Next generation sequencing technologies in the genetic diagnosis of congenital myasthenic syndrome

S196

Abstracts 2017 / Neuromuscular Disorders 27 (2017) S96–S249

P.330 A national French consensus on gene lists for NGS-based diagnosis of myopathies M. Krahn 1, V. Biancalana 2, L. Michel-Calemard 3, J. Nectoux 4, F. Leturcq 4, C. Bouchet Seraphin 5, C. Bourdain-Acquaviva 6, R. Froissart 6, J. Melki 7, J. Urtizberea 8, A. Molon 9, E. Campana-Salort 9, J. Pouget 9, J. Rendu 10, F. Petit 11, C. Metay 12, N. Seta 5, D. Sternberg 12, J. Faure 10, M. Cossée 13 1 Aix Marseille Univ, Inserm, GMGF, Marseille, France; 2 Diagnostic Genetic Laboratory, New Civil Hospital, Regional University Hospital Center, Strasbourg, France; 3 Service d’Endocrinologie Moléculaire et Maladies Rares, Centre de Biologie et Pathologie Est, Bron, France; 4 APHP, Service de Biochimie et Génétique Moléculaire, Cochin Hospital, Paris, France; 5 AP-HP, Hôpital Bichat Claude Bernard, Département de Génétique, Paris, France; 6 Service Maladies Héréditaires du Métabolisme et Dépistage Néonatal, Centre de Biologie et de Pathologie Est CHU de Lyon, Lyon, France; 7 Inserm UMR-1169, Université Paris Sud, Paris, France; 8 Hôpital Marin Centre Neuromusculaire GNMH, Henday, France; 9 APHM, Hôpital La Timone, Centre de référence des maladies neuromusculaires et de la SLA, Marseille, France; 10 Centre Hospitalier Régional Universitaire de Grenoble, Hôpital Michallon, Biochimie Génétique et Moléculaire, Grenoble, France; 11 Laboratoire de Génétique moléculaire, APHP – GH Antoine Béclère, Clamart, France; 12 Centre de génétique moléculaire et chromosomique AP-HP, Hôpital Pitié-Salpêtrière, Paris, France; 13 CHRU Montpellier, Laboratoire de Génétique moléculaire, Montpellier, France More than 200 genes have been reported to date as being implicated in myopathies. The use of Next-generation sequencing (NGS) for diagnostics allows for simultaneous analysis of lists of genes (gene-panels). In the field of myopathies, strategies set up at the international level are currently extremely diverse, which complicates inter-laboratory exchange of data and expertise, and blurs the visibility of the diagnostic offers available for referring neurologists (“Where do I have to send my sample for which indication?”). Through the genetic diagnosis section of the french national network for rare neuromuscular diseases (Filière Nationale des Maladies Rares Neuromusculaires, FILNEMUS), we initiated in 2016 a nationwide working-group with the objective of homogenizing the genetic diagnosis strategies using NGS for myopathies at the national level. A consultation of the nine french genetic diagnosis laboratories using NGS for myopathies allowed identifying consensual key points as the foundation for a nationwide homogenization. A consensual diagnostic strategy has been set up, based on the determination of 14 clinical entry-diagnosis groups. For each group, the strategy consists either in the NGS-analysis of a unique exhaustive gene list, or a successive NGS-analysis of a “main-genes” list followed in case of a negative result by the analysis of an “exhaustive” gene list. This consensual strategy has been validated by an expert clinician group, and is currently being adopted by all implicated laboratories, which will declare their respective lists analyzed, on the FILNEMUS website, therefore clarifying the nationwide diagnostic offer. Moreover, the homogenization will allow for efficient data exchange between laboratories, including consensual variant classifications and inter-laboratory external quality assessments. The consensual gene-lists will regularly be updated, and are being included in diagnostic decision-trees which a currently being developed within FILNEMUS. http://dx.doi.org/10.1016/j.nmd.2017.06.370

