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Correlation between mutation size and cardiac involvement in myotonic dystrophy type 1: An analysis of the DM1-heart registry
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Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212
IHC findings for each of the three groups studied suggest that the histopathological findings may help to guide genetic testing. http://dx.doi.org/10.1016/j.nmd.2016.06.389
P.361 Analysis of the autophagic pathway during in vitro muscle differentiation in X-linked myopathy with excessive autophagy S. Fernandes 1, C. Almeida 1, P. Onofre-Oliveira 1, A. Bigot 2, V. Mouly 2, M. Vainzof 1 1 Human Genome and Stem Cell Research Center, University of Sao Paulo, Sao Paulo, Brazil; 2 Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Paris, France X-linked myopathy with excessive autophagy (XMEA) is an inherited, slowly progressive myopathy, characterized by sarcoplasmic vacuoles in muscle fibers. XMEA is caused by mutations in the vacuolar membrane ATPase 21 gene (Vma21), resulting in a reduction of both mRNA and protein VMA21 levels, elevating lysosomal pH, blocking partially the final degradation step of autophagy and increasing the formation of autolysosomes. We recently identified a Brazilian family with XMEA caused by a unique small insertion-deletion in the Vma21 gene. Here, we studied immortalized myoblasts isolated from muscle biopsies from one XMEA patient and one control. Differentiation was induced, and myotubes were analyzed after three and six days of differentiation. Through qPCR, the relative expression of autophagy-related genes was evaluated, and Myog analysis confirmed myotube formation. In controls, the autophagy genes Lc3b, Vps34 and Atg12 showed a similar pattern of expression in undifferentiated cells as well as after myotube differentiation. Interestingly, in the XMEA patient, these genes showed a lower expression in myoblasts, but a higher expression after myotube formation, suggesting a more activated autophagic gene induction. Immunofluorescence analysis using anti-LC3 antibody confirmed at the protein level the gene expression data. In the control, a similar number of LC3 puncta was observed before and after differentiation, while in the XMEA patient, less LC3 puncta than the control in the undifferentiated cells and more in the differentiated ones were observed. Remarkably, Bnip3 gene was only slightly altered in all conditions, indicating that mitophagy may not be dramatically affected. Our results show that the increase in autophagy that characterizes XMEA may arise after cells start to differentiate, and that in progenitor cells such as myoblasts, the signaling may be affected in a distinct way. http://dx.doi.org/10.1016/j.nmd.2016.06.390
MYOTONIC DYSTROPHY AND CHANNELOPATHIES P.362 Correlation between mutation size and cardiac involvement in myotonic dystrophy type 1: An analysis of the DM1-heart registry K. Wahbi 1, C. Chong-Nguyen 2, V. Algalarrondo 3, P. Laforet 4, D. Furling 4, T. Stojkovic 4, G. Bassez 4, A. Behin 4, B. Eymard 4, D. Duboc 2 1 Cochin and Pitié-Salpetrière Hospitals, Paris, France; 2 Cochin Hospital, Paris, France; 3 Antoine Beclere Hospital, Clamart, France; 4 Pitié Salpêtrière Hospital, Paris, France In myotonic dystrophy type 1 (DM1), the correlation between the number of CTG repeats and the severity of the cardiac involvement remains controversial. To determine the relationship between the amplification mutation size in DM1 and the prevalence of the cardiac events and long-term cardiac prognosis. The DM1 heart registry from the Neurological Unit of the Myology Institute of Pitié Salpêtrière Hospital and the Cardiology Department of Cochin Hospital in Paris included consecutive patients with genetically confirmed DM1 between January 2000 and December 2015. The association between the number of CTG repeats and the following end-point was studied: overall survival, sudden death, conduction system disease requiring the implantation of a permanent pacemaker,
