- Email: [email protected]
G.P.18
Abstracts / Neuromuscular Disorders 24 (2014) 791–924 Outcomes (including a 3 Tesla quantitative MR imaging of the upper leg muscles) were assessed at baseline, immediately following 16 weeks of intervention, and after a 12-week follow-up. A linear mixed model for repeated measurements was used to study the estimated group differences. Following treatment, both the AET and CBT intervention groups had significantly less fatigue relative to the UC group, with a difference of 9.1 for AET (95%CI: 12.4 to 5.8) and 13.3 for CBT (95%CI: 16.5 to 10.2). These beneficial effects lasted through the follow-up period, with a difference of 8.2 for AET (95%CI: 12.4 to 5.8) and 10.2 for CBT (95%CI 16.5 to 10.1). The patients who received CBT had an increase in registered and experienced physical activity, sleep quality, and social participation. The patients who received AET had an increase in registered physical activity only. The increase in registered physical activity in both groups and the improvement in social participation following CBT were still present at follow-up. MRI determined fat fractions showed decelerated progression of fatty infiltration, most prominently in the adductor magnus muscle. In this muscle fat percentage increased with 1.56% monthly (UC) with a difference of 1.21 for AET (95%CI: 1.99 to 0.34) and 1.73 for CBT (95%CI: 2.56 to 0.87). This is the first RCT showing that chronic fatigue can be ameliorated in patients with a muscular dystrophy. AET and/or CBT reduce fatigue and slow progression of fatty infiltration in patients with FSHD. http://dx.doi:10.1016/j.nmd.2014.06.029
G.P.16 MR-guided muscle biopsy: A novel technique for the collection of muscle biopsies S. Lassche, B.H. Janssen, N.C. Voermans, J.J. Futterer, B.G.M. van Engelen Radboud University Medical Center, Nijmegen, Netherlands Muscle biopsy remains an important procedure in the diagnosis and research of muscle disorders. Traditional methods such as open or Bergstro¨m needle biopsy can be hampered by sampling error, especially in multifocal disorders such as inflammatory myopathies and facioscapulohumoral muscular dystrophy. Here we describe a novel technique to accurately obtain affected muscle tissue using a MR-guided approach. MR-guided biopsy of the vastus lateralis muscle was performed in 10 FSHD patients with varying degrees of disease severity. Fatty infiltration of vastus lateralis ranged from <5% to >90% of muscle area. The targeted area of muscle biopsy and needle trajectory were determined by an experienced neurologist and intervention radiologist using 3D T1 images. A MR-compatible needle was placed, after which another series of 3D T1 images were made to confirm that the needle was inserted at the region of interest. A vacuum-assisted biopsy was taken and a verification image with the stylet left in situ obtained. Despite the large amounts of fatty infiltration in some participants, muscle tissue was successfully acquired in all patients. A small non-symptomatic intramuscular hematoma (12 24 mm) was observed in 1 patient, a large symptomatic intramuscular hematoma (32 44 mm) was observed in another. However, in this patient a very lateral biopsy site was chosen, and the increased vascular supply to this area likely increased the risk of bleeding. MR-guided muscle biopsy is a safe and feasible method which enables the collection of muscle tissue from specific regions of interest, e.g. focal areas of inflammation. MRguided biopsy can be performed in all muscles, however we advise to consider local vascular and nerve supply when choosing a target biopsy site. A titanium marker can be left in situ to perform follow-up imaging and/or biopsies of the same target area. Although not part of the
799
current study, the technique can also be used to deliver therapeutic compounds. http://dx.doi:10.1016/j.nmd.2014.06.030
