- Email: [email protected]
NeurOmics: EU-funded-omics research for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases
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Abstracts / Neuromuscular Disorders 25 (2015) S184–S316
Department of Neuromuscular Research, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan We take advantage of using high-throughput sequencers to make a genetic diagnosis of muscle disease. We made four target-resequencing panels each of which covers all exonic regions of genes known to be causative for specific groups of muscle diseases; 1) muscular dystrophy, 2) congenital myopathy/ congenital myasthenic syndrome, 3) metabolic myopathy, and 4) myopathy with protein aggregation/rimmed-vacuole. Target gene numbers and sizes are 65, 42, 45, and 36 genes and 502, 352, 422 and 242 kb, respectively. The coverage rates of the targets (exons and franking regions) are 96.8%, 97.2%, 97.8% and 96.7%, respectively. We chose one panel for each case based upon the clinical and muscle pathological features, and performed high-throughput sequencing using IonPGM sequencer. We identified possible or definitely causative gene mutations in ~25% of the patients. However, more than 70% of the cases tested remained undiagnosed. It is challenging to prove the pathogenicity of novel gene mutations because most of the cases are sporadic and the functional analysis of mutated genes is not always feasible. We performed whole exome sequencing using Illumina HiSeq1000 in almost all the undiagnosed cases to build an exome database of genetically-undiagnosed myopathy that would enable us to find novel diseases. In conclusion, high-throughput sequencing technique is useful and effective for mutation screening in muscular disease. http://dx.doi.org/10.1016/j.nmd.2015.06.398
G.P.375 Novel variant blossom: From pathology to next generation sequencing to cellular biology W. Zhu *,1, S. Mitsuhashi 1, W. Liang 2, T. Ito 3, I. Nishino 1 1 Department of Neuromuscular Research, National Center of Neurology and Psychiatry, Tokyo, Japan; 2 Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; 3 Department of Neurology, Matsudo City Hospital, Chiba, Japan With the great technical progress in the last few decades, next generation sequencing (NGS) becomes more and more feasible to myology specialists or even general physicians. However, the high throughput of NGS leads to a novel variant blossom, which might blur the culprit mutation. Furthermore, cellular biological evidence is needed before a candidate variant could be regarded as pathogenic. An algorism of targeted enrichment sequencing and subsequent functional analysis is followed to maximize the diagnostic yield of inherited muscular disorders in our center. Case 1: A 4-year-old girl was presented with delayed motor and speech development, hyperCKemia, bilateral cataracts, and hepatic steatosis. Muscle biopsy revealed dystrophic changes with mildly decreased alpha-dystroglycan. Targeted NGS revealed compound heterozygous mutations in TRAPPC11, c.2938G>A [p.Gly980Arg] and c.661-1G>T. The former was reported while the latter was novel, which resulted in two mutant transcripts that both led to truncated proteins. Complete deficiency of Trappc11 in the skeletal muscle was demonstrated by WB, thus limb-girdle muscular dystrophy type 2S was diagnosed. Case 2: A 35-year-old man presented with a 10-year-history of bilateral ankle joint pain and difficulty in climbing stairs. Muscle MRI showed a distinct involvement pattern while muscle biopsy showed extraordinarily disorganized myofibrillar networks with rimmed vacuoles and cytoplasmic inclusions. Targeted NGS revealed a heterozygous mutation in MATR3, c.A2155G [p.Lys719Glu]. The mutant protein and the MATR3 hotspot mutation, p.Ser85Cys, were transfected into HeLa cells, in which an abnormal giant nucleus was found. In conclusion, muscle pathology is nonetheless an important clue to guide the targeted enrichment NGS. Meanwhile, the high throughput of NGS also leads to tremendous work to discriminate pathogenic variants from neutral ones, which every myologist will encounter in the NGS era. http://dx.doi.org/10.1016/j.nmd.2015.06.399
