P.3.13 Gene expression profiling in Welander distal myopathy

Abstracts / Neuromuscular Disorders 23 (2013) 738–852 gotes with novel TTN frameshift mutations combined with previously reported TMD mutations. One LGMD2J patient was heterozygous for the FINmaj TMD mutation with a likely second yet undiscovered mutation. The unequal expression levels of TTN transcripts in these four probands suggested that the expression of the frameshifted allele was severely reduced, probably through nonsense-mediated decay, explaining the observed more severe phenotypes. One Portuguese patient was homozygote for a previously known Spanish TMD mutation. This TMD mutation seems to cause a more severe TMD rather than LGMD2J when homozygous. One Finnish patient had FINmaj mutation combined with a novel missense mutation in the A-band region of titin causing a new phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism of phenotype variability in muscular dystrophies. http://dx.doi:10.1016/j.nmd.2013.06.436

P.3.12 Characterization of CAPN3-dependent proteolysis of C-terminal titin J. Sarparanta 1, K. Charton 2, H. Luque 1, P.H. Jonson 1, I. Richard 2, B. Udd 1 1 Folkha¨lsan Institute of Genetics, Dept. of Medical Genetics, University of Helsinki, Helsinki, Finland; 2 Ge´ne´thon, Evry, France The titinopathies tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the C-terminus of titin, residing in the sarcomeric M-band. Mutations identified so far affect the last Ig domain M10 or the preceding is7 region. The FINmaj mutation, underlying TMD/LGMD2J in patients of Finnish origin, causes the exchange of four amino acids in M10 and presumably leads to domain unfolding. The other known mutations cause missense changes of single amino acids or truncation of the ultimate C-terminus. Loss of C-terminal titin epitopes in IF microscopy and reduced amount of C-terminal titin fragments in western blotting suggest that increased or abnormal proteolytic turnover of mutant titin may contribute to the pathomechanism. The muscle-specific protease calpain 3 (CAPN3) binds M-band titin at the is7 region; the interaction is thought to regulate the autolytic activation of CAPN3. LGMD2J patients and FINmaj knock-in mice show secondary CAPN3 deficiency, reflecting loss of the binding site and consequent dysregulation of CAPN3 activity. To elucidate the proteolytic events involved in the pathogenesis of Mband titinopathies, we investigated the cleavage of C-terminal titin by CAPN3. Cleavage fragments generated by CAPN3 were identified by coexpressing various wild-type and mutant titin constructs with active or inactive CAPN3 in cell culture, and comparing the resulting fragment patterns. Active CAPN3 produced several titin fragments, which were characterized by western blotting, protein sequencing and mass spectrometry for identification of cleavage sites. Furthermore, targeted mutagenesis of predicted CAPN3 recognition sites was utilized for understanding the sequence determinants of CAPN3 cleavage and studying the order of cleavage events. http://dx.doi:10.1016/j.nmd.2013.06.437

P.3.13 Gene expression profiling in Welander distal myopathy M. Screen 1, P. Hackman 1, P. Johson 1, S. Huovinen 2, B. Udd 1 1 Folkha¨lsan Institute of Genetics, University of Helsinki, Helsinki, Finland; 2 Neuromuscular Research Unit, Tampere University Hospital and Tampere University, Helsinki, Finland

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Welander distal myopathy (WDM) is a late onset disease caused by a mutation in the C-terminal region of the TIA1 gene. The secondary molecular events resulting from the causative TIA1 mutations to the preterm death of muscle cells in WDM are mostly unknown. In order to identify downstream pathogenetic mechanisms we explored WDM biopsies genetic profile by expression profiling. We compared the changes to the expression profile of the phenotypically overlapping tibial muscular dystrophy (TMD). Six WDM patient biopsies and five healthy control muscle biopsies were used and the expression data was analyzed using Ingenuity Pathway Analysis. We identified biochemical and genetic pathway changes distinctive to WDM, such as 14-3-3 mediated apoptosis, cleavage and polyadenylation of pre-mRNA, protein trafficking and transport, and oxidative phosphorylation pathway changes. We also identified shared changes with TMD that result in the similarity seen in both these distal myopathies, such as SAP-JNK apoptosis, P70S6 mTOR signalling, mitochondrial dysfunction, and protein ubiquitination pathway changes. TIA1 is a key component of stress granules, a mechanism used to protect other cellular mechanisms during stress. The unique oxidative phosphorylation changes we identified by expression profiling may be associated with increased oxidative stress in WDM. TIA1 is also involved in polyadenylation and splicing of mRNA and the identified changes in these pathways in our study suggest mutated TIA1 affects these pathways in WDM. In addition, both WDM and TMD have common pathological changes in P70S6-mTOR signalling, SAP-JNK apoptosis and protein ubiquitination signalling pathways, which have been reported in other rimmed vacuolar myopathies. http://dx.doi:10.1016/j.nmd.2013.06.438

CONGENITAL MYOPATHIES P.4.1 PIP kinases, muscle development, and the pathogenesis of myotubular myopathy A. Reifler 1, D. Michele 2, A. Archambeau 2, X. Li 3, J.J. Dowling 3 1 University of Michigan, Neuroscience, Ann Arbor, United States; 2 University of Michigan, Physiology, Ann Arbor, United States; 3 University of Michigan, Pediatrics, Ann Arbor, United States Myotubular myopathy (MTM) is a severe congenital myopathy with no currently identified treatment. MTM is caused by mutations in MTM1, a phosphatase that dephosphorylates 3-position phosphoinositides. Through the use of vertebrate model systems, the consequences (s) of MTM1 mutation in skeletal muscle in vivo are beginning to be unraveled. In particular, work from several laboratories (including our own) has demonstrated that loss of MTM1 (1) increases the levels of PI3P in skeletal muscle and (2) results in the disruption of the structure and function of the EC coupling apparatus. Based on these data, one hypothesis to explain MTM is that pathologic elevation of PI3P results in aberrant EC coupling, which in turn causes muscle weakness and severe neurologic disability. A correlate hypothesis is that lowering PI3P levels in MTM will enable normal muscle structure and will thus ameliorate the disease. These hypotheses lead to 2 critical questions: What is the consequence of reducing PI3P levels on normal skeletal muscle development? What is the impact of reducing PI3P levels on MTM pathogenesis? The goal of this study is to address these questions by studying models with reduced PI3P. We have created knockout mice that lack either Pik3c2b or Pik3c3, the kinases responsible for PI3P production in skeletal muscle. Using these animals, we have identified unique and non-overlapping functions for these kinases, including the observation that loss of Pik3c3 results in a muscular dystrophy phenotype. Furthermore, we have tested the concept of PI3P reduction as a therapeutic strategy for MTM by crossbreeding individual