SEQUENCING AND NEUROMUSCULAR STUDIES G.P.17 TTN a challenge for next generation sequencing P. Hackman 1, A. Evila¨ 1, B. Udd 2 1 Folkha¨lsan Institute of Genetics/University of Helsinki, Helsinki, Finland; 2 Tampere University Hospital, Tampere, Finland Tibial muscular dystrophy (TMD) is caused by mutations in the TTN gene. TTN is transcribing a >100 kb long mRNA, coding for the sarcomeric, largest known human protein, titin, spanning one half sarcomere in striated muscle. Titin (TTN) binds many other proteins and has important structural, mechanical and regulatory roles in muscles. The first TMD causing mutation, FINmaj a deletion/insertion of eleven base pairs, in the last 363:rd exon, Mex6, was reported by our group in 2002. Several different TTN mutations in the last two exons causing TMD have since been reported in European families. Due to its giant size and complexity analysis of TTN by conventional Sanger sequencing has been difficult and laborous. Usually only certain selected parts of the gene has been screened. Therefore the general spectrum and incidence of titinopathies has been underestimated. Next generation sequencing (NGS) methods have enabled an extensive increase in identification of mutations, and more than 120 TTN mutations have been reported in patients with at least 10 different conditions. In addition to using exome sequencing we have developed a targeted NGS custom panel, MyoCap, with the coding exons and UTRs of 236 muscular dystrophy related genes, including all exons of TTN. Due to the giant size of the gene, rare and novel TTN variants are now identified in many individuals analysed. Thus we have found more than ten patients with previously un-described phenotypes, with potential disease causing variants. This puts challenges in assessing the relevance and real correlation of these mutations with a certain disease phenotype. Since TTN is very repetitive, some regions are not well covered by NGS techniques. Insertion/deletion mutations may be missed in the variant calling. All this adds challenges to the development of the NGS methods and analysis of the results. Moreover, usage of the NGS for TTN requires large repertoire of RNA and protein techniques for the assessment of new variants. http://dx.doi:10.1016/j.nmd.2014.06.031
G.P.18 Neurogenetic disease diagnostics by targeted capture and next generation sequencing K. Yau 1, R. Allcock 2, K. Mina 3, G. Ravenscroft 1, M. Cabrera 1, R. Gooding 3, C. Wise 3, P. Sivadorai 3, D. Trajanoski 3, V. Atkinson 2, S. Wagner 2, K. Nowak 1, R. Duff 1, P. Lamont 4, M. Davis 3, N. Laing 1,2,3,4 1 Centre for Medical Research, University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Australia; 2 Lotterywest State Biomedical Facility Genomics and School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia; 3 Neuromusculars Unit, Department of Diagnostic Genomics, PathWest Laboratory Medicine, Department of Health, Western Australia, Perth, Australia; 4 Neuromuscular Unit, Royal Perth Hospital, Western Australia, Perth, Australia
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Abstracts / Neuromuscular Disorders 24 (2014) 791–924
Two problems for molecular diagnosis of neurogenetic disorders are high levels of genetic heterogeneity and involvement of large genes, e.g. titin. These problems result in many patients not being diagnosed by current Sanger-sequencing based diagnostics. To offset these problems we have developed a TargetSeq (Life Technologies) capture panel consisting of 335 disease genes with mutations detectable by next generation sequencing (NGS). The genes were 276 neurogenetic and 59 cardiomyopathy genes, targeted together because of clinical overlap. The 335 panel genes were captured from pools of 16 bar-coded patient DNA samples and sequenced 16 patients at a time using Ion Protone (Life Technologies) sequencing. Variant analysis was by either custom Annovar or Cartagenia (Cartagenia, Inc.) based pipelines. Testing the panel identified 88% of the known small-scale mutations in 28 positive controls. Known mutations deliberately chosen in regions not sequenced well by NGS were not detected. Using the FishingCNV package, we identified 90% of CNV controls, including 100% of CMT1A/HNPP duplication/deletion controls. Although one single exon CNV was detected, not all small CNVs were detected. We have screened >300 molecularly undiagnosed probands, identifying mutations in 59 disease genes, many of which were not previously analysed by the diagnostic laboratory due to Sanger sequencing costs. Using the targeted panel is not ethically different to traditional sequential Sanger sequencing diagnostics. The panel needs updating regularly with new disease gene discoveries. We now receive samples from all round Australasia for analysis of the 335-gene panel, which is cheaper than Sanger. http://dx.doi:10.1016/j.nmd.2014.06.032
