title: Validation of ambiguous MLPA results by targeted next-generation sequencing discloses a nonsense mutation in the DMD gene.
abstract: BACKGROUND
  Duchenne muscular dystrophy (DMD) is the most common inherited muscular disease and caused by mutations in the DMD gene on the X-chromosome. Multiplex ligation-dependent probe amplification (MLPA) is recognized as a convenient and reliable technique to detect exon deletion/duplication mutations in the DMD gene. Here, we applied targeted semi-conductor next-generation sequencing to clarify the cause of ambiguous MLPA results.
  
  
  METHODS
  Targeted semi-conductor next-generation sequencing was carried out using the Inherited Disease Panel (IDP) on the Ion Torrent Personal Genome Machine (PGM).
  
  
  RESULTS
  MLPA analysis disclosed unclassifiable relative peak ratio of exon 18 in a DMD boy. His female cousin was indicated to have exon 18 deletion in one allele. To validate these incompatible results, targeted next-generation sequencing was conducted. A nucleotide change, C.2227 C>T creating a premature stop codon, was in exon 18. Concomitantly, both C and T nucleotides were identified in his cousin's genome. Ambiguous values of the relative peak ratio in MLPA were considered due to the one nucleotide mismatch between the genomic sequence and the probe used in MLPA.
  
  
  CONCLUSIONS
  Analysis using IDP on PGM disclosed a nonsense mutation in the DMD gene as a cause of ambiguous results of MLPA.
authors.affiliation: Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, 518 Arise, Ikawadani, Nishi, Kobe, 651-2180, Japan. Electronic address: emma@reha.kobegakuin.ac.jp.
authors.forename: Emma Tabe Eko
authors.initials: ET
authors.lastname: Niba
authors.affiliation: Center for Gene and Protein Research, Hanoi Medical University, Hanoi, Vietnam. Electronic address: tranvankhanh@hmu.edu.vn.
authors.forename: Van Khanh
authors.initials: VK
authors.lastname: Tran
authors.affiliation: Center for Gene and Protein Research, Hanoi Medical University, Hanoi, Vietnam. Electronic address: tuanpla@hus.edu.vn.
authors.forename: Le Anh
authors.initials: le A
authors.lastname: Tuan-Pham
authors.affiliation: Department of Medical Genetics, Metabolism & Endocrinology and Clinical Research's Division in Research Institute for Child Health Vietnam, National Hospital of Pediatrