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P2.13 Proteolytic processing of C-terminal titin
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Abstracts / Neuromuscular Disorders 20 (2010) 596–680
them. The raw data of both distal myopathies was processed and analysed using Chipster and Ingenuity Pathway Analysis. Results: Sets of unique biochemical and genetic changes characteristic for TMD and WDM, such as ATM signalling, EIF2 signalling and clathrin mediated Endocytosis signalling in TMD and melatonin signalling, GNRH signalling and purine metabolism in WDM were uncovered. We also identified shared changes that result in the similarity seen in these distal myopathies, such as mitochondrial dysfunction, protein Ubiquitination and oxidative phosphorylation changes. We report heterogenous dysregulation in two pathways in both disorders (SAP-Jnk apoptosis) and in TMD (unfolded protein response linked ER stress) by semi-quantitive RT-PCR and western blotting. Conclusion: TMD has previously been reported to have NF-KB linked apoptotic changes in dystrophic tissue. We have identified a range of significantly upregulated apoptotic pathway molecules in TMD and we show that SAP-Jnk apoptotic signalling is common to the pathology of TMD and WDM. FINMaj mutations in TMD also causes induction of ER stress markers (BIP) and lysosomal components (LAMP2). doi:10.1016/j.nmd.2010.07.084
P2.13 Proteolytic processing of C-terminal titin J. Sarparanta, A. Vihola, P.H. Jonson, B. Udd Folkhälsan Institute of Genetics and Dept. of Medical Genetics, University of Helsinki, Helsinki, Finland Mutations in the extreme C-terminal part of titin, residing in the sarcomeric M-band, lead to tibial muscular dystrophy (TMD), a dominant late-onset distal myopathy. In homozygotes, the same mutations cause the more severe recessive limb-girdle muscular dystrophy 2J (LGMD2J). The FINmaj mutation, underlying the titinopathies TMD/LGMD2J in Finnish patients, causes an exchange of four consecutive amino acids in the Ig-like domain M10, the most C-terminal domain of titin. Seven other missense or truncating mutations, all affecting M10 or the preceding is 7 region, are known to cause TMD/LGMD2J in other European populations. The molecular pathomechanisms of the titinopathies are not known in detail, but a number of findings suggest that proteolytic cleavage of titin might play a role in the pathogenesis. The loss of C-terminal titin epitopes and the secondary calpain 3 deficiency in LGMD2J muscle indicate that the entire mutant titin C-terminus may be absent. Moreover, characteristic patterns of small protein fragments originating from the titin C-terminus are seen in western blots of normal muscle extracts. These fragments are altered in TMD and LGMD2J muscles, further emphasizing the possible role of aberrant proteolysis in the diseases. We have used several complementary approaches for studying the proteolytic processing of C-terminal titin. Expression of epitope-tagged and untagged C-terminal titin constructs in different mammalian cell types has shown that titin constructs are fragmented in a characteristic way, with some differences between wild-type and FINmaj mutant titin. High-resolution western blotting with a wide panel of antibodies – including three new monoclonal antibodies raised against the M10 domain – has allowed comparison of the fragments produced in cell cultures and in muscle. For detailed characterization of some fragments, titin fragments produced in cell culture have been purified and analyzed by mass spectrometry. doi:10.1016/j.nmd.2010.07.085
P2.14 Familial myopathy with early respiratory failure and sharing of a large haplotype at chromosome 2q31 M. Ohlsson 1, B. Brådvik 2, C. Lindberg 3, H. Tajsharghi 1, T. Martinsson 4, A. Oldfors 1 1 University of Gothenburg, Institute of Biomedicine, Department of Pathology, Gothenburg, Sweden, 2 Lund University Hospital, Department of Neurology, Lund, Sweden, 3 University of Gothenburg, Institute of Neuroscience and Physiology, Department of Clinical Neuroscience and Rehabilitation, Gothenburg, Sweden, 4 University of Gothenburg, Insitute of Biomedicine, Department of Medical Genetics, Gothenburg, Sweden