G.P.376 Collaboration in NeurOmics: Enabling effective data-sharing and maximising impact in neuromuscular disease C. Turner *,1, K. Bushby 1, L. Johnston 1, H. Lochmüller 1, O. Riess 2, B. Wirth 3, V. Straub 1, R. Thompson 1, G. van Ommen 4 1 John Walton Muscular Dystrophy Research Centre, Newcastle University, Newcastle upon Tyne, UK; 2 Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany; 3 Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany; 4 Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands Within the NeurOmics project 1100 samples from across 10 rare neurodegenerative and neuromuscular diseases will undergo whole exome sequencing. In addition patients will be deep phenotyped, RNAseq will be carried out and biomarker studies are in progress – this will lead to improved understanding of the conditions, causative and modifier gene discovery, more diagnoses and the identification of potential therapeutic targets. For these ambitious aims to be realised, both omics and phenotypic data should be accessible for study by NeurOmics partners across the disease groups. In order to enable this, the NeurOmics project has built an online clinical database in which all phenotypic data are mapped to the Human Phenotype Ontology (HPO), and has established data sharing policies and procedures in close collaboration with RD-Connect. This means that partners are committed to collaborative working within the consortium now and to wider data-sharing via the RD-Connect platform and the European Genome-phenome Archive (EGA) in future, according to agreed timelines that ensure all NeurOmics data are ultimately accessible to researchers worldwide. This model of successful datasharing in order to promote collaborative research and accelerate research could be applied across the rare disease field. It is important to consider how the impact of the outputs from NeurOmics will be ensured and maximised across the neuromuscular community. This means translating the research outcomes and model of data-sharing into everyday scientific, clinical and industry practice as well as into national and international policymaking. Partners in the project are proactively engaged in activities to ensure this is achieved. http://dx.doi.org/10.1016/j.nmd.2015.06.400
G.P.377 NeurOmics: EU-funded-omics research for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases C. Turner *,1, A. Brice 2, K. Bushby 1, O. Riess 3, M. Hanna 4, G. van Ommen 5, F. Muntoni 6, T. Klockgether 7, B. Wirth 8, H. Lochmüller 1, V. Timmerman 9, L. Schoells 3, V. Straub 1, S. Tabrizi 10 1 John Walton Muscular Dystrophy Research Centre, Newcastle University, Newcastle upon Tyne, UK; 2 Institut du Cerveau et de la Moelle Epinière (ICM), Groupe Hospitalier Pitié Salpêtrière, Paris, France; 3 Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany; 4 The MRC Centre for Neuromuscular Diseases, University College London, London, UK; 5 Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands; 6 Dubowitz Neuromuscular Centre, University College London, London, UK; 7 German Center for Neurodegenerative Disease, University of Bonn, Bonn, Germany; 8 Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany; 9 Department of Molecular Genetics, University of Antwerp, Antwerp, Belgium; 10 Institute of Neurology, University College London, London, UK NeurOmics is an EU-funded translational research project which has the primary aim of greatly improving understanding of neuromuscular and neurodegenerative diseases. The research is studying around 1100 exomes and/or genomes from patients in an aim to discover novel disease-causing and disease-modifying genes and to identify potential new therapeutic targets. Halfway through the project, more than 500 exomes have been sequenced, over 50 novel disease causing genes have been identified and 39 were published by March 2015. Gene panels combined with WES analysis have resulted in the identification of the genetic cause of patients’ phenotypes in around 50% of those tested. Partners have also undertaken deep-phenotyping of patients using
Abstracts / Neuromuscular Disorders 25 (2015) S184–S316 human phenotype ontology (HPO) terms and there are around 1000 clinical data sets relating to patients and their unaffected relatives already entered into the NeurOmics database. Agreements are in place to allow the secure sharing of this standardised clinical information along with the WES and other -omics data within NeurOmics and with the wider rare-disease field. The project focuses on 10 rare, genetic neuromuscular and neurodegenerative disease groups: frontotemporal lobe degeneration; Huntington’s disease; ataxia; hereditary spastic paraplegia; spinal muscular atrophy; hereditary motor neuropathy; congenital myasthenic syndrome; muscular dystrophy and muscular channelopathy. This poster summarises the aims and methods used in the NeurOmics project and reports on results and progress so far. It highlights how Neuromics will contribute significantly to the ambitious goals of the International Rare Diseases Research Consortium (IRDiRC): deciphering the genetic causes of all rare diseases and the development of 200 new therapies by 2020. http://dx.doi.org/10.1016/j.nmd.2015.06.401
G.P.378 Omics approach and novel biostatistic tools identified RPL3L as potential genetic modifier of clinical severity in female carriers of Duchenne muscle dystrophy M. Neri 1, C. Scotton 1, C. Scapoli 2, A. Carrieri 2, F. Di Raimo 1, M. Bovolenta 1, S. Gherardi 1, A. Armaroli 1, C. Passarelli 3, A. D’Amico 3, E. Bertini 3, M. Pane 4, E. Mercuri 4, G. Pesole 5, L. Wenyan 6, F. Mingyan 6, F. Gualandi 1, E. Schwartz 7, A. Yuryev 7, A. Ferlini *,1 1 UOL Medical Genetics, Università Ferrara, Ferrara, Italy; 2 Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy; 3 Bambino Gesù Children’s Hospital, Rome, Italy; 4 Department of Paediatric Neurology, Catholic University, Rome, Italy; 5 Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy; 6 BGI-SHENZHEN, Shenzhen, China; 7 Ariadne Diagnostics, Rockville, USA Duchenne muscular dystrophy (DMD) female carriers are usually clinically asymptomatic. Nevertheless exceptions are reported and manifesting carriers can develop symptoms, varying from a mild muscle weakness to a DMD-like phenotype. The molecular mechanism underlying the clinical heterogeneity in female carriers is unknown and other modifiers, either genetics or environmental, could play a role. We adopted a combined approach based on omics studies associated with bioinformatics and novel statistical tools, in order to identify genetic modifiers influencing the symptomatic phenotype in a group of DMD female carriers. Using whole exome sequencing, RNA-seq and interactome mapping we identified a cohort of single nucleotide polymorphisms (SNPs) in genes possibly influencing the phenotype in carriers. Multivariate statistical approaches on selected SNPs mainly based on discriminant analysis of principal component (DAPC) followed by univariate association tests allowed us to prioritize two SNPs in the 3′ untranslated regions of the RPL3L gene which could represent genetic modifiers of the symptomatic/asymptomatic phenotype. These novel statistical tools, applied to gene discovery based on omics tools, identified SNPs within the ribosomal protein L3-like gene (RPL3L gene) we propose as potential modifiers of the symptomatic status. Our approach could be very useful for the investigation of small cohorts of patients affected by rare diseases ad widely applied in omics output analysis. http://dx.doi.org/10.1016/j.nmd.2015.06.402
G.P.379 Whole exome sequencing at the Institute of Myology in the context of the Myocapture project to identify novel genes of myopathies I. Nelson *,1, R. BenYaou 2, J. Nectoux 3, C. Masson 4, F. Leturq 3, P. Richard 5, T. Stojkovic 2, A. Behin 2, P. Laforêt 2, V. Allamand 1, B. Eymard 2, G. Bonne 1 1 UPMC – Inserm UMRS 974, CNRS FRE3617, Centre of Research in Myology, Paris, France; 2 Centre de Référence de Pathologie Neuromusculaire, AP-HP
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Groupe Hospitalier Pitié-Salpêtrière Paris-Est, Paris, France; Laboratoire de Biochimie et Génétique Moléculaire, AP-HP Groupe Hospitalier Cochin-Broca-Hôtel Dieu, Paris, France; 4 Plateforme de Bioinformatique, Université Paris Descartes, Paris, France; 5 AP-HP, U.F. Cardiogénétique et Myogénétique, Service de Biochimie Métabolique, Paris, France According to the GeneTable, over 400 genes have been associated with neuromuscular diseases while 76 mapped loci are awaiting gene identification. At least 25% of patients remain without molecular diagnosis. The Myocapture project was designed to identify new genes responsible for myopathies by exome sequencing of 1000 individuals. In this context, we selected clinically well-characterised patients with limb girdle muscular dystrophies, congenital muscular dystrophies and hereditary sensory-motor neuropathies. The known genes had been previously excluded through routine diagnosis strategy. For each patient, we gathered all available information (biological analyses, electromyography, imaging and muscle biopsy histology). Out of 111 families, DNA samples from 161 patients and their relatives have been collected. Agilent SureSelectXT Human All Exon V5 was used for DNA capture before sequencing on Illumina HighSeq sequencer. Variants have been selected and filtered with the Polyweb software. We identified mutations in known genes (CAPN3, COL6A1, COL6A2 and TTN) that had either been missed or not screened during previous molecular investigations. Likewise, putative mutations in POMT2, RYR1 and BIN1 are awaiting further confirmation. A mutation in ASAH1 previously described in SMA with myoclonic epilepsy has been associated with a new phenotype. For the remaining cases, variants in non-previously reported genes for myopathies are under evaluation based on prioritisation pipeline focusing on genes encoding either skeletal muscle proteins or interacting with known disease proteins or implicated in biological processes related to striated muscle. New candidate genes were selected that are under confirmation based on functional studies. Overall, exome sequencing leads to 1) identification of new genes responsible for myopathies, 2) description of new phenotypic entities due to genes previously involved in other conditions and 3) identification of 10% of “missed” diagnoses. http://dx.doi.org/10.1016/j.nmd.2015.06.403