G.P.19 Global gene expression profile in different forms of murine muscular dystrophies C.F. Almeida, P.C.M. Martins, P.C.G. Onofre-Oliveira, M. Vainzof Genetics and Evolutionary Biology Department, Institute of Biosciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil The muscular dystrophies are a clinically and histologically well characterized group of genetic diseases, with predominantly monogenic etiologies. Many causative gene mutations have been already identified, but the underlying pathophysiological pathways and phenotypic variability in each form are much more complex, suggesting the involvement of many other genes. Thus, study the whole genome expression profile can significantly contribute to: identify altered biological functions which could contribute to the understanding of the disease; identify possible prognostic biomarkers and find points for therapeutic intervention. Here, we present the first global gene expression analysis of the muscle of MD mouse models: Largemyd (CMD1D), Dmdmdx/Largemyd (double mutant for DMD and CMD1D) compared to Dmdmdx (model for Duchenne MD) and normal C57BL, in three different ages: 21 days, 3 and 6 months. Five animals from each group were analyzed using DNA microarrays. The data were preprocessed and normalized in Expression Console software and screening of DEGs was done with SAM method. In general, a predominance of upregulated than downregulated genes was observed in the dystrophic muscles, compatible with high rates of protein turnover that could occur due to the subsequent activation of regeneration process. The Largemyd and Dmdmdx/Largemyd mice present a progressive increase in the number of differentially expressed genes (DEGs) with aging. The analysis done between Dmdmdx/Largemyd and its parental lineages Dmdmdx and Largemyd showed the smallest number of DEGs, suggesting that the reason of its worst phenotype is controlled by the expression of a few but very important genes. Preliminary functional analyses in IPA and Expander softwares showed a high proportion of genes involved in immune and inflammatory
pathways in all lineages that could be explained by the intense dystrophic process. http://dx.doi:10.1016/j.nmd.2014.06.033
G.P.20 A targeted next-generation sequencing panel for diagnostic use in primary myopathies A. Evila 1, B. Udd 2, P. Hackman 1 1 Folkhalsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland; 2 Neuromuscular Research Center, University of Tampere and Tampere University Hospital, Tampere, Finland Myopathies are a clinically and genetically heterogeneous group of disorders that cause progressive weakness and atrophy of muscles. Mutations in more than 200 different genes are known to cause many of these disorders and in less obvious disorders several genes may have to be sequenced in order to identify the correct diagnosis in a patient. The large number of possible candidate genes, overlapping phenotypes as well as an enormous size of some of the genes e.g. DMD, TTN and NEB constitute difficult challenges for clinical diagnostics. Molecular characterization is nevertheless important for the final diagnosis and accurate management of the diseases. Targeted next-generation sequencing (NGS) is an efficient and cost-effective method to sequence several genes simultaneously. A targeted NGS custom panel with a total size of 1.3 Mb was designed for the coding exons and UTRs of 180 myopathy related genes. For DNA capture a custom NimbleGen SeqCap EZ Choice Library was designed. Sequencing was performed at The Institute for Molecular Medicine Finland (FIMM) using Illumina HiSeq 1500 with sequencing depth of 100, and files were processed with their Variant Calling Pipeline. In this part of the study DNA samples of 67 patients negative for previous candidate gene approaches were sequenced. According to preliminary results definite diagnosis based on disease-causing mutations was obtained directly in 15 patients and probable disease-causing mutations were found in another 12 patients. Ten patients had potential diseasecausing TTN mutations with previously undescribed phenotypes and the in depth study of the results is still ongoing. http://dx.doi:10.1016/j.nmd.2014.06.034
