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Original Investigation |

Use of Whole-Exome Sequencing for Diagnosis of Limb-Girdle Muscular Dystrophy Outcomes and Lessons Learned

Roula Ghaoui, FRACP1,2; Sandra T. Cooper, PhD1,2; Monkol Lek, PhD3; Kristi Jones, FRACP, PhD1,2; Alastair Corbett, FRACP, MD4; Stephen W. Reddel, FRACP, PhD4; Merrilee Needham, FRACP, PhD5; Christina Liang, FRACP6; Leigh B. Waddell, PhD1,2; Garth Nicholson, FRACP, PhD7; Gina O’Grady, FRACP1,2; Simranpreet Kaur, MSc, MPhil1,2; Royston Ong, BSc8; Mark Davis, PhD, FFSc(RCPA)9; Carolyn M. Sue, FRACP, PhD6; Nigel G. Laing, PhD, FFSc(RCPA)8; Kathryn N. North, MD, FRACP10; Daniel G. MacArthur, PhD3; Nigel F. Clarke, FRACP, PhD1,2
[+] Author Affiliations
1Institute for Neuroscience and Muscle Research, Kid’s Research Institute, Children’s Hospital at Westmead, Sydney, New South Wales, Australia
2Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
3Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston
4Department of Neurology, Concord General Repatriation Hospital, Sydney, New South Wales, Australia
5Western Australian Neurosciences Research Institute, University of Western Australia, Perth, Western Australia, Australia
6Department of Neurology, Royal North Shore Hospital, Sydney, New South Wales, Australia
7University of Sydney ANZAC Research Institute and Molecular Medicine Laboratory, Concord Hospital, Sydney, New South Wales, Australia
8Centre for Medical Research, University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia
9Department of Diagnostic Genomics, Pathwest Laboratory Medicine, Perth, Western Australia, Australia
10Murdoch Children’s Research Institute, The Royal Children’s Hospital, Melbourne, Victoria, Australia
JAMA Neurol. 2015;72(12):1424-1432. doi:10.1001/jamaneurol.2015.2274.
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Importance  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.

Design, Setting, and Participants  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.

Main Outcomes and Measures  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 and Relevance  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.

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Selection of Families With Limb-Girdle Muscular Dystrophy (LGMD) for Inclusion in Whole-Exome Sequencing (WES) and the Diagnoses Achieved

Of 237 families referred to the Institute for Neuroscience and Muscle Research, 83 had LGMDs that were diagnosed using standard techniques of immunohistochemistry (IHC), Western blotting (WB), candidate gene sequencing, and screening for type 2 myotonic dystrophy (DM2) and type 1 facioscapulohumeral muscular dystrophy (FSHD1). Based on the clinical indication, IHC was performed for merosin, α-dystroglycan, α-sarcoglycan, γ-sarcoglycan, β-sarcoglycan, δ-sarcoglycan, dysferlin, caveolin-3, desmin, myotilin, and type VI collagen, and/or WB was performed for dystrophin, α-dystroglycan, lamin A/C, emerin, caveolin-3, dysferlin, and calpain-3. Candidate genetic testing was then performed for LGMD1A-related MYOT mutations, LGMD1B-related LMNA mutations, LGMD1C-related CAV3 mutations, LGMD1E-related DES mutations, LGMD2A-related CAPN3 mutations, LGMD2B-related DYSF mutations, LGMD2C-related γ-sarcoglycan mutations, LGMD2D-related α-sarcoglycan mutations, LGMD2E-related β-sarcoglycan mutations, LGMD2F-related δ-sarcoglycan mutations, LGMD2I-related FKRP mutations, LGMD2K-related POMT1 mutations, LGMD2M-related FKTN mutations, LGMD2N-related POMT2 mutations, FSHD1, and DM2. Consent and DNA samples were obtained from 60 of the families with undiagnosed LGMDs for WES. Diagnosis was achieved for 27 families, including variants in genes not typically considered LGMD-related genes. Neurogenetic subexomic supercapture (NSES) confirmed all the sequence variants identified by WES and did not reveal any new pathogenic variants. There were 2 families for whom pathogenicity could not be confirmed in known myopathy-related genes.

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