G.P.380 Efficacy of next-generation sequencing in molecular diagnosis of archived DNA samples S. Beecroft *,1, R. Ong 1, K. Yau 2, R. Duff 1, R. Allcock 2, M. Davis 3, P. Lamont 4, N. Laing 1 1 Neurogenetic Diseases Laboratory, Harry Perkins Institute for Medical Research, Nedlands, Australia; 2 School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, Australia; 3 Neurogenetics Unit, PathWest Laboratory Medicine, Nedlands, Australia; 4 Division of Neurosciences, Royal Perth Hospital, Perth, Australia Inherited neurogenetic disorders include some of the most debilitating genetic diseases. Their clinical heterogeneity makes diagnosis difficult without genetic screening. The recent advent of affordable next-generation DNA sequencing (NGS) now makes this fast and inexpensive. Utilising this advance, we previously developed a neurogenetic disease gene sub-exomic screening panel (NSES) that has been validated and used for prospective diagnostic samples since 2013. Previously undiagnosed patients with archived DNA samples pre-dating this innovation might now be solved via NSES. However, the DNA of the banked sample may be unviable because of degradation, and new samples may not be available. This pilot study aimed to test the efficacy of NSES on archived DNA samples (obtained 1995–2012) from patients that had received no diagnosis from previous genetic testing methods. NSES was performed on 143 archived samples. A subset of archived samples (n = 110) had measurements of DNA and NGS quality compared against 116 prospective control NSES samples obtained between 2013 and 2014. Diagnostic success was compared to prospective NSES rates. DNA and NGS quality were found to decrease slightly with age, but not enough to be a hindrance. 23% of the archived cases (n = 33) had a diagnosis confirmed and 6% (n = 9) had variants
Abstracts / Neuromuscular Disorders 25 (2015) S184–S316
Department of Neuromuscular Research, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan We take advantage of using high-throughput sequencers to make a genetic diagnosis of muscle disease. We made four target-resequencing panels each of which covers all exonic regions of genes known to be causative for specific groups of muscle diseases; 1) muscular dystrophy, 2) congenital myopathy/ congenital myasthenic syndrome, 3) metabolic myopathy, and 4) myopathy with protein aggregation/rimmed-vacuole. Target gene numbers and sizes are 65, 42, 45, and 36 genes and 502, 352, 422 and 242 kb, respectively. The coverage rates of the targets (exons and franking regions) are 96.8%, 97.2%, 97.8% and 96.7%, respectively. We chose one panel for each case based upon the clinical and muscle pathological features, and performed high-throughput sequencing using IonPGM sequencer. We identified possible or definitely causative gene mutations in ~25% of the patients. However, more than 70% of the cases tested remained undiagnosed. It is challenging to prove the pathogenicity of novel gene mutations because most of the cases are sporadic and the functional analysis of mutated genes is not always feasible. We performed whole exome sequencing using Illumina HiSeq1000 in almost all the undiagnosed cases to build an exome database of genetically-undiagnosed myopathy that would enable us to find novel diseases. In conclusion, high-throughput sequencing technique is useful and effective for mutation screening in muscular disease. http://dx.doi.org/10.1016/j.nmd.2015.06.398
