title: Genetic diagnosis of Duchenne/Becker muscular dystrophy using next-generation sequencing: validation analysis of DMD mutations.
abstract: Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited neuromuscular disease. The genetic diagnosis is not easily made because of the large size of the dystrophin gene, complex mutational spectrum and high number of tests patients undergo for diagnosis. Multiplex ligation-dependent probe amplification (MLPA) has been used as the initial diagnostic test of choice. Although MLPA can diagnose 70% of DMD/BMD patients having deletions/duplications, the remaining 30% of patients with small mutations require further analysis, such as Sanger sequencing. We applied a high-throughput method using Ion Torrent next-generation sequencing technology and diagnosed 92% of patients with DMD/BMD in a single analysis. We designed a multiplex primer pool for DMD and sequenced 67 cases having different mutations: 37 with deletions/duplications and 30 with small mutations or short insertions/deletions in DMD, using an Ion PGM sequencer. The results were compared with those from MLPA or Sanger sequencing. All deletions were detected. In contrast, 50% of duplications were correctly identified compared with the MLPA method. Small insertions in consecutive bases could not be detected. We estimated that Ion Torrent sequencing could diagnose ~92% of DMD/BMD patients according to the mutational spectrum of our cohort. Our results clearly indicate that this method is suitable for routine clinical practice providing novel insights into comprehensive genetic information for future molecular therapy.
authors.affiliation: Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan.|Department of Child Neurology, National Center Hospital, NCNP, Tokyo, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Okubo
authors.affiliation: Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan.|Department of Genome Medicine Development, Medical Genome Center, NCNP, Tokyo, Japan.|Division of Genetic Diagnosis, Department of Laboratory Medicine, National Center Hospital, NCNP, Tokyo, Japan.
authors.forename: Narihiro
authors.initials: N
authors.lastname: Minami
authors.affiliation: Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan.|Department of Genome Medicine Development, Medical Genome Center, NCNP, Tokyo, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Goto
authors.affiliation: Department of Genome Medicine Development, Medical Genome Center, NCNP, Tokyo, Japan.|Division of Genetic Diagnosis, Department of Laboratory Medicine, National Center Hospital, NCNP, Tokyo, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Goto
authors.affiliation: Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Noguchi
authors.affiliation: Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan.|Department of Genome Medicine Development, Medical Genome Center, NCNP, Tokyo, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Mitsuhashi
authors.affiliation: Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan.|Department of Genome Medicine Development, Medical Genome Center, NCNP, Tokyo, Japan.
authors.forename: Ichizo
authors.identifier: http://orcid.org/0000-0001-9452-112X
authors.initials: I
authors.lastname: Nishino
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D016022:Case-Control Studies; D016189:Dystrophin / Q000235:genetics*; D005091:Exons; D018628:Gene Dosage; D020440:Gene Duplication; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007438:Introns; D020388:Muscular Dystrophy, Duchenne / Q000175:diagnosis* / Q000235:genetics*; D009154:Mutation; D017384:Sequence Deletion
chemical_list: D016189:Dystrophin