G.P.272

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Abstracts / Neuromuscular Disorders 24 (2014) 791–924

G.P.269 CAP-disease not-related to ACTA1, TPM2 or TPM3 genes M. Garibaldi 1, E. Malfatti 1, G. Brochier 1, J.M. Cuisset 2, C.A. Maurage 2, N. Monnier 3, B. Eymard 4, J. Laporte 5, M. Fardeau 1, N.B. Romero 1 1 GHU Pitie´-Salpeˆtrie`re, Researcher Centre, Myology Institute, Paris, France; 2 CHU de Lille, Lille, France; 3 CHU de Grenoble, Grenoble, France; 4 GHU Pitie´-Salpeˆtrie`re, AP-HP, Paris, France; 5 IGBMC, Illkirch, France CAP-disease is a rare congenital myopathy characterized by the presence of demarcated areas of subsarcolemmal myofibrillar disorganization that are devoid of myosin ATPase, stained with NADH-TR and strongly immunolabeled for desmin. Mutations in ACTA1, TPM2 or TPM3 have been reported so far. We report on 5 cases: 1 sporadic severe case and four patients from 2 families, respectively. In family 1 mother and daughter were affected; in family 2, two siblings manifested the disease. Mutations in the three documented genes were excluded. Age at onset ranged from birth to childhood. The main clinical phenotype consisted of clear dysmorphic features with elongated face and high-arched palate associated with axial, lower and upper limbs muscle weakness. The most severe cases presented early respiratory distress, and feeding difficulties. One patient had arthrogryposis. Muscle biopsies performed at different ages (from newborn to adult age) showed severe changes with marked variation of fiber size, increased internal nuclei, and type I fiber predominance. Several fibers contained a variable number of CAP in subsarcolemal areas. By electron microscopy, well-delineated CAP structures were constantly encountered. These showed disorganization of myofibrils, loss of thick filaments, and thickened fragments of Z bands without nemaline rod bodies. CAP contained segments of sarcomeres constituted by fragments of Z-lines from where arising thin filaments (”butterfly-like” aspect). Furthermore dense thin filamentous material was found in the subsarcolemmal region and less frequently between the myofibrils. In longitudinal sections, the Z lines appeared occasionally jagged and partially misaligned in some myofibrils. No basal membrane abnormalities were observed. To identify the causative mutation whole exome sequencing is ongoing. In conclusion, we attempt to characterize the clinical and morphological particularities of five cases presenting CAP disease, not-related to known genes. http://dx.doi:10.1016/j.nmd.2014.06.345

G.P.270 A large genomic rearrangement affecting TPM3 causing severe nemaline myopathy V.L. Lehtokari 1, K. Kiiski 1, K. Pelin 2, C. Wallgren-Pettersson 3 1 Folkha¨lsan Institute of Genetics and University of Helsinki, Faculty of Medicine, Helsinki, Finland; 2 Folkha¨lsan Institute of Genetics and University of Helsinki, Faculty of Biological and Environmental Sciences, Helsinki, Finland; 3 Folkha¨lsan Institute of Genetics and University of Helsinki, Faculty of Medicine, 00014 HU, Finland Using our self-designed NM-CGH microarray, which we have developed to identify large copy number variations in the currently known nine nemaline myopathy (NM) genes, we identified the first large (>20 kb) aberration in the alpha-tropomyosin gene (TPM3). This homozygous mutation deletes the promoter as well as the muscle- specific exons 1 and 2 of TPM3. The promoter and exon 1 of the non-muscle isoform seem to be intact. The deletion also spans the micro-RNA-encoding MIR190B gene and the last two exons of the C1orf189 gene upstream of TPM3. Our attempts to sequence the promoter and exon 1 of TPM3 have constantly failed, supporting the array result, showing an approximately 6 kb deletion including the musclespecific exons 1 and 2 as well as regions upstream from the gene. Before analyzing the patient sample using our array, we had analyzed

other NM genes including nebulin (NEB), in which we identified a heterozygous nonsense mutation in exon 42. No second mutation, however, was identified in NEB and this nonsense mutation likely has a modifying impact on the phenotype of the patient. We also performed whole-exome sequencing, but no other pathogenic mutations were revealed. The patient was born to consanguineous parents. He deceased at the age of approximately 1.5 years. His biopsy confirmed the diagnosis of NM. Unfortunately, to date, no further clinical details are available. We have screened 250 samples from 185 NM and related myopathy families using the NM-CGH-array and identified nine large aberrations in NEB in nine families, but we have not detected such mutations in other NM genes except the one discussed here. Therefore large aberrations in known NM genes other than NEB are likely a very rare cause of NM. The NM-CGH microarray method is currently available for mutation analysis in our laboratory. http://dx.doi:10.1016/j.nmd.2014.06.346