G.P.375 Novel variant blossom: From pathology to next generation sequencing to cellular biology W. Zhu *,1, S. Mitsuhashi 1, W. Liang 2, T. Ito 3, I. Nishino 1 1 Department of Neuromuscular Research, National Center of Neurology and Psychiatry, Tokyo, Japan; 2 Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; 3 Department of Neurology, Matsudo City Hospital, Chiba, Japan With the great technical progress in the last few decades, next generation sequencing (NGS) becomes more and more feasible to myology specialists or even general physicians. However, the high throughput of NGS leads to a novel variant blossom, which might blur the culprit mutation. Furthermore, cellular biological evidence is needed before a candidate variant could be regarded as pathogenic. An algorism of targeted enrichment sequencing and subsequent functional analysis is followed to maximize the diagnostic yield of inherited muscular disorders in our center. Case 1: A 4-year-old girl was presented with delayed motor and speech development, hyperCKemia, bilateral cataracts, and hepatic steatosis. Muscle biopsy revealed dystrophic changes with mildly decreased alpha-dystroglycan. Targeted NGS revealed compound heterozygous mutations in TRAPPC11, c.2938G>A [p.Gly980Arg] and c.661-1G>T. The former was reported while the latter was novel, which resulted in two mutant transcripts that both led to truncated proteins. Complete deficiency of Trappc11 in the skeletal muscle was demonstrated by WB, thus limb-girdle muscular dystrophy type 2S was diagnosed. Case 2: A 35-year-old man presented with a 10-year-history of bilateral ankle joint pain and difficulty in climbing stairs. Muscle MRI showed a distinct involvement pattern while muscle biopsy showed extraordinarily disorganized myofibrillar networks with rimmed vacuoles and cytoplasmic inclusions. Targeted NGS revealed a heterozygous mutation in MATR3, c.A2155G [p.Lys719Glu]. The mutant protein and the MATR3 hotspot mutation, p.Ser85Cys, were transfected into HeLa cells, in which an abnormal giant nucleus was found. In conclusion, muscle pathology is nonetheless an important clue to guide the targeted enrichment NGS. Meanwhile, the high throughput of NGS also leads to tremendous work to discriminate pathogenic variants from neutral ones, which every myologist will encounter in the NGS era. http://dx.doi.org/10.1016/j.nmd.2015.06.399
G.P.376 Collaboration in NeurOmics: Enabling effective data-sharing and maximising impact in neuromuscular disease C. Turner *,1, K. Bushby 1, L. Johnston 1, H. Lochmüller 1, O. Riess 2, B. Wirth 3, V. Straub 1, R. Thompson 1, G. van Ommen 4 1 John Walton Muscular Dystrophy Research Centre, Newcastle University, Newcastle upon Tyne, UK; 2 Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany; 3 Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany; 4 Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands Within the NeurOmics project 1100 samples from across 10 rare neurodegenerative and neuromuscular diseases will undergo whole exome sequencing. In addition patients will be deep phenotyped, RNAseq will be carried out and biomarker studies are in progress – this will lead to improved understanding of the conditions, causative and modifier gene discovery, more diagnoses and the identification of potential therapeutic targets. For these ambitious aims to be realised, both omics and phenotypic data should be accessible for study by NeurOmics partners across the disease groups. In order to enable this, the NeurOmics project has built an online clinical database in which all phenotypic data are mapped to the Human Phenotype Ontology (HPO), and has established data sharing policies and procedures in close collaboration with RD-Connect. This means that partners are committed to collaborative working within the consortium now and to wider data-sharing via the RD-Connect platform and the European Genome-phenome Archive (EGA) in future, according to agreed timelines that ensure all NeurOmics data are ultimately accessible to researchers worldwide. This model of successful datasharing in order to promote collaborative research and accelerate research could be applied across the rare disease field. It is important to consider how the impact of the outputs from NeurOmics will be ensured and maximised across the neuromuscular community. This means translating the research outcomes and model of data-sharing into everyday scientific, clinical and industry practice as well as into national and international policymaking. Partners in the project are proactively engaged in activities to ensure this is achieved. http://dx.doi.org/10.1016/j.nmd.2015.06.400