supraventricular tachyarrhythmia. 855 patients were included, with 420 males (49%) and a median age of 37 years. Over a 10 years median follow-up, 210 patients died (25%), 32 suddenly. 166 patients (19%) developed supraventricular tachyarrhythmia and 181 (21%) had a permanent pacemaker implanted. In multivariate analysis, the severity of the cardiac involvement increases with the number of CTG repeats. Patients with a larger mutation size had a worse survival (HR 1.54 IC95% [1.35–1.76] p < 0.001) and a higher incidence of all cardiac events: sudden death (HR 1.56 IC95% [1.13–2.17] p = 0.0076), supraventricular tachyarrhythmia (HR 1.20 [1.01–1.41] p = 0.0334), pacemaker (HR 1.33 [1.17– 1.52] p < 0.0001). Male gender developed more cardiac events and had a worse cardiac prognosis. The CTG repeat size is an age and gender independent factor of the cardiac severity involvement and prognosis. http://dx.doi.org/10.1016/j.nmd.2016.06.391
P.363 DM2-linked myopathy caused by uninterrupted short (CCTG)50–70 repeat expansion in CNBP T. Suominen 1, L. Bachinski 2, O. Raheem 1, H. Haapasalo 3, W. Kress 4, R. Krahe 2, B. Udd 5 1 University of Tampere, Tampere, Finland; 2 University of Texas MD Anderson Cancer Center, Houston, USA; 3 Fimlab Laboratories, Tampere University Hospital, Tampere, Finland; 4 University of Wuerzburg, Biozentrum, Am Hubland, Wuerzburg, Germany; 5 Neuromuscular Research Center, Tampere University Hospital, Tampere, Finland Myotonic dystrophy type 2 (DM2) is one of the most common adult-onset muscular dystrophies in Europe. It is a dominantly inherited multisystemic disease caused by a repeat expansion in intron 1 of CNBP gene. The diseasecausing repeat expansions consist of 75–11,000 (CCTG) repeats. In this study, we characterize a new type of DM2-linked mild myopathy in several patients without myotonia associated with very short (CCTG)50–70 repeat expansions. We have identified eleven individuals carrying a short (CCTG) repeat expansion. They have been analyzed by repeat-primed PCR (RP-PCR) and haplotype analysis. Seven has been clinically evaluated and muscle biopsy analyzed in three patients. For one of the patient, an index patient of a larger family, the mutation has been studied further by Southern blot, sequencing, allele specific expression, in situ hybridization, Western blot and analysis of fetal splice isoforms of DM2 effector proteins. Clinical features are mild, including myalgic pain, muscle weakness and stiffness. Some patients hardly had any clinical symptoms. Even though the clinical symptoms are milder than in DM2, the muscle biopsy findings show DM2-like changes, including highly atrophic type 2 fibers, nuclear clump fibers and internal nuclei. PCR-based methods and Southern blot indicated a repeat expansion of (CCTG)50–70 for the first index patient. Sequencing of the repeat region will be performed also for additional five samples. In contrast to DM2, ribonuclear foci of accumulated RNA were not detected, splicing of DM2 effector proteins was normal and CNBP protein level was normal. Finding of CNBP repeat expansion mutations containing only (CCTG)50–70 repeats in mildly symptomatic myopathy patients without myotonia suggests that even shorter than previously reported expansions may cause a disease, although with a different phenotype. The molecular findings indicate a unique pathomechanism and the term ‘myotonic dystrophy’ cannot be applied to this new disease. http://dx.doi.org/10.1016/j.nmd.2016.06.392
P.364 The brain diffusion tensor and voxel based morphometry imaging correlates with the phenotypic severity in myotonic dystrophy type 1 J. Park 1, J. Shin 2, H. Song 3, S. Lee 4, Y. Chang 3 1 Kyungpook National University School of Medicine, Daegu, Republic of Korea; 2 Pusan National University Yangsan hospital, Yangsan, Republic of Korea; 3 Kyungpook National University and Hospital, Daegu, Republic of Korea; 4 Kyungpook National University Medical Center, Daegu, Republic of Korea
Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212
IHC findings for each of the three groups studied suggest that the histopathological findings may help to guide genetic testing. http://dx.doi.org/10.1016/j.nmd.2016.06.389
P.361 Analysis of the autophagic pathway during in vitro muscle differentiation in X-linked myopathy with excessive autophagy S. Fernandes 1, C. Almeida 1, P. Onofre-Oliveira 1, A. Bigot 2, V. Mouly 2, M. Vainzof 1 1 Human Genome and Stem Cell Research Center, University of Sao Paulo, Sao Paulo, Brazil; 2 Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Paris, France X-linked myopathy with excessive autophagy (XMEA) is an inherited, slowly progressive myopathy, characterized by sarcoplasmic vacuoles in muscle fibers. XMEA is caused by mutations in the vacuolar membrane ATPase 21 gene (Vma21), resulting in a reduction of both mRNA and protein VMA21 levels, elevating lysosomal pH, blocking partially the final degradation step of autophagy and increasing the formation of autolysosomes. We recently identified a Brazilian family with XMEA caused by a unique small insertion-deletion in the Vma21 gene. Here, we studied immortalized myoblasts isolated from muscle biopsies from one XMEA patient and one control. Differentiation was induced, and myotubes were analyzed after three and six days of differentiation. Through qPCR, the relative expression of autophagy-related genes was evaluated, and Myog analysis confirmed myotube formation. In controls, the autophagy genes Lc3b, Vps34 and Atg12 showed a similar pattern of expression in undifferentiated cells as well as after myotube differentiation. Interestingly, in the XMEA patient, these genes showed a lower expression in myoblasts, but a higher expression after myotube formation, suggesting a more activated autophagic gene induction. Immunofluorescence analysis using anti-LC3 antibody confirmed at the protein level the gene expression data. In the control, a similar number of LC3 puncta was observed before and after differentiation, while in the XMEA patient, less LC3 puncta than the control in the undifferentiated cells and more in the differentiated ones were observed. Remarkably, Bnip3 gene was only slightly altered in all conditions, indicating that mitophagy may not be dramatically affected. Our results show that the increase in autophagy that characterizes XMEA may arise after cells start to differentiate, and that in progenitor cells such as myoblasts, the signaling may be affected in a distinct way. http://dx.doi.org/10.1016/j.nmd.2016.06.390
MYOTONIC DYSTROPHY AND CHANNELOPATHIES P.362 Correlation between mutation size and cardiac involvement in myotonic dystrophy type 1: An analysis of the DM1-heart registry K. Wahbi 1, C. Chong-Nguyen 2, V. Algalarrondo 3, P. Laforet 4, D. Furling 4, T. Stojkovic 4, G. Bassez 4, A. Behin 4, B. Eymard 4, D. Duboc 2 1 Cochin and Pitié-Salpetrière Hospitals, Paris, France; 2 Cochin Hospital, Paris, France; 3 Antoine Beclere Hospital, Clamart, France; 4 Pitié Salpêtrière Hospital, Paris, France In myotonic dystrophy type 1 (DM1), the correlation between the number of CTG repeats and the severity of the cardiac involvement remains controversial. To determine the relationship between the amplification mutation size in DM1 and the prevalence of the cardiac events and long-term cardiac prognosis. The DM1 heart registry from the Neurological Unit of the Myology Institute of Pitié Salpêtrière Hospital and the Cardiology Department of Cochin Hospital in Paris included consecutive patients with genetically confirmed DM1 between January 2000 and December 2015. The association between the number of CTG repeats and the following end-point was studied: overall survival, sudden death, conduction system disease requiring the implantation of a permanent pacemaker,