G.P.21 Whole exome sequencing as a diagnostic tool in neuromuscular disorders S. Penttila¨ 1, B. Udd 2 1 University of Tampere, Tampere, Finland; 2 University and University Hospital of Tampere, Tampere, Finland Diagnosing neuromuscular disorders is often very challenging. The phenotypes of the diseases are often overlapping and furthermore, the symptoms of the patient may be more or less atypical. In normal diagnostic procedure the clinical evaluation and muscle biopsy, MRI and ENMG findings lead to a suspicion of a certain genetic background and genetic testing. Sometimes, however, the clinical findings are too indefinite or deceptive for defining which genetic test(s) should be performed. Furthermore, quite often the number of possible disease causing genes is too high and/or the size of them too big for traditional genetic testing. In these cases next generation sequencing seems to be an adequate tool for finding the disease causing gene. In our study we performed whole exome sequencing for 12 NMD-patients whose diagnose had remained unsolved despite extensive studies. From the sequencing data we extracted variants lying in candidate genes compatible with the phenotype of the patient. Of these variants were further selected those that had a frequency lower than
G.P.16 MR-guided muscle biopsy: A novel technique for the collection of muscle biopsies S. Lassche, B.H. Janssen, N.C. Voermans, J.J. Futterer, B.G.M. van Engelen Radboud University Medical Center, Nijmegen, Netherlands Muscle biopsy remains an important procedure in the diagnosis and research of muscle disorders. Traditional methods such as open or Bergstro¨m needle biopsy can be hampered by sampling error, especially in multifocal disorders such as inflammatory myopathies and facioscapulohumoral muscular dystrophy. Here we describe a novel technique to accurately obtain affected muscle tissue using a MR-guided approach. MR-guided biopsy of the vastus lateralis muscle was performed in 10 FSHD patients with varying degrees of disease severity. Fatty infiltration of vastus lateralis ranged from <5% to >90% of muscle area. The targeted area of muscle biopsy and needle trajectory were determined by an experienced neurologist and intervention radiologist using 3D T1 images. A MR-compatible needle was placed, after which another series of 3D T1 images were made to confirm that the needle was inserted at the region of interest. A vacuum-assisted biopsy was taken and a verification image with the stylet left in situ obtained. Despite the large amounts of fatty infiltration in some participants, muscle tissue was successfully acquired in all patients. A small non-symptomatic intramuscular hematoma (12 24 mm) was observed in 1 patient, a large symptomatic intramuscular hematoma (32 44 mm) was observed in another. However, in this patient a very lateral biopsy site was chosen, and the increased vascular supply to this area likely increased the risk of bleeding. MR-guided muscle biopsy is a safe and feasible method which enables the collection of muscle tissue from specific regions of interest, e.g. focal areas of inflammation. MRguided biopsy can be performed in all muscles, however we advise to consider local vascular and nerve supply when choosing a target biopsy site. A titanium marker can be left in situ to perform follow-up imaging and/or biopsies of the same target area. Although not part of the
799
current study, the technique can also be used to deliver therapeutic compounds. http://dx.doi:10.1016/j.nmd.2014.06.030
SEQUENCING AND NEUROMUSCULAR STUDIES G.P.17 TTN a challenge for next generation sequencing P. Hackman 1, A. Evila¨ 1, B. Udd 2 1 Folkha¨lsan Institute of Genetics/University of Helsinki, Helsinki, Finland; 2 Tampere University Hospital, Tampere, Finland Tibial muscular dystrophy (TMD) is caused by mutations in the TTN gene. TTN is transcribing a >100 kb long mRNA, coding for the sarcomeric, largest known human protein, titin, spanning one half sarcomere in striated muscle. Titin (TTN) binds many other proteins and has important structural, mechanical and regulatory roles in muscles. The first TMD causing mutation, FINmaj a deletion/insertion of eleven base pairs, in the last 363:rd exon, Mex6, was reported by our group in 2002. Several different TTN mutations in the last two exons causing TMD have since been reported in European families. Due to its giant size and complexity analysis of TTN by conventional Sanger sequencing has been difficult and laborous. Usually only certain selected parts of the gene has been screened. Therefore the general spectrum and incidence of titinopathies has been underestimated. Next generation sequencing (NGS) methods have enabled an extensive increase in identification of mutations, and more than 120 TTN mutations have been reported in patients with at least 10 different conditions. In addition to using exome sequencing we have