G.P.377 NeurOmics: EU-funded-omics research for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases C. Turner *,1, A. Brice 2, K. Bushby 1, O. Riess 3, M. Hanna 4, G. van Ommen 5, F. Muntoni 6, T. Klockgether 7, B. Wirth 8, H. Lochmüller 1, V. Timmerman 9, L. Schoells 3, V. Straub 1, S. Tabrizi 10 1 John Walton Muscular Dystrophy Research Centre, Newcastle University, Newcastle upon Tyne, UK; 2 Institut du Cerveau et de la Moelle Epinière (ICM), Groupe Hospitalier Pitié Salpêtrière, Paris, France; 3 Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen, Germany; 4 The MRC Centre for Neuromuscular Diseases, University College London, London, UK; 5 Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands; 6 Dubowitz Neuromuscular Centre, University College London, London, UK; 7 German Center for Neurodegenerative Disease, University of Bonn, Bonn, Germany; 8 Institute of Human Genetics, University Hospital of Cologne, Cologne, Germany; 9 Department of Molecular Genetics, University of Antwerp, Antwerp, Belgium; 10 Institute of Neurology, University College London, London, UK NeurOmics is an EU-funded translational research project which has the primary aim of greatly improving understanding of neuromuscular and neurodegenerative diseases. The research is studying around 1100 exomes and/or genomes from patients in an aim to discover novel disease-causing and disease-modifying genes and to identify potential new therapeutic targets. Halfway through the project, more than 500 exomes have been sequenced, over 50 novel disease causing genes have been identified and 39 were published by March 2015. Gene panels combined with WES analysis have resulted in the identification of the genetic cause of patients’ phenotypes in around 50% of those tested. Partners have also undertaken deep-phenotyping of patients using
Abstracts / Neuromuscular Disorders 25 (2015) S184–S316 human phenotype ontology (HPO) terms and there are around 1000 clinical data sets relating to patients and their unaffected relatives already entered into the NeurOmics database. Agreements are in place to allow the secure sharing of this standardised clinical information along with the WES and other -omics data within NeurOmics and with the wider rare-disease field. The project focuses on 10 rare, genetic neuromuscular and neurodegenerative disease groups: frontotemporal lobe degeneration; Huntington’s disease; ataxia; hereditary spastic paraplegia; spinal muscular atrophy; hereditary motor neuropathy; congenital myasthenic syndrome; muscular dystrophy and muscular channelopathy. This poster summarises the aims and methods used in the NeurOmics project and reports on results and progress so far. It highlights how Neuromics will contribute significantly to the ambitious goals of the International Rare Diseases Research Consortium (IRDiRC): deciphering the genetic causes of all rare diseases and the development of 200 new therapies by 2020. http://dx.doi.org/10.1016/j.nmd.2015.06.401
G.P.378 Omics approach and novel biostatistic tools identified RPL3L as potential genetic modifier of clinical severity in female carriers of Duchenne muscle dystrophy M. Neri 1, C. Scotton 1, C. Scapoli 2, A. Carrieri 2, F. Di Raimo 1, M. Bovolenta 1, S. Gherardi 1, A. Armaroli 1, C. Passarelli 3, A. D’Amico 3, E. Bertini 3, M. Pane 4, E. Mercuri 4, G. Pesole 5, L. Wenyan 6, F. Mingyan 6, F. Gualandi 1, E. Schwartz 7, A. Yuryev 7, A. Ferlini *,1 1 UOL Medical Genetics, Università Ferrara, Ferrara, Italy; 2 Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy; 3 Bambino Gesù Children’s Hospital, Rome, Italy; 4 Department of Paediatric Neurology, Catholic University, Rome, Italy; 5 Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy; 6 BGI-SHENZHEN, Shenzhen, China; 7 Ariadne Diagnostics, Rockville, USA Duchenne muscular dystrophy (DMD) female carriers are usually clinically asymptomatic. Nevertheless exceptions are reported and manifesting carriers can develop symptoms, varying from a mild muscle weakness to a DMD-like phenotype. The molecular mechanism underlying the clinical heterogeneity in female carriers is unknown and other modifiers, either genetics or environmental, could play a role. We adopted a combined approach based on omics studies associated with bioinformatics and novel statistical tools, in order to identify genetic modifiers influencing the symptomatic phenotype in a group of DMD female carriers. Using whole exome sequencing, RNA-seq and interactome mapping we identified a cohort of single nucleotide polymorphisms (SNPs) in genes possibly influencing the phenotype in carriers. Multivariate statistical approaches on selected SNPs mainly based on discriminant analysis of principal component (DAPC) followed by univariate association tests allowed us to prioritize two SNPs in the 3′ untranslated regions of the RPL3L gene which could represent genetic modifiers of the symptomatic/asymptomatic phenotype. These novel statistical tools, applied to gene discovery based on omics tools, identified SNPs within the ribosomal protein L3-like gene (RPL3L gene) we propose as potential modifiers of the symptomatic status. Our approach could be very useful for the investigation of small cohorts of patients affected by rare diseases ad widely applied in omics output analysis. http://dx.doi.org/10.1016/j.nmd.2015.06.402
