 4; A. d’Amico 4; M. Roberts 5; E. Ricci 1; F. De Benedetti 4; R. Barresi 2; E. Bertini 2; V. Straub 2 1 Catholic University Rome, Institute of Neurology, Rome, Italy; 2 Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, UK; 3 IRCCS

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CSS-Mendel Institute, Rome, Italy; 4 Bambino Gesu’ Research Children’s Hospital, Unit of Molecular Medicine, Rome, Italy; 5 Hope Hospital, Greater Manchester Neurosciences Centre, Salford, UK Rippling muscle disease (RMD) has been associated with mutations in the caveolin 3 gene and with immune-mediated myasthenia gravis. Here we report two patients with RMD and acquired caveolin 3 deficiency due to immune-mediated conditions of unknown origin. Both patients showed neither mutations in the CAV3 gene nor antibodies against the acetylcholine receptor or MuSK. Following respiratory infection, one patient developed pronounced RMD, myalgia and limb girdle weakness at around 33 years of age. Electrophysiological investigations did not show signs of a myopathy or neuropathy and the rippling was electrically silent. His serum CK activity was about 400 U/l. He showed a mosaic caveolin 3 expression pattern in his muscle biopsy with subtle histological abnormalities. After two years the patient recovered spontaneously from his symptoms. A second biopsy showed an improved caveolin 3 expression but more severe histological changes and signs of inflammation. A second patient experienced first symptoms of limb girdle weakness and myalgia at 20 years of age. She subsequently developed RMD and myoedema of all skeletal muscles including her facial muscles. Electrophysiological investigations showed a myopathic EMG pattern with no spontaneous activity and normal nerve conduction velocity. Her serum CK activity was persistently elevated 6–8 fold above normal. A muscle biopsy showed moderate dystrophic features, with no inflammation. Immunofluorescence analysis also showed a mosaic expression pattern of caveolin 3. Weakness in the second patient progressed slowly and Holter monitoring detected a 2nd degree atrioventricular block. Myalgia improved with dantrolene treatment. Under the assumption of an immune-mediated disorder, the patient was treated with plasmapheresis followed by steroids and cyclosporine and is since then getting better. Interestingly a consecutive muscle biopsy showed features of an inflammatory myopathy. We conclude that in patients with acquired RMD, partial caveolin 3 deficiency can be immune-mediated and of temporary nature. It can also be effectively treated like an autoimmune disorder. The patients will be closely followed.

D.P.3.12 Autosomal recessive Ala93Thr mutation of caveolin-3 gene: A new family F. Magri; C. Lamperti; D. Ronchi; E. Fassone; N. Grimoldi; M. Moggio; N. Bresolin; G.P. Comi University of Milan, Milan, Italy Caveolin-3 is the muscle-specific protein of the caveolin family. It is expressed both in cardiac and skeletal muscles and it is the principal integral membrane component of caveolae, small vescicular invaginations of the plasma membrane implicated in cell signalling. Mutations in the caveolin-3 gene (CAV3) are associated with four different phenotypes: limb girdle muscular dystrophy 1C (LGMD1C), rippling muscle disease (RMD), distal myopathy and familial iperCKemia. All phenotypes are usually inherited with an autosomal dominant pattern of inheritance. We describe an Italian family which presented an autosomal recessive pattern of inheritance. A 30-year-old man presented with rippling, myotonic discharges at electromyography and elevated serum CK levels. His 34-year-old sister had normal muscular strength but referred rippling after strong exercise. DM1 and DM2 gene analysis was normal. Muscle biopsy was obtained from brachial biceps after written informed consent. Caveolin-3 immunohistochemistry (IHC) analysis and full gene sequencing were ultimately performed. Muscle biopsy showed a mild dystrophic pattern and a partial deficit of caveolin-3 at IHC analysis. Mutation analysis showed the homozygous mutation c.277G>A (Ala93Thr) in the CAV3 gene in both probands. Genetic analysis was extended to parents. They carried the same mutation in heterozygosis, were completely asymptomatic and showed normal cardiac function. The same mutation had been previously described by Kubisch et al. (2005) in homozygosis associated with a severe form of RMD. Our patients did not presented a more severe phenotype that usually seen. We confirmed the presence of intrafamilial variability. So far few missense mutations of the caveolin-3 gene behave as recessive. Genetic confirmation in independent families strengthens the reliability of genetic assessment. The mechanism preserving the integrity of caveolin oligomerisation and sarcolemmal localization in the presence of heterozygous A93T mutant caveolin-3 remains to be understood. doi:10.1016/j.nmd.2008.06.154

doi:10.1016/j.nmd.2008.06.152

D.P.3.11 Influences of caveolin-3 mutations on canonical signaling pathways E. Brauers; K. Reiss; M. Esser; J. Weis; A. Kru¨ttgen University Hospital, RWTH Aachen, Institute of Neuropathology, Aachen, Germany Caveolins are structural and functional proteins localized in flaskshaped plasmamembrane-invaginations called caveolae. These proteins have been implicated in several signal transduction and trafficking processes. Specific point mutations in caveolin-3, which is predominantly, expressed in skeletal and cardiac muscle cells, cause different muscle diseases like rippling muscle disease, limb-girdle muscular dystrophy (LGMD) or hyperCKemia. The pathomechanisms of these diseases are incompletely understood. We analysed various point mutations in caveolin-3 regarding their subcellular localization and function after transient transfection into different cell lines. Like it has already been shown for the mutation R26Q [Carozzi et al. (2002)], the amino acid changes P28L, A45T and G55S lead to a mislocalization of the plasma membrane. Interestingly, the mutations seem to influence the signaling and trafficking of Trk neurotrophin receptors, but have no apparent effect on STAT-3 signaling, which constitutes a major pathway downstream of IL-6, LIF and other neurocytokines. doi:10.1016/j.nmd.2008.06.153

D.P.3.13 A small-molecule inhibitor targeting transforming growth factor-b type I receptor kinase ameliorates muscular atrophy in a mouse model of caveolin3-deficient muscular dystrophy Y. Ohsawa; T. Okada; A. Kuga; S. Hayashi; T. Murakami; Y. Sunada Kawasaki Medical School, Division of Neurology, Department of Internal Medi, Kurashiki-City, Okayama, Japan Transforming growth factor (TGF)-b plays important roles in the pathogenesis of various disorders including cancer and muscular dystrophy. Small-molecule adenosine triphosphate (ATP)-competitive inhibitors of TGF-b type I receptor (TbRI) kinase have recently been developed to prevent the progression of cancer in advanced stages. Myostatin (GDF8) is a muscle-specific TGF-b superfamily member and plays an essential role in the negative regulation of skeletal muscle growth. Myostatin inhibition has drawn attention because a blocking antibody against myostatin ameliorates dystrophic phenotypes in various models of muscular dystrophy. Here, we evaluated the specificity and therapeutic potency of small-molecule TbRI kinase inhibitors for the suppression of myostatin signaling. TbRI kinase inhibitors suppressed myostatin-induced intracellular signaling, as well as TGF-b1, GDF11, activin A signalings in vitro. In addition, a TbRI kinase inhibitor enhanced in vitro myogenesis and attenuated myostatin-induced impairment of myoblast differentiation. Moreover, oral administration of the TbRI kinase inhibitor eventually suppressed myostatin activity in mouse sera. Long-term oral administration of the TbRI kinase inhibitor attenuated body-weight reduction, muscular atrophy, muscular weakness, and the increased expression of a