- Email: [email protected]
P2.32 The frequency of myotonic dystrophy type 2 (DM2) and type 1 (DM1) mutations in the population
628
Abstracts / Neuromuscular Disorders 20 (2010) 596–680
symptoms directly related to DM1. All incident cases are invited to participate in the cohort study. Phone interviews, standardized questionnaires and reports from the primary physician are used for follow-up data. Clinical information and quality of life data is recorded in the cohort phase. Results: There were a total of 119 cases reported, with 38 confirmed as CDM. Neonatal data was available for 36 cases with a mean number of trinucleotide repeat of 1355. Twenty two of the children were the index cases for their families. The mean length of hospital stay was 48 days. Twentynine (29/36) children received naso-gastric feeding therapy for a mean of 49 days. Twenty (20/36) children received invasive ventilation for a mean of 37 days. Seven (7/36) children died during the neonatal period, four due to withdraw of life support at a mean of 28 days, two of the children died in the first 24 h with full support. Other complications included: 14/36 central nervous system complications; 10/36 cardiac complications. Eighteen children are currently enrolled in the cohort study, one child has completed the 5 year surveillance. Conclusion: Surveillance and prospective examination of CMD at a population level is important, as the impact of this rare disease is systemic, chronic and associated with significant morbidity and mortality in the newborn period. doi:10.1016/j.nmd.2010.07.103
P2.32 The frequency of myotonic dystrophy type 2 (DM2) and type 1 (DM1) mutations in the population T. Suominen 1, L.L. Bachinski 2, S. Auvinen 3, P. Hackman 4, C. Angelini 5, L. Peltonen 6, R. Krahe 2, B. Udd 7 1 University of Tampere, Neuromuscular Research Unit, Neurogenetics, Tampere, Finland, 2 University of Texas M. D. Anderson Cancer Center, Department of Genetics, Houston, United States, 3 Central Hospital of Jyväskylä, Department of Neurology, Jyväskylä, Finland, 4 University of Helsinki and Folkhälsan Institute of Genetics, Department of Medical Genetics, Helsinki, Finland, 5 University of Padova, Department of Neurosciences, Padova, Italy, 6 University of Helsinki, Department of Medical Genetics, Helsinki, Finland, 7 Tampere University Hospital and Medical School, Department of Neurology, Tampere, Finland
The prevalence of DM1 disease is estimated to be 1 in 8000 based on clinical assessment and genetic confirmation, but for DM2 the prevalence has not been established. Our clinical experience indicated that DM2 disease is at least as common as DM1 which prompted for a large scale population based study. Such studies have not been carried out to determine the frequency of the mutations. To study the frequency of DM2 and DM1 mutations we analyzed 4532 Finnish population control DNA samples obtained from anonymous blood donors, and extended this with 1003 Finnish samples consisting of 827 patients with various neuromuscular disorders without myotonia and 176 healthy relatives. We also analyzed a cohort of 93 Italian patients with undetermined proximal myopathy for DM2. ZNF9 and DMPK gene alleles were sized using PCR and fragment analysis. Samples showing only one allele were further analyzed by repeat-primed PCR to identify possible existence of expansion mutation. Inconclusive samples in the NMD cohort were sequenced. Results for allele sizes were obtained from 4511 samples for DM2 and 4520 for DM1. Two DM2 and two DM1 mutation positive samples were identified. In the NMD cohort of 991 samples with reliable results there were no DM1 mutations. Among 988 samples with reliable results for DM2, one mutation was found. In the cohort of 93 Italian proximal myopathy patients one DM2 mutation was found. Our results indicate a frequency of 1 in 1850 for DM2 and 1 in 2750 for DM1 mutations. The frequency of DM1 mutations is not
surprising as there should be at least one mutation carrier with short expansion and subclinical symptoms per symptomatic proband. The surprising result is for the DM2 mutation which is much more frequent than previously suspected and confirm our clinical experience that DM2 is more common than DM1. The results also indicate that the vast majority of DM2 patients is still not diagnosed in our clinics and that DM2 may be the most common single muscle disease doi:10.1016/j.nmd.2010.07.104
P2.33 Ribonuclear foci in differentiated tissues of patients with DM2 Z. Lukas 1, I. Falk 1, M. Falk 2, R. Hrabalkova 1, J. Zaoralkova 1 Faculty Hospital Brno, Pathology, Brno, Czech Republic, 2 Institute of Biophysics, Academy of Sciences, Czech Republic, Brno, Czech Republic 1