G.P.271 Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies M. Marttila, V.L. Lehtokari, S.B. Marston, T.A. Nyman, C. Barnerias, ¨ . Ceyhan-Birsoy, P. Cintas, M. Gerard, A.H. Beggs, E. Bertin, O B. Gilbert-Dussardier, J.S. Hogue, C. Longman, B. Eymard, M. Frydman, P.B. Kang, L. Klinge, H. Kolski, H. Lochmu¨ller, L. Magy, V. Manel, M. Mayer, K.N. North, S. Peudenier-Robert, H. Pihko, F.J. Probst, R. Reisin, W. Stewart, A.L. Taratuto, M. de Visser, E. Wilichowski, J. Winer, K. Nowak, N.G. Lain, T.L. Winder, N. Monnier, N.F. Clarke, K. Pelin, M. Gro¨nholm, C. Wallgren-Pettersson The Folkha¨lsan Institute of Genetics, Helsinki, Finland Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy (NM), cap myopathy, core-rod myopathy, congenital fibre-type disproportion, core-rod myopathy, distal arthrogryposes and Escobar syndrome. Here we correlate the clinical picture of these diseases with novel (16) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. A total of 27 Twenty-seven distinct pathogenic variants of TPM2, and 20 of TPM3, have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca2 + sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotypes more often had contractures of the limb joints (18/19) and jaw (6/19) than those with non-hypercontractile ones (2/22 and 1/22), while patients with the non-hypercontractile molecular phenotypes more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin–actin association or tropomyosin head-to-tail binding. http://dx.doi:10.1016/j.nmd.2014.06.347

G.P.272 Novel deletions in TPM3 define a hypercontractile phenotype with marked congenital muscle stiffness: Expanding the spectrum of TPM3 related disease S. Donkervoort 1, M. Neu 1, J. Kirschner 2, M.L. Yang 3, S.B. Marston 4, M.A. Gibbons 5, Y. Hu 1, J.M. de Winter 6, C.A.C. Ottenheijm 6,

Abstracts / Neuromuscular Disorders 24 (2014) 791–924 A. Rutkowski 7, M. Kru¨ger 2, E. McNamara 8, R. Ong 8, K. Nowak 8, N.F. Clarke 9, C.G. Bo¨nnemann 1 1 National Institutes of Health, Bethesda, USA; 2 University Medical Center Freiburg, Freiburg, Germany; 3 University of Colorado School of Medicine, Aurora, USA; 4 Imperial College London, London, UK; 5 University of Colorado Denver School of Medicine, Aurora, USA; 6 VU University Medical Center, Amsterdam, Netherlands; 7 Kaiser SCPMG, Cure CMD, Olathe, USA; 8 The University of Western Australia, Crawley, Australia; 9 The Children’s Hospital at Westmead, University of Sydney, Sydney, USA Tropomyosin 3 encoded by the TPM3 gene is a member of the acting binding tropomyosin family, a component of the sarcomeric thin filaments troponin tropomyosin complex that is essential in muscle contraction by regulating the calcium dependent binding of the myosin head to the actin filament in anticipation of the force generating power stroke. Mutations in TPM3 cause a clinical and histopathological heterogeneous group of neuromuscular disorders characterized by progressive weakness that includes CAP myopathy, congenital fiber type disproportion and nemaline myopathy. Recent studies of the deltaK7 mutation in TPM2 suggest that the pathogenic mechanism underlying tropomyosin related disease with progressive muscle contractures may be a gain of function change, leading to elevated Ca2+ sensitivity of force generation, while this has not yet been shown conclusively for TPM3. Here we report two unrelated patients with a de novo TPM3 single glutamic acid deletions resulting in a much more significant contractile phenotype with marked congenital muscle stiffness associated with ventilator failure in one case. We hypothesize that these single amino acid deletions result in increased Ca2+ sensitivity of the troponin tropomyosin complex and a consequently hypercontractile sarcomere. We thus expand the clinical, phenotypic and pathophysiological spectrum of TPM3 mutations, which now join recessive aB-crystallin mutations and specific ACTA1 and TPM2 mutations as a myogenic cause for neonatal muscle rigidity. http://dx.doi:10.1016/j.nmd.2014.06.348