developed a targeted NGS custom panel, MyoCap, with the coding exons and UTRs of 236 muscular dystrophy related genes, including all exons of TTN. Due to the giant size of the gene, rare and novel TTN variants are now identified in many individuals analysed. Thus we have found more than ten patients with previously un-described phenotypes, with potential disease causing variants. This puts challenges in assessing the relevance and real correlation of these mutations with a certain disease phenotype. Since TTN is very repetitive, some regions are not well covered by NGS techniques. Insertion/deletion mutations may be missed in the variant calling. All this adds challenges to the development of the NGS methods and analysis of the results. Moreover, usage of the NGS for TTN requires large repertoire of RNA and protein techniques for the assessment of new variants. http://dx.doi:10.1016/j.nmd.2014.06.031
G.P.18 Neurogenetic disease diagnostics by targeted capture and next generation sequencing K. Yau 1, R. Allcock 2, K. Mina 3, G. Ravenscroft 1, M. Cabrera 1, R. Gooding 3, C. Wise 3, P. Sivadorai 3, D. Trajanoski 3, V. Atkinson 2, S. Wagner 2, K. Nowak 1, R. Duff 1, P. Lamont 4, M. Davis 3, N. Laing 1,2,3,4 1 Centre for Medical Research, University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Australia; 2 Lotterywest State Biomedical Facility Genomics and School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia; 3 Neuromusculars Unit, Department of Diagnostic Genomics, PathWest Laboratory Medicine, Department of Health, Western Australia, Perth, Australia; 4 Neuromuscular Unit, Royal Perth Hospital, Western Australia, Perth, Australia
800
Abstracts / Neuromuscular Disorders 24 (2014) 791–924
Two problems for molecular diagnosis of neurogenetic disorders are high levels of genetic heterogeneity and involvement of large genes, e.g. titin. These problems result in many patients not being diagnosed by current Sanger-sequencing based diagnostics. To offset these problems we have developed a TargetSeq (Life Technologies) capture panel consisting of 335 disease genes with mutations detectable by next generation sequencing (NGS). The genes were 276 neurogenetic and 59 cardiomyopathy genes, targeted together because of clinical overlap. The 335 panel genes were captured from pools of 16 bar-coded patient DNA samples and sequenced 16 patients at a time using Ion Protone (Life Technologies) sequencing. Variant analysis was by either custom Annovar or Cartagenia (Cartagenia, Inc.) based pipelines. Testing the panel identified 88% of the known small-scale mutations in 28 positive controls. Known mutations deliberately chosen in regions not sequenced well by NGS were not detected. Using the FishingCNV package, we identified 90% of CNV controls, including 100% of CMT1A/HNPP duplication/deletion controls. Although one single exon CNV was detected, not all small CNVs were detected. We have screened >300 molecularly undiagnosed probands, identifying mutations in 59 disease genes, many of which were not previously analysed by the diagnostic laboratory due to Sanger sequencing costs. Using the targeted panel is not ethically different to traditional sequential Sanger sequencing diagnostics. The panel needs updating regularly with new disease gene discoveries. We now receive samples from all round Australasia for analysis of the 335-gene panel, which is cheaper than Sanger. http://dx.doi:10.1016/j.nmd.2014.06.032
G.P.19 Global gene expression profile in different forms of murine muscular dystrophies C.F. Almeida, P.C.M. Martins, P.C.G. Onofre-Oliveira, M. Vainzof Genetics and Evolutionary Biology Department, Institute of Biosciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil The muscular dystrophies are a clinically and histologically well characterized group of genetic diseases, with predominantly monogenic etiologies. Many causative gene mutations have been already identified, but the underlying pathophysiological pathways and phenotypic variability in each form are much more complex, suggesting the involvement of many other genes. Thus, study the whole genome expression profile can significantly contribute to: identify altered biological functions which could contribute to the understanding of the disease; identify possible prognostic biomarkers and find points for therapeutic intervention. Here, we present the first global gene expression analysis of the muscle of MD mouse models: Largemyd (CMD1D), Dmdmdx/Largemyd (double mutant for DMD and CMD1D) compared to Dmdmdx (model for Duchenne MD) and normal C57BL, in three different ages: 21 days, 3 and 6 months. Five animals from each group were analyzed using DNA microarrays. The data were preprocessed and normalized in Expression Console software and screening of DEGs was done with SAM method. In general, a predominance of upregulated than downregulated genes was observed in the dystrophic muscles, compatible with high rates of protein turnover that could occur due to the subsequent activation of regeneration process. The Largemyd and Dmdmdx/Largemyd mice present a progressive increase in the number of differentially expressed genes (DEGs) with aging. The analysis done between Dmdmdx/Largemyd and its parental lineages Dmdmdx and Largemyd showed the smallest number of DEGs, suggesting that the reason of its worst phenotype is controlled by the expression of a few but very important genes. Preliminary functional analyses in IPA and Expander softwares showed a high proportion of genes involved in immune and inflammatory
pathways in all lineages that could be explained by the intense dystrophic process. http://dx.doi:10.1016/j.nmd.2014.06.033
G.P.20 A targeted next-generation sequencing panel for diagnostic use in primary myopathies A. Evila 1, B. Udd 2, P. Hackman 1 1 Folkhalsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland; 2 Neuromuscular Research Center, University of Tampere and Tampere University Hospital, Tampere, Finland Myopathies are a clinically and genetically heterogeneous group of disorders that cause progressive weakness and atrophy of muscles. Mutations in more than 200 different genes are known to cause many of these disorders and in less obvious disorders several genes may have to be sequenced in order to identify the correct diagnosis in a patient. The large number of possible candidate genes, overlapping phenotypes as well as an enormous size of some of the genes e.g. DMD, TTN and NEB constitute difficult challenges for clinical diagnostics. Molecular characterization is nevertheless important for the final diagnosis and accurate management of the diseases. Targeted next-generation sequencing (NGS) is an efficient and cost-effective method to sequence several genes simultaneously. A targeted NGS custom panel with a total size of 1.3 Mb was designed for the coding exons and UTRs of 180 myopathy related genes. For DNA capture a custom NimbleGen SeqCap EZ Choice Library was designed. Sequencing was performed at The Institute for Molecular Medicine Finland (FIMM) using Illumina HiSeq 1500 with sequencing depth of 100, and files were processed with their Variant Calling Pipeline. In this part of the study DNA samples of 67 patients negative for previous candidate gene approaches were sequenced. According to preliminary results definite diagnosis based on disease-causing mutations was obtained directly in 15 patients and probable disease-causing mutations were found in another 12 patients. Ten patients had potential diseasecausing TTN mutations with previously undescribed phenotypes and the in depth study of the results is still ongoing. http://dx.doi:10.1016/j.nmd.2014.06.034
G.P.21 Whole exome sequencing as a diagnostic tool in neuromuscular disorders S. Penttila¨ 1, B. Udd 2 1 University of Tampere, Tampere, Finland; 2 University and University Hospital of Tampere, Tampere, Finland Diagnosing neuromuscular disorders is often very challenging. The phenotypes of the diseases are often overlapping and furthermore, the symptoms of the patient may be more or less atypical. In normal diagnostic procedure the clinical evaluation and muscle biopsy, MRI and ENMG findings lead to a suspicion of a certain genetic background and genetic testing. Sometimes, however, the clinical findings are too indefinite or deceptive for defining which genetic test(s) should be performed. Furthermore, quite often the number of possible disease causing genes is too high and/or the size of them too big for traditional genetic testing. In these cases next generation sequencing seems to be an adequate tool for finding the disease causing gene. In our study we performed whole exome sequencing for 12 NMD-patients whose diagnose had remained unsolved despite extensive studies. From the sequencing data we extracted variants lying in candidate genes compatible with the phenotype of the patient. Of these variants were further selected those that had a frequency lower than