G.P.21

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

Abstracts / Neuromuscular Disorders 24 (2014) 791–924 1 % in normal population. With this method we managed to find the causative gene in four patients. Among these were the first PFKM, COL6A2 and CACNA1S mutations identified in Finland. One patient had compound heterozygous mutations in NEB gene. For three patients a probable causative gene was found but further evaluations are needed to confirm the findings. In the rest of the patients there were a number of possible causative mutations. However, none of the variants identified were known disease causing mutations nor were any of the genes obviously a better candidate then the others. By exome sequencing we were able to solve 30% of our cases. It seems that whole exome sequencing may be really effective but in most cases selecting the right mutation from the list of many seems to require further studies. http://dx.doi:10.1016/j.nmd.2014.06.035

G.P.22 Utilising next-generation sequencing to determine the genetic basis of recurrent rhabdomyolysis R.S. Scalco 1, R.D.S. Pitceathly 1, A. Gardiner 1, C. Woodward 1, J.M. Polke 1, M.G. Sweeney 1, S.E. Olpin 2, R. Kirk 2, E. Murphy 1, D. Hilton-Jones 3, H. Jungbluth 4, H. Houlden 1, M.G. Hanna 1, R. Quinlivan 1, R.S.G. Rhabdomyolysis Study Group 5 1 UCL Institute of Neurology, National Hospital for Neurology and Neurosurgery, Queen Square, London, UK; 2 Sheffield Children’s NHS Foundation Trust, Sheffield, UK; 3 John Radcliffe Hospital, Oxford, UK; 4 Evelina Childrens Hospital, Guy’s & St. Thomas’ NHS Foundation Trust, London, UK; 5 International Collaboration, UK Recurrent rhabdomyolysis complicates a number of inherited muscle and metabolic disorders and represents a serious, potentially life-threatening condition which frequently requires critical care. Identification of the underlying genetic cause has traditionally relied upon detailed history and examination findings which subsequently guide the investigative work-up. However, in many cases the causative molecular defect remains undetermined. This study aims to investigate whether utilising next-generation sequencing (NGS) technology early in the diagnostic pathway might offer a rapid, cost-effective tool for the diagnosis of patients with recurrent attacks of rhabdomyolysis when a genetic aetiology is suspected. We have designed a “rhabdomyolysis gene panel” comprised of 48 genes known or predicted to cause rhabdomyolysis using NGS technology. Over 200 patients have been recruited. In addition, array CGH and whole exome sequencing may be used. A pilot study of 53 patients with a panel of sequenced 35 rhabdomyolysis genes using an amplicon based sequencing panel on an Illumina MiSeq was performed. 52 of the first 53 first evaluated patients have a variant in at least 1 gene. 49 patients have heterozygous variants in at least two different genes. We identified 15 cases out of 52 with probable pathogenic mutations using this approach. The pilot study showed that the rhabdomyolysis genetic panel is a potentially useful way to identify genetic alterations in patients with rhabdomyolysis. The high number of symptomatic patients with mutations identified in more than one gene associated with rhabdomyolysis suggests that gene–gene interaction(s) may play an important role. We are currently preparing a new extended panel of 48 genes. http://dx.doi:10.1016/j.nmd.2014.06.036

G.P.23 Diagnostic application of targeted NGS in early onset myopathies A. Cho 1, V. Vasta 2, B.C. Lim 3, J.S. Lee 3, S.H. Eun 4, K.J. Kim 3, Y.S. Hwang 3, S. Hahn 2, J.H. Chae 3 1 Ewha Womans University School of Medicine, Seoul, Republic of Korea; 2 Seattle Children’s Hospital, Seattle, WA, USA; 3 Seoul National

University Children’s Hospital, Seoul, Republic of Korea; Ansan Hospital, Gyeonggi-do, Republic of Korea

801 4

University

Congenital myopathies and congenital muscular dystrophies are groups of clinically, pathologically, and genetically heterogeneous disorders. Even for the experienced clinicians, an accurate genetic diagnosis has often been challenging due to the heterogeneity and complexity of these groups of disorders. One gene can cause a wide variety of clinical and/or pathological features, while similar clinical features can be caused by mutations in different genes. Since next generation sequencing (NGS) is an effective diagnostic tool for the parallel investigation of a large number of genes, it has been increasingly used in recent clinical practices to diagnose these genetically and phenotypically heterogeneous diseases. Here, we present the result of a targeted NGS panel analysis in early onset myopathies. We selected 703 known pathogenic genes causing congenital myopathies, congenital muscular dystrophies, metabolic and mitochondrial myopathies, distal myopathies, channelopathies, neuromuscular junction disorders, and diseases of peripheral nerve. Total 42 infants or children with early onset (<5 years) hypotonia and/or weakness were included and 14 patients (33.3%) has been genetically confirmed up to date: COL6A1 (5), COL6A3 (1), LMNA (3), ACTA1 (2), MTM1 (1), DOK7 (1), and GARS (1). Our results suggest that targeted NGS has a significant potential to synergy with clinical and pathological analysis for an effective diagnosis of primary myopathies. http://dx.doi:10.1016/j.nmd.2014.06.037

G.P.24 Cardiomyopathy in childhood: Results from a single tertiary care center E. Batu, M. Pehlivanturk, G. Haliloglu, E. Utine, N. Ulgen Tekerek, F. Kara Eroglu, I. Ertugrul, G. Hizal, K. Boduroglu, H. Topaloglu, T. Coskun, H. Ozen, G. Kale, D. Alehan Hacettepe University Children’s Hospital, Ankara, Turkey Cardiomyopathy (CMP) in childhood is an etiologically heterogeneous group of cardiac disease, including many genetic, metabolic, neuromuscular and environmental causes. The goal of this study is to determine the underlying causes and clinical characteristics of children presenting with CMP to a tertiary medical center in Turkey. We analyzed the data of 109 patients retrospectively, who presented with CMP as an initial feature, and without a specific diagnosis, between May 2007 and May 2012. Patients who already developed CMP during the course of a diagnosed disease were excluded from the study. Among 109 patients, 57 were male, and 52 were female. Most of the patients were symptomatic (n = 59) in the first year of life (54.1%). The patients were subdivided into three groups, as dilated CMP (n = 69, 63.3%), hypertrophic CMP (n = 32, 9.4%), and restrictive CMP (n = 8, 7.3%). The etiology remained unknown in 68.8% of all cases. Inborn errors of metabolism (n = 16) and cardiac diseases (n = 10) comprised the main etiological diagnosis, while dysmorphic syndromes (n = 4), immunological diseases (n = 2), neuromuscular diseases (n = 2) were responsible from minority of cases. Cardiac transplantation was possible for 4.6% of patients (n = 5), whereas 29.4% of all patients died during a median of 13-month follow-up. In the era of advanced molecular genetic testing, approach to CMP in childhood is still challenging, and 57–68% patients remain idiopathic. The reported success rate of finding an etiology of CMP is around 30% in literature, and 31.2% in this group. This study highlights the importance of detailed clinical evaluation as a first-step. http://dx.doi:10.1016/j.nmd.2014.06.038