P.331 Utility of a next-generation sequencing (NGS)-based neuromuscular disease gene panel in an investigation of 30 families with early-onset presentation from a tertiary pediatric neuromuscular clinic D. Ardicli 1, K. Nowak 2, G. Haliloglu 1, H. Goullee 3, M. Davis 4, B. Talim 1, N. Laing 5, H. Topalog˘lu 1 1 Hacettepe University Children’s Hospital, Ankara, Turkey; 2 School of Biomedical Sciences, UWA, Harry Perkins Institute of Medical Research, Perth, Australia; 3 Harry Perkins Institute of Medical Research, Centre for Medical Research, UWA, Perth, Australia; 4 Pathwest, QEII Medical Centre, Western Australia, Perth, Australia; 5 Centre for Medical Research, UWA, Harry Perkins Institute of Medical Research, Diagnostic Genoimcs, Pathwest, Perth, Australia

We investigated the utility of NGS diagnostics in a cohort of patients with neuromuscular disease from a single pediatric clinic. According to clinical and pathological features, 30 Turkish families were grouped as congenital myopathy, congenital myasthenic syndrome, congenital muscular dystrophy or unclassified. We used a NGS-based gene panel of >450 neuromuscular disease genes and did not pre-filter variants based on the gene. There were 35 patients from 30 families, aged between 18 months to 20 years, and a male/female ratio of 21/14. There were 5 affected siblings, and consanguinity in 20 families. Based on index cases, the mean age at first symptoms was 14 months (range 1 month - 6 years). Presentation was in the newborn period (n = 10), <2 years (n=13), and ≥2 years of age (n=7). Clinical findings suggested a congenital myopathy in 80% (24/30). The disease nature was characterized by facial involvement (n=12), ophthalmoparesis (n=3), joint contractures (n=5), cervical weakness (n=8), distal laxity (n=9), scoliosis (n=2), and a progressive course (n=6). Muscle biopsy revealed diagnostic (n=12), and nonspecific changes (n=13). According to internationally accepted classification criteria, pathogenic mutations were detected in 12/29 (41.4%) families (1 proband’s DNA sample was too degraded for analysis). Variants of unknown significance were identified in 8/29 (27.6%) families, however these variants were consistent with known genotype/phenotype correlations, rare or absent in normal databases, fitted inheritance patterns, and were predicted to be pathogenic. Mutations in RYR1 (n=4 families) were the most prevalent disease cause, followed by NEB (n=3) and PYROXD1 (n=2). A definitive or probable molecular genetic diagnosis was made in 69% of families. Therefore NGS-based disease-specific targeted panels are a highly effective first choice diagnostic approach. http://dx.doi.org/10.1016/j.nmd.2017.06.371

P.332 Contribution of the NGS analysis to the hyperCKemia P. Marti, N. Muelas, T. Jaijo, J. Millan, I. Azorin, J. Vilchez Hospital Universitari i Politècnic La Fe, Valencia, Spain Genetic study using new generation techniques (NGS) is a tool that is increasingly being used in the diagnosis of myopathies. However, its usefulness in hyperCKemia (HCK) is not known. The aim is to evaluate the diagnostic performance of a panel of genes potentially involved in HCK. We studied 138 patients that remained undiagnosed after performing all the steps of the EFNS guidelines over a series of 371cases with pauci-or-asymptomatic HCK. Ion Torrent technology was applied to a home designpanel of 40 genes (LGMD / myofibrillar / glycosylation),which allows to study the genecoding and intronic flanking regions. Significant changes were found in 45% of patients; 16% of them were ofpathological significance (reported mutations or new/uncertain variants expressing biopsy marker); the other 29% represented possible pathogenic changes (heterozygous dominant and recessive mutations as well as new or uncertain variantswithoutbiopsy marker). As a whole this NGS procedure increment on a 6–16% the rate of 30% of detection yielded by the classical EFNS algorithm applied tothe total series of HCK patients.This increment corresponds mainly to genes not screened byconventional methods. This study proves that the NGS is a very useful tool in HCK investigation. It can permit to change the algorithm of HCK investigation increasing the rate of diagnoses and reducing the proportion of muscle biopsies when used as first tier. However this procedure provides a deal of raw data that requires expertise and sometimes biological analysis on tissue or cells to confirm pathogenicity. http://dx.doi.org/10.1016/j.nmd.2017.06.372