We describe seven individuals from two apparently unrelated Swedish families with adult-onset autosomal dominant myopathy characterized by limb-girdle muscle weakness and early respiratory failure. All patients had neck flexor, ankle dorsiflexor and trunk muscle weakness together with prominent calf hypertrophy. Three of the patients also had mild facial weakness. Examination of lung function showed decreased vital capacity in all patients (range 20–74% of predicted value). Two patients had ventilatory support at night. Electrocardiography and echocardiography were normal in all patients. Muscle histopathological features included fiber size variability, prominent fiber fragmentation, increased internal nucleation, rimmed vacuoles and frequent cytoplasmic bodies. Some muscles were severely atrophic with connective and fat tissue replacement. Array data using single nucleotide polymorphism (SNP) markers including only definitely affected individuals from the two families demonstrated that they shared a large haploptype at chromosome 2q31, indicating that the gene defect responsible for the disease is located in this region. The results from our studies on two families with myopathy with early respiratory failure demonstrate striking clinical and morphological similarities with a myopathy previously described by Edström et al. (1990) in several Swedish families. Recent linkage and sequence analyses in two of these families demonstrated a heterozygous mutation in the titin kinase domain of the titin gene (TTN), which is located on chromosome 2q31. TTN was the obvious candidate gene in our patients but sequencing analysis did not reveal any mutation in the titin kinase domain. Therefore this type of familial myopathy with early respiratory failure and distinct histopathological changes is probably genetically heterogeneous. doi:10.1016/j.nmd.2010.07.086
P2.15 Oculopharyngodistal myopathy – a possible association with cardiomyopathy D. Hilton-Jones 1, W. Thevathasan 1, W. Squier 1, D. MacIver 2, D. Hilton 3, E. Fathers 2 1 John Radcliffe Hospital, Oxford, United Kingdom, 2 Musgrove Park Hospital, Taunton, United Kingdom, 3 Derriford Hospital, Plymouth, United Kingdom
Oculopharyngodistal myopathy (OPDM) is an uncommon myopathy characterised clinically by cranial and distal limb muscle weakness. Here we describe two siblings with autosomal dominant OPDM apparently associated with dilated cardiomyopathy, which in one case progressed to ventricular hypertrabeculation/noncompaction. Electrocardiographic screening was normal and the cardiomyopathy was detected only with echocardiography. Our findings suggest that patients with OPDM should be screened for
Abstracts / Neuromuscular Disorders 20 (2010) 596–680
them. The raw data of both distal myopathies was processed and analysed using Chipster and Ingenuity Pathway Analysis. Results: Sets of unique biochemical and genetic changes characteristic for TMD and WDM, such as ATM signalling, EIF2 signalling and clathrin mediated Endocytosis signalling in TMD and melatonin signalling, GNRH signalling and purine metabolism in WDM were uncovered. We also identified shared changes that result in the similarity seen in these distal myopathies, such as mitochondrial dysfunction, protein Ubiquitination and oxidative phosphorylation changes. We report heterogenous dysregulation in two pathways in both disorders (SAP-Jnk apoptosis) and in TMD (unfolded protein response linked ER stress) by semi-quantitive RT-PCR and western blotting. Conclusion: TMD has previously been reported to have NF-KB linked apoptotic changes in dystrophic tissue. We have identified a range of significantly upregulated apoptotic pathway molecules in TMD and we show that SAP-Jnk apoptotic signalling is common to the pathology of TMD and WDM. FINMaj mutations in TMD also causes induction of ER stress markers (BIP) and lysosomal components (LAMP2). doi:10.1016/j.nmd.2010.07.084
P2.13 Proteolytic processing of C-terminal titin J. Sarparanta, A. Vihola, P.H. Jonson, B. Udd Folkhälsan Institute of Genetics and Dept. of Medical Genetics, University of Helsinki, Helsinki, Finland Mutations in the extreme C-terminal part of titin, residing in the sarcomeric M-band, lead to tibial muscular dystrophy (TMD), a dominant late-onset distal myopathy. In homozygotes, the same mutations cause the more severe recessive limb-girdle muscular dystrophy 2J (LGMD2J). The FINmaj mutation, underlying the titinopathies TMD/LGMD2J in Finnish patients, causes an exchange of four consecutive amino acids in the Ig-like domain M10, the most C-terminal domain of titin. Seven other missense or truncating mutations, all affecting M10 or the preceding is 7 region, are known to cause TMD/LGMD2J in other European populations. The molecular pathomechanisms of the titinopathies are not known in detail, but a number of findings suggest that