supraventricular tachyarrhythmia. 855 patients were included, with 420 males (49%) and a median age of 37 years. Over a 10 years median follow-up, 210 patients died (25%), 32 suddenly. 166 patients (19%) developed supraventricular tachyarrhythmia and 181 (21%) had a permanent pacemaker implanted. In multivariate analysis, the severity of the cardiac involvement increases with the number of CTG repeats. Patients with a larger mutation size had a worse survival (HR 1.54 IC95% [1.35–1.76] p < 0.001) and a higher incidence of all cardiac events: sudden death (HR 1.56 IC95% [1.13–2.17] p = 0.0076), supraventricular tachyarrhythmia (HR 1.20 [1.01–1.41] p = 0.0334), pacemaker (HR 1.33 [1.17– 1.52] p < 0.0001). Male gender developed more cardiac events and had a worse cardiac prognosis. The CTG repeat size is an age and gender independent factor of the cardiac severity involvement and prognosis. http://dx.doi.org/10.1016/j.nmd.2016.06.391
P.363 DM2-linked myopathy caused by uninterrupted short (CCTG)50–70 repeat expansion in CNBP T. Suominen 1, L. Bachinski 2, O. Raheem 1, H. Haapasalo 3, W. Kress 4, R. Krahe 2, B. Udd 5 1 University of Tampere, Tampere, Finland; 2 University of Texas MD Anderson Cancer Center, Houston, USA; 3 Fimlab Laboratories, Tampere University Hospital, Tampere, Finland; 4 University of Wuerzburg, Biozentrum, Am Hubland, Wuerzburg, Germany; 5 Neuromuscular Research Center, Tampere University Hospital, Tampere, Finland Myotonic dystrophy type 2 (DM2) is one of the most common adult-onset muscular dystrophies in Europe. It is a dominantly inherited multisystemic disease caused by a repeat expansion in intron 1 of CNBP gene. The diseasecausing repeat expansions consist of 75–11,000 (CCTG) repeats. In this study, we characterize a new type of DM2-linked mild myopathy in several patients without myotonia associated with very short (CCTG)50–70 repeat expansions. We have identified eleven individuals carrying a short (CCTG) repeat expansion. They have been analyzed by repeat-primed PCR (RP-PCR) and haplotype analysis. Seven has been clinically evaluated and muscle biopsy analyzed in three patients. For one of the patient, an index patient of a larger family, the mutation has been studied further by Southern blot, sequencing, allele specific expression, in situ hybridization, Western blot and analysis of fetal splice isoforms of DM2 effector proteins. Clinical features are mild, including myalgic pain, muscle weakness and stiffness. Some patients hardly had any clinical symptoms. Even though the clinical symptoms are milder than in DM2, the muscle biopsy findings show DM2-like changes, including highly atrophic type 2 fibers, nuclear clump fibers and internal nuclei. PCR-based methods and Southern blot indicated a repeat expansion of (CCTG)50–70 for the first index patient. Sequencing of the repeat region will be performed also for additional five samples. In contrast to DM2, ribonuclear foci of accumulated RNA were not detected, splicing of DM2 effector proteins was normal and CNBP protein level was normal. Finding of CNBP repeat expansion mutations containing only (CCTG)50–70 repeats in mildly symptomatic myopathy patients without myotonia suggests that even shorter than previously reported expansions may cause a disease, although with a different phenotype. The molecular findings indicate a unique pathomechanism and the term ‘myotonic dystrophy’ cannot be applied to this new disease. http://dx.doi.org/10.1016/j.nmd.2016.06.392
P.364 The brain diffusion tensor and voxel based morphometry imaging correlates with the phenotypic severity in myotonic dystrophy type 1 J. Park 1, J. Shin 2, H. Song 3, S. Lee 4, Y. Chang 3 1 Kyungpook National University School of Medicine, Daegu, Republic of Korea; 2 Pusan National University Yangsan hospital, Yangsan, Republic of Korea; 3 Kyungpook National University and Hospital, Daegu, Republic of Korea; 4 Kyungpook National University Medical Center, Daegu, Republic of Korea