P.333 Next generation sequencing technologies in the genetic diagnosis of congenital myasthenic syndrome A. Topf 1, Y. Azuma 1, S. Gorokhova 2, E. O’Connor 1, A. Porter 1, E. Harris 1, T. Evangelista 1, D. Cox 1, P. Lorenzoni 3, G. McMacken 1, M. Bartoli 2,

Abstracts 2017 / Neuromuscular Disorders 27 (2017) S96–S249 4

5

6

7

1

D. McArthur , O. Magnusson , A. Abicht , J. Senderek , A. Roos , A. Abicht 6, H. Lochmüller 1 1 John Walton Muscular Dystrophy Research Centre, Newcastle upon Tyne, UK; 2 Universite Aix Marseille, Marseille, France; 3 Neurologia Hospital de Clinicas, Curitiba, Brazil; 4 Analytic and Translational Genetics Unit, Boston, USA; 5 deCODE Genetics, Reykjavik, Iceland; 6 Medical Genetics Center, Munich, Germany; 7 Friedrich-Baur-Institute, Ludwig-Maximilians-University, Munich, Germany Congenital myasthenic syndrome (CMS) is a clinical and genetic heterogeneous condition caused by defect in the neuromuscular junction and characterised by fatigable muscle weakness, bulbar and ocular symptoms. Over 20 disease causative genes have been reported, however 30% of CMS patients remain genetically undiagnosed, suggesting that many novel CMS are yet to be identified. After a hotspot pre-screening step, we have applied next generation sequencing techniques, including target panel, whole exome (WES) and whole genome sequencing (WGS) to investigate a cohort of clinically diagnosed CMS patients (n = 54; 43 pedigrees). We have identified 17 disease causing variants in known CMS genes in 11 pedigrees. Twelve of these variants are novel. Thanks to the deep coverage of WGS and NGS panel data, we were able to detect a homozygous deletion in COLQ, as well as a large heterozygous intragenic deletion in DOK7. Surprisingly, we have also found variants in genes that had not been anticipated (POLG, CPL1, CRLF1, SLC2A1 and PTPN11). In addition, two new CMS genes were identified, one of them in two independent families (SCL25A1 and MYO9A, respectively). From the remaining unsolved cases, three strong candidate and modifier genes have emerged and functional investigation is ongoing. NGS technologies have allowed to genetically diagnose 19 CMS pedigrees (45%) as well highlighted new candidate genes and pathways. This is of extreme value as CMS has effective treatment that depends on the causative defect, and therefore genetic diagnosis has a direct impact on patients’ quality of life. http://dx.doi.org/10.1016/j.nmd.2017.06.373

P.334 Genetic diagnosis of inherited peripheral neuropathies using gene panel testing: The utility of follow up familial testing in 4 years of experience N. Forrester 1, C. Buxton 1, M. Greenslade 1, K. Vijayakumar 2, A. Majumdar 2 1 Bristol Genetics Laboratory, Bristol, UK; 2 University Hospital Bristol, Bristol, UK Inherited peripheral neuropathy (IPN) encompasses a clinically and genetically heterogeneous group of disorders. The Bristol Genetics Laboratory has provided a specialist UK Genetic Testing Network (UKGTN) service for 4 years, testing 56 genes associated with IPN using Next Generation Sequencing (NGS) technology. In this time we have tested approximately 1,200 patients with IPN. A genetic diagnosis provides a definite clinical classification, guides the prognosis and provides accurate genetic risk assessment for the patient, as well as offering predictive information for their family. The use of NGS is a powerful tool for the genetic diagnosis of heterogeneous disorders such as IPN, providing a higher diagnostic yield in a significantly shorter time and at a lower cost compared to sequential testing of individual genes. One challenge faced with NGS testing is the analysis and interpretation of a large number of sequence variants. Variants are classified into 5 groups ranging from benign to pathogenic according to ACGS guidelines, a proportion of variants are classified as variants of uncertain clinical significance (VUS). These ambiguous VUS results cause confusion and uncertainty for patients and in the absence of functional studies they are often not resolved. Segregation testing in both affected and unaffected family members can provide evidence for the reclassification of VUS. To date we have performed familial testing for 115 patients with VUS identified on the IPN NGS panel. Testing in this way has led to downgrading (VUS to likely benign) of 72 variants (65%). Additionally, 11 VUS (10%) were reclassified as likely pathogenic variants. We present the most up-to-date audit data from our IPN panel including detection rates and findings in rare genes. We also demonstrate the advantages of testing other family