G.P.273 Nemaline myopathy 8 and KLHL40 in diseased and normal skeletal muscle G. Ravenscroft 1, E.J. Todd 1, K.S. Yau 1, C.A. Sewry 2, C.A. McLean 3, M.M. Ryan 4, R.J. Allcock 5, N.G. Laing 1 1 University of Western Australia, Nedlands, Australia; 2 UCL Institute of Child Health and Great Ormond Street Hospital for Children, London, UK; 3 Alfred Hospital, Melbourne, Australia; 4 Royal Children’s Hospital, Melbourne, Australia; 5 University of Western Australia, Perth, Australia Mutations in the gene (KLHL40), encoding Kelch-like family member 40, were recently identified in patients presenting with severe autosomal recessive nemaline myopathy (NEM8). Using exome sequencing or targeted re-sequencing of 276 known neuromuscular disease genes through a TargetSeq (Life Technologies) capture panel, we have identified two additional cases with KLHL40 mutations. Both were from consanguineous kindreds. Case 1 was diagnosed with severe SMA and congenital fractures. We identified a homozygous nonsense mutation in KLHL40 (c.46C>T, p. Gln16*). Muscle biopsy identified variation in fibre size and miliary nemaline bodies by electron microscopy seen as very fine scattered granules upon Gomori trichrome staining. Case 2 presented with severe nemaline myopathy, the biopsy showing numerous tiny nemaline bodies. We identified a homozygous missense mutation in KLHL40 (c.931C>A, p. Arg311Ser). Neither of the variants was present in 1000genomes or EVS databases. These data further suggest that muscle biopsy may be useful to inform genetic testing and that KLHL40 should be considered in patients presenting with severe miliary nemaline myopathy. Nemaline myopathy should also be considered in the differential diagnosis of severe SMA, especially in patients presenting

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with congenital fractures and contractures. The normal biological function of KLHL40 is largely unknown and therefore the basis of the severe muscle disease resulting from mutations of KLHL40 is also unknown. In investigations of control and diseased mouse and patient muscle, we show that KLHL40 is more abundant in the mouse soleus and diaphragm (muscles that comprise oxidative type I and IIA myofibres) than in the EDL, gastrocnemius and quadriceps muscles (type II glycolytic predominant). In addition we show that KLHL40 is increased in dystrophic mouse (mdx) and regenerating patient muscle (including DMD), suggesting that it may serve as a novel marker of skeletal muscle regeneration. http://dx.doi:10.1016/j.nmd.2014.06.349

G.P.274 Cold shock domain protein A – A novel nemaline myopathy-causing gene? J. Laitila 1, V.L. Lehtokari 1, K. Kiiski 1, C. Wallgren-Pettersson 1, K. Pelin 2 1 The Folkha¨lsan Institute of Genetics, Helsinki, Finland; 2 University of Helsinki, Helsinki, Finland Mutation analysis of the known nemaline myopathy (NM)-causing genes in a Finnish patient with an unusual form of NM, with legs clearly stronger than arms, did not reveal the cause of the disease. A heterozygous TPM3 splice site mutation, delTAGG, in intron 1 was identified, inherited from the healthy father. The mutation was predicted to be recessive, but no TPM3 mutation was found on the maternal allele. Large copy number variations affecting the known nine NM genes were excluded by NM-CGH array analysis. Whole-exome sequencing revealed a heterozygous RYR1 missense mutation, p.Leu2031Phe, predicted to be probably damaging by PolyPhen-2, but the healthy father also carried this mutation. No other putative pathogenic mutation in RYR1 was found in the patient, and in myoblasts of the patient, epigenetic silencing of the maternal allele was ruled out. Exome sequencing did, however, disclose two putative pathogenic mutations in the cold shock domain protein A encoding gene, CSDA. The CSDA gene has 11 exons and encodes three different protein isoforms: 303 aa, 372 aa and 200 aa. CSDA is highly expressed in skeletal muscle and heart. Our patient was compound heterozygous for two missense mutations, p.Ser34Arg and p.Arg129Trp, in CSDA, each inherited from one of the healthy parents. The mutations are not listed in the EVS database (http://evs.gs.washington.edu/EVS/). Ser34 and Arg129 are highly conserved between species, and reside in the DNA-binding domain (cold shock domain) of CSDA. Furthermore, Ser34 is phosphorylated by ERK2 and GSK3b, and the phosphorylation is thought to be important for the formation of nuclear CSDA complexes and binding of CSDA to single-stranded DNA. Western blot analysis of cultured myotubes from the patient confirms CSDA expression on the protein level. Further analyses are ongoing in order to quantify the expression levels and determine the intracellular location of CSDA in myoblasts from the patient and healthy controls. http://dx.doi:10.1016/j.nmd.2014.06.350

LGMD2 G.P.275 Expanding the clinical and genetic spectrum of FKRP-related myopathies in Argentinean pediatric patients F. Lubieniecki 1, S. Monges 1, J. Mozzoni 1, A. Moresco 1, V. Aguerre 1, M.F. de Castro 1, N. Pozzo 1, E. Foncuberta 1, C. Bouchet Seraphin 2, N.B. Romero 3, A.L. Taratuto 4