DM2 is caused by an expansion of CCTG repeat in the gene encoding ZNF9 protein. A model for the pathogenesis of DM2 has been proposed: the mutant gene is transcribed giving rise to a transcript that contains an expanded CCUG repeat and accumulates in the cell nucleus as RNA foci; retained expanded transcripts consequently sequester RNA binding proteins, including muscleblind protein 1(MBNL1), and alter the activity of splicing factors; this leads to an abnormal alternative splicing of a sub-group of pre-mRNAs. Expression of inappropriate splicing products is then responsible for DM2 symptoms. The presence of RNA foci in cross-striated muscle may direct the diagnosis to myotonic dystrophy, but was only rarely studied in other tissues. An importance of ribonuclear foci for the pathogenesis of DM1 and 2 in non-muscle cells is therefore largely unknown. We show here that CUG and CCUG intranuclear foci form also in vascular endothelia, smooth muscle cells of media, fibroblasts, adipocytes, Schwann and some other cells; this indicates that transcription of DMPK and ZNF9 genes and nuclear retention of their expanded transcripts take place also in differentiated non-muscle tissues, present in muscle biopsies from DM1 and DM2 patients, respectively. In addition, as confirmed by high-resolution confocal microscopy, ribonuclear foci colocalized with MBNL1 protein in tissues examined, which indicates that alteration of nuclear processes might be similar to muscle cells and that also non-muscle tissues might participate on DM pathogenesis. Interestingly, some MBNL1 intranuclear foci without RNA transcripts were detected in patients with DM2, so not all MBNL1 protein must be necessarily sequestered by expanded transcripts. It seems thus that the disturbance of normal RNA splicing and its consequences may depend on many factors, like the cell type, level of MBNL1 expression, size of CUG/CCUG expansion and other parameters. These conclusions will be discussed in our presentation doi:10.1016/j.nmd.2010.07.105
MYOTONIA; POSTER PRESENTATIONS P2.34 A myotonia caused by aberrant splicing of voltage-gated sodium channel due to an intronic mutation of SCN4A M.P. Takahashi 1, T. Kubota 1, Y. Kokunai 1, T. Kimura 2, S. Sakoda 1 1 Osaka University Graduate School of Medicine, Department of Neurology, Suita, Japan, 2 Hyogo College of Medicine, Internal Medicine (Division of Neurology), Nishinomiya, Japan
Abstracts / Neuromuscular Disorders 20 (2010) 596–680
symptoms directly related to DM1. All incident cases are invited to participate in the cohort study. Phone interviews, standardized questionnaires and reports from the primary physician are used for follow-up data. Clinical information and quality of life data is recorded in the cohort phase. Results: There were a total of 119 cases reported, with 38 confirmed as CDM. Neonatal data was available for 36 cases with a mean number of trinucleotide repeat of 1355. Twenty two of the children were the index cases for their families. The mean length of hospital stay was 48 days. Twentynine (29/36) children received naso-gastric feeding therapy for a mean of 49 days. Twenty (20/36) children received invasive ventilation for a mean of 37 days. Seven (7/36) children died during the neonatal period, four due to withdraw of life support at a mean of 28 days, two of the children died in the first 24 h with full support. Other complications included: 14/36 central nervous system complications; 10/36 cardiac complications. Eighteen children are currently enrolled in the cohort study, one child has completed the 5 year surveillance. Conclusion: Surveillance and prospective examination of CMD at a population level is important, as the impact of this rare disease is systemic, chronic and associated with significant morbidity and mortality in the newborn period. doi:10.1016/j.nmd.2010.07.103
P2.32 The frequency of myotonic dystrophy type 2 (DM2) and type 1 (DM1) mutations in the population T. Suominen 1, L.L. Bachinski 2, S. Auvinen 3, P. Hackman 4, C. Angelini 5, L. Peltonen 6, R. Krahe 2, B. Udd 7 1 University of Tampere, Neuromuscular Research Unit, Neurogenetics, Tampere, Finland, 2 University of Texas M. D. Anderson Cancer Center, Department of Genetics, Houston, United States, 3 Central Hospital of Jyväskylä, Department of Neurology, Jyväskylä, Finland, 4 University of Helsinki and Folkhälsan Institute of Genetics, Department of Medical Genetics, Helsinki, Finland, 5 University of Padova, Department of Neurosciences, Padova, Italy, 6 University of Helsinki, Department of Medical Genetics, Helsinki, Finland, 7 Tampere University Hospital and Medical School, Department of Neurology, Tampere, Finland