S197

members for VUS and discuss scenarios in which this approach is particularly beneficial. http://dx.doi.org/10.1016/j.nmd.2017.06.374

P.335 Two different NGS approaches to address molecular diagnosis of congenital neuromuscular diseases L. Gonzalez-Quereda 1, M. Rodriguez 2, A. Nascimento 3, C. Ortez 3, C. Jou 3, J. Milisenda 4, J. Diaz-Manera 2, I. Jerico 5, I. Tejada 6, P. Gallano 1 1 U705 CIBERER, Hospital de Sant Pau, Barcelona, Spain; 2 Hospital de Sant Pau, Barcelona, Spain; 3 Hospital Sant Joan de Deu, Barcelona, Spain; 4 Hospital Clinic, Barcelona, Spain; 5 Hospital V. del Camino, Pamplona, Spain; 6 Hospital de Cruces, Barakaldo, Spain Congenital neuromuscular diseases are early onset muscle disorders encompassing great clinical and genetic heterogeneity so that reaching and accurate genetic diagnosis is still a challenge. We aim to evaluate the diagnostic advantage of different NGS approaches in a cohort of congenital neuromuscular disorders and to validate an efficient and cost-effective diagnostic strategy to be incorporated to the National health system. Sixty-two gDNA samples from patients with clinical suspicion of congenital neuromuscular diseases were analysed by NGS. A custom panel including 118 genes (Nextera Rapid Capture, Illumina) and TruSight One Panel (Clinical Exome, Illumina) were employed. Sequencing data were analysed by Variant Studio and DNA Nexus. All candidate variants were confirmed by sanger sequencing and pathogenicity was analysed by using alamut software (Interactive Biosoftwares) and LOVD database. The genetic cause of the disease was elucidated in 11/25 samples (44%) by the clinical exome and in 28/48 samples (58%) by the custom panel. Eleven samples from the second group (custom panel) had previously been analysed by the clinical exome without positive results. Mutations in RYR1 were the most common cause of disease in our cohort. Variants in genes related to Congenital myasthenic syndromes were identified in 8 patients. This was crucial to choose the appropriate treatment, which depends on the particular molecular defect. The custom panel design has proved to be a more useful tool than the clinical exome to provide a molecular diagnosis of congenital neuromuscular diseases. http://dx.doi.org/10.1016/j.nmd.2017.06.375

P.336 MNDcap gene panel as a diagnostic tool in motor neuron disorders S. Penttilä 1, M. Arumilli 2, P. Hackman 2, M. Sainio 3, E. Ylikallio 3, H. Tyynismaa 3, B. Udd 1 1 University of Tampere, Tampere, Finland; 2 Folkhälsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland; 3 University of Helsinki, Helsinki, Finland Motor neuron disorders (MNDs) are a heterogeneous group of diseases that result from degeneration of motor neurons. Clinical and genetic overlap of MNDs is significant, however, and therefore diagnostics is challenging. MNDcap is a gene panel targeted to the exons of 275 genes that are known or predicted to cause motor neuron disease, hereditary neuropathy, spastic paraplegia or myasthenic syndrome. In our study, we have used MNDcap as a diagnostic tool for 157 MND patients whose diagnose had remained unsolved despite extensive studies. The sequencing data was processed using an in-house developed next-generation sequencing pipeline and the variants found were compared to the clinical findings. The results were classified in five categories: 1) diagnostic finding; 2) likely diagnostic finding; 3) possibly disease-related finding; 4) unlikely disease-related finding, and 5) no findings. In our patient cohort, the final diagnosis was reached in 20 patients (13 %), and in ten patients (6 %) the finding was likely diagnostic. Twenty-five patients (16 %) had a possibly disease-related finding, whereas 25 patients