G.P.379 Whole exome sequencing at the Institute of Myology in the context of the Myocapture project to identify novel genes of myopathies I. Nelson *,1, R. BenYaou 2, J. Nectoux 3, C. Masson 4, F. Leturq 3, P. Richard 5, T. Stojkovic 2, A. Behin 2, P. Laforêt 2, V. Allamand 1, B. Eymard 2, G. Bonne 1 1 UPMC – Inserm UMRS 974, CNRS FRE3617, Centre of Research in Myology, Paris, France; 2 Centre de Référence de Pathologie Neuromusculaire, AP-HP
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Groupe Hospitalier Pitié-Salpêtrière Paris-Est, Paris, France; Laboratoire de Biochimie et Génétique Moléculaire, AP-HP Groupe Hospitalier Cochin-Broca-Hôtel Dieu, Paris, France; 4 Plateforme de Bioinformatique, Université Paris Descartes, Paris, France; 5 AP-HP, U.F. Cardiogénétique et Myogénétique, Service de Biochimie Métabolique, Paris, France According to the GeneTable, over 400 genes have been associated with neuromuscular diseases while 76 mapped loci are awaiting gene identification. At least 25% of patients remain without molecular diagnosis. The Myocapture project was designed to identify new genes responsible for myopathies by exome sequencing of 1000 individuals. In this context, we selected clinically well-characterised patients with limb girdle muscular dystrophies, congenital muscular dystrophies and hereditary sensory-motor neuropathies. The known genes had been previously excluded through routine diagnosis strategy. For each patient, we gathered all available information (biological analyses, electromyography, imaging and muscle biopsy histology). Out of 111 families, DNA samples from 161 patients and their relatives have been collected. Agilent SureSelectXT Human All Exon V5 was used for DNA capture before sequencing on Illumina HighSeq sequencer. Variants have been selected and filtered with the Polyweb software. We identified mutations in known genes (CAPN3, COL6A1, COL6A2 and TTN) that had either been missed or not screened during previous molecular investigations. Likewise, putative mutations in POMT2, RYR1 and BIN1 are awaiting further confirmation. A mutation in ASAH1 previously described in SMA with myoclonic epilepsy has been associated with a new phenotype. For the remaining cases, variants in non-previously reported genes for myopathies are under evaluation based on prioritisation pipeline focusing on genes encoding either skeletal muscle proteins or interacting with known disease proteins or implicated in biological processes related to striated muscle. New candidate genes were selected that are under confirmation based on functional studies. Overall, exome sequencing leads to 1) identification of new genes responsible for myopathies, 2) description of new phenotypic entities due to genes previously involved in other conditions and 3) identification of 10% of “missed” diagnoses. http://dx.doi.org/10.1016/j.nmd.2015.06.403
G.P.380 Efficacy of next-generation sequencing in molecular diagnosis of archived DNA samples S. Beecroft *,1, R. Ong 1, K. Yau 2, R. Duff 1, R. Allcock 2, M. Davis 3, P. Lamont 4, N. Laing 1 1 Neurogenetic Diseases Laboratory, Harry Perkins Institute for Medical Research, Nedlands, Australia; 2 School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, Australia; 3 Neurogenetics Unit, PathWest Laboratory Medicine, Nedlands, Australia; 4 Division of Neurosciences, Royal Perth Hospital, Perth, Australia Inherited neurogenetic disorders include some of the most debilitating genetic diseases. Their clinical heterogeneity makes diagnosis difficult without genetic screening. The recent advent of affordable next-generation DNA sequencing (NGS) now makes this fast and inexpensive. Utilising this advance, we previously developed a neurogenetic disease gene sub-exomic screening panel (NSES) that has been validated and used for prospective diagnostic samples since 2013. Previously undiagnosed patients with archived DNA samples pre-dating this innovation might now be solved via NSES. However, the DNA of the banked sample may be unviable because of degradation, and new samples may not be available. This pilot study aimed to test the efficacy of NSES on archived DNA samples (obtained 1995–2012) from patients that had received no diagnosis from previous genetic testing methods. NSES was performed on 143 archived samples. A subset of archived samples (n = 110) had measurements of DNA and NGS quality compared against 116 prospective control NSES samples obtained between 2013 and 2014. Diagnostic success was compared to prospective NSES rates. DNA and NGS quality were found to decrease slightly with age, but not enough to be a hindrance. 23% of the archived cases (n = 33) had a diagnosis confirmed and 6% (n = 9) had variants