The prevalence of DM1 disease is estimated to be 1 in 8000 based on clinical assessment and genetic confirmation, but for DM2 the prevalence has not been established. Our clinical experience indicated that DM2 disease is at least as common as DM1 which prompted for a large scale population based study. Such studies have not been carried out to determine the frequency of the mutations. To study the frequency of DM2 and DM1 mutations we analyzed 4532 Finnish population control DNA samples obtained from anonymous blood donors, and extended this with 1003 Finnish samples consisting of 827 patients with various neuromuscular disorders without myotonia and 176 healthy relatives. We also analyzed a cohort of 93 Italian patients with undetermined proximal myopathy for DM2. ZNF9 and DMPK gene alleles were sized using PCR and fragment analysis. Samples showing only one allele were further analyzed by repeat-primed PCR to identify possible existence of expansion mutation. Inconclusive samples in the NMD cohort were sequenced. Results for allele sizes were obtained from 4511 samples for DM2 and 4520 for DM1. Two DM2 and two DM1 mutation positive samples were identified. In the NMD cohort of 991 samples with reliable results there were no DM1 mutations. Among 988 samples with reliable results for DM2, one mutation was found. In the cohort of 93 Italian proximal myopathy patients one DM2 mutation was found. Our results indicate a frequency of 1 in 1850 for DM2 and 1 in 2750 for DM1 mutations. The frequency of DM1 mutations is not
surprising as there should be at least one mutation carrier with short expansion and subclinical symptoms per symptomatic proband. The surprising result is for the DM2 mutation which is much more frequent than previously suspected and confirm our clinical experience that DM2 is more common than DM1. The results also indicate that the vast majority of DM2 patients is still not diagnosed in our clinics and that DM2 may be the most common single muscle disease doi:10.1016/j.nmd.2010.07.104
P2.33 Ribonuclear foci in differentiated tissues of patients with DM2 Z. Lukas 1, I. Falk 1, M. Falk 2, R. Hrabalkova 1, J. Zaoralkova 1 Faculty Hospital Brno, Pathology, Brno, Czech Republic, 2 Institute of Biophysics, Academy of Sciences, Czech Republic, Brno, Czech Republic 1
DM2 is caused by an expansion of CCTG repeat in the gene encoding ZNF9 protein. A model for the pathogenesis of DM2 has been proposed: the mutant gene is transcribed giving rise to a transcript that contains an expanded CCUG repeat and accumulates in the cell nucleus as RNA foci; retained expanded transcripts consequently sequester RNA binding proteins, including muscleblind protein 1(MBNL1), and alter the activity of splicing factors; this leads to an abnormal alternative splicing of a sub-group of pre-mRNAs. Expression of inappropriate splicing products is then responsible for DM2 symptoms. The presence of RNA foci in cross-striated muscle may direct the diagnosis to myotonic dystrophy, but was only rarely studied in other tissues. An importance of ribonuclear foci for the pathogenesis of DM1 and 2 in non-muscle cells is therefore largely unknown. We show here that CUG and CCUG intranuclear foci form also in vascular endothelia, smooth muscle cells of media, fibroblasts, adipocytes, Schwann and some other cells; this indicates that transcription of DMPK and ZNF9 genes and nuclear retention of their expanded transcripts take place also in differentiated non-muscle tissues, present in muscle biopsies from DM1 and DM2 patients, respectively. In addition, as confirmed by high-resolution confocal microscopy, ribonuclear foci colocalized with MBNL1 protein in tissues examined, which indicates that alteration of nuclear processes might be similar to muscle cells and that also non-muscle tissues might participate on DM pathogenesis. Interestingly, some MBNL1 intranuclear foci without RNA transcripts were detected in patients with DM2, so not all MBNL1 protein must be necessarily sequestered by expanded transcripts. It seems thus that the disturbance of normal RNA splicing and its consequences may depend on many factors, like the cell type, level of MBNL1 expression, size of CUG/CCUG expansion and other parameters. These conclusions will be discussed in our presentation doi:10.1016/j.nmd.2010.07.105
MYOTONIA; POSTER PRESENTATIONS P2.34 A myotonia caused by aberrant splicing of voltage-gated sodium channel due to an intronic mutation of SCN4A M.P. Takahashi 1, T. Kubota 1, Y. Kokunai 1, T. Kimura 2, S. Sakoda 1 1 Osaka University Graduate School of Medicine, Department of Neurology, Suita, Japan, 2 Hyogo College of Medicine, Internal Medicine (Division of Neurology), Nishinomiya, Japan