proteolytic cleavage of titin might play a role in the pathogenesis. The loss of C-terminal titin epitopes and the secondary calpain 3 deficiency in LGMD2J muscle indicate that the entire mutant titin C-terminus may be absent. Moreover, characteristic patterns of small protein fragments originating from the titin C-terminus are seen in western blots of normal muscle extracts. These fragments are altered in TMD and LGMD2J muscles, further emphasizing the possible role of aberrant proteolysis in the diseases. We have used several complementary approaches for studying the proteolytic processing of C-terminal titin. Expression of epitope-tagged and untagged C-terminal titin constructs in different mammalian cell types has shown that titin constructs are fragmented in a characteristic way, with some differences between wild-type and FINmaj mutant titin. High-resolution western blotting with a wide panel of antibodies – including three new monoclonal antibodies raised against the M10 domain – has allowed comparison of the fragments produced in cell cultures and in muscle. For detailed characterization of some fragments, titin fragments produced in cell culture have been purified and analyzed by mass spectrometry. doi:10.1016/j.nmd.2010.07.085
P2.14 Familial myopathy with early respiratory failure and sharing of a large haplotype at chromosome 2q31 M. Ohlsson 1, B. Brådvik 2, C. Lindberg 3, H. Tajsharghi 1, T. Martinsson 4, A. Oldfors 1 1 University of Gothenburg, Institute of Biomedicine, Department of Pathology, Gothenburg, Sweden, 2 Lund University Hospital, Department of Neurology, Lund, Sweden, 3 University of Gothenburg, Institute of Neuroscience and Physiology, Department of Clinical Neuroscience and Rehabilitation, Gothenburg, Sweden, 4 University of Gothenburg, Insitute of Biomedicine, Department of Medical Genetics, Gothenburg, Sweden
We describe seven individuals from two apparently unrelated Swedish families with adult-onset autosomal dominant myopathy characterized by limb-girdle muscle weakness and early respiratory failure. All patients had neck flexor, ankle dorsiflexor and trunk muscle weakness together with prominent calf hypertrophy. Three of the patients also had mild facial weakness. Examination of lung function showed decreased vital capacity in all patients (range 20–74% of predicted value). Two patients had ventilatory support at night. Electrocardiography and echocardiography were normal in all patients. Muscle histopathological features included fiber size variability, prominent fiber fragmentation, increased internal nucleation, rimmed vacuoles and frequent cytoplasmic bodies. Some muscles were severely atrophic with connective and fat tissue replacement. Array data using single nucleotide polymorphism (SNP) markers including only definitely affected individuals from the two families demonstrated that they shared a large haploptype at chromosome 2q31, indicating that the gene defect responsible for the disease is located in this region. The results from our studies on two families with myopathy with early respiratory failure demonstrate striking clinical and morphological similarities with a myopathy previously described by Edström et al. (1990) in several Swedish families. Recent linkage and sequence analyses in two of these families demonstrated a heterozygous mutation in the titin kinase domain of the titin gene (TTN), which is located on chromosome 2q31. TTN was the obvious candidate gene in our patients but sequencing analysis did not reveal any mutation in the titin kinase domain. Therefore this type of familial myopathy with early respiratory failure and distinct histopathological changes is probably genetically heterogeneous. doi:10.1016/j.nmd.2010.07.086
P2.15 Oculopharyngodistal myopathy – a possible association with cardiomyopathy D. Hilton-Jones 1, W. Thevathasan 1, W. Squier 1, D. MacIver 2, D. Hilton 3, E. Fathers 2 1 John Radcliffe Hospital, Oxford, United Kingdom, 2 Musgrove Park Hospital, Taunton, United Kingdom, 3 Derriford Hospital, Plymouth, United Kingdom
Oculopharyngodistal myopathy (OPDM) is an uncommon myopathy characterised clinically by cranial and distal limb muscle weakness. Here we describe two siblings with autosomal dominant OPDM apparently associated with dilated cardiomyopathy, which in one case progressed to ventricular hypertrabeculation/noncompaction. Electrocardiographic screening was normal and the cardiomyopathy was detected only with echocardiography. Our findings suggest that patients with OPDM should be screened for