title: Use of Whole-Exome Sequencing for Diagnosis of Limb-Girdle Muscular Dystrophy: Outcomes and Lessons Learned.
abstract: OBJECTIVE
  To our knowledge, the efficacy of transferring next-generation sequencing from a research setting to neuromuscular clinics has never been evaluated.
  
  
  OBJECTIVE
  To translate whole-exome sequencing (WES) to clinical practice for the genetic diagnosis of a large cohort of patients with limb-girdle muscular dystrophy (LGMD) for whom protein-based analyses and targeted Sanger sequencing failed to identify the genetic cause of their disorder.
  
  
  METHODS
  We performed WES on 60 families with LGMDs (100 exomes). Data analysis was performed between January 6 and December 19, 2014, using the xBrowse bioinformatics interface (Broad Institute). Patients with LGMD were ascertained retrospectively through the Institute for Neuroscience and Muscle Research Biospecimen Bank between 2006 and 2014. Enrolled patients had been extensively investigated via protein studies and candidate gene sequencing and remained undiagnosed. Patients presented with more than 2 years of muscle weakness and with dystrophic or myopathic changes present in muscle biopsy specimens.
  
  
  METHODS
  The diagnostic rate of LGMD in Australia and the relative frequencies of the different LGMD subtypes. Our central goals were to improve the genetic diagnosis of LGMD, investigate whether the WES platform provides adequate coverage of known LGMD-related genes, and identify new LGMD-related genes.
  
  
  RESULTS
  With WES, we identified likely pathogenic mutations in known myopathy genes for 27 of 60 families. Twelve families had mutations in known LGMD-related genes. However, 15 families had variants in disease-related genes not typically associated with LGMD, highlighting the clinical overlap between LGMD and other myopathies. Common causes of phenotypic overlap were due to mutations in congenital muscular dystrophy-related genes (4 families) and collagen myopathy-related genes (4 families). Less common myopathies included metabolic myopathy (2 families), congenital myasthenic syndrome (DOK7), congenital myopathy (ACTA1), tubular aggregate myopathy (STIM1), myofibrillar myopathy (FLNC), and mutation of CHD7, usually associated with the CHARGE syndrome. Inclusion of family members increased the diagnostic efficacy of WES, with a diagnostic rate of 60% for "trios" (an affected proband with both parents) vs 40% for single probands. A follow-up screening of patients whose conditions were undiagnosed on a targeted neuromuscular disease-related gene panel did not improve our diagnostic yield.
  
  
  CONCLUSIONS
  With WES, we achieved a diagnostic success rate of 45.0% in our difficult-to-diagnose cohort of patients with LGMD. We expand the clinical phenotypes associated with known myopathy genes, and we stress the importance of accurate clinical examination and histopathological results for interpretation of WES, with many diagnoses requiring follow-up review and ancillary investigations of biopsy specimens or serum samples.
authors.affiliation: Institute for Neuroscience and Muscle Research, Kid's Research Institute, Children's Hospital at Westmead, Sydney, New South Wales, Australia2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales.
authors.forename: Roula
authors.initials: R
authors.lastname: Ghaoui
authors.affiliation: Institute for Neuroscience and Muscle Research, Kid's Research Institute, Children's Hospital at Westmead, Sydney, New South Wales, Australia2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales.
authors.forename: Sandra T
authors.initials: ST
authors.lastname: Cooper
authors.affiliation: Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston.
authors.forename: Monkol
authors.initials: M
authors.lastname: Lek
authors.affiliation: Institute for Neuroscience and Muscle Research, Kid's Research Institute, Children's Hospital at Westmead, Sydney, New South Wales, Australia2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales.
authors.forename: Kristi
authors.initials: K
authors.lastname: Jones
authors.affiliation: Department of Neurology, Concord General Repatriation Hospital, Sydney, New South Wales, Australia.
authors.forename: Alastair
authors.initials: A
authors.lastname: Corbett
authors.affiliation: Department of Neurology, Concord General Repatriation Hospital, Sydney, New South Wales, Australia.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Reddel
authors.affiliation: Western Australian Neurosciences Research Institute, University of Western Australia, Perth, Western Australia, Australia.
authors.forename: Merrilee
authors.initials: M
authors.lastname: Needham
authors.affiliation: Department of Neurology, Royal North Shore Hospital, Sydney, New South Wales, Australia.
authors.forename: Christina
authors.initials: C
authors.lastname: Liang
authors.affiliation: Institute for Neuroscience and Muscle Research, Kid's Research Institute, Children's Hospital at Westmead, Sydney, New South Wales, Australia2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales.
authors.forename: Leigh B
authors.initials: LB
authors.lastname: Waddell
authors.affiliation: University of Sydney ANZAC Research Institute and Molecular Medicine Laboratory, Concord Hospital, Sydney, New South Wales, Australia.
authors.forename: Garth
authors.initials: G
authors.lastname: Nicholson
authors.affiliation: Institute for Neuroscience and Muscle Research, Kid's Research Institute, Children's Hospital at Westmead, Sydney, New South Wales, Australia2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales.
authors.forename: Gina
authors.initials: G
authors.lastname: O'Grady
authors.affiliation: Institute for Neuroscience and Muscle Research, Kid's Research Institute, Children's Hospital at Westmead, Sydney, New South Wales, Australia2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales.
authors.forename: Simranpreet
authors.initials: S
authors.lastname: Kaur
authors.affiliation: Centre for Medical Research, University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia.
authors.forename: Royston
authors.initials: R
authors.lastname: Ong
authors.affiliation: Department of Diagnostic Genomics, Pathwest Laboratory Medicine, Perth, Western Australia, Australia.
authors.forename: Mark
authors.initials: M
authors.lastname: Davis
authors.affiliation: Department of Neurology, Royal North Shore Hospital, Sydney, New South Wales, Australia.
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Sue
authors.affiliation: Centre for Medical Research, University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia.
authors.forename: Nigel G
authors.initials: NG
authors.lastname: Laing
authors.affiliation: Murdoch Children's Research Institute, The Royal Children's Hospital, Melbourne, Victoria, Australia.
authors.forename: Kathryn N
authors.initials: KN
authors.lastname: North
authors.affiliation: Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: MacArthur
authors.affiliation: Institute for Neuroscience and Muscle Research, Kid's Research Institute, Children's Hospital at Westmead, Sydney, New South Wales, Australia2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales.
authors.forename: Nigel F
authors.initials: NF
authors.lastname: Clarke
mesh_terms: D001315:Australia; D019295:Computational Biology; D059472:Exome / Q000235:genetics*; D005192:Family Health; D005260:Female; D005820:Genetic Testing; D006801:Humans; D008297:Male; D049288:Muscular Dystrophies, Limb-Girdle / Q000175:diagnosis* / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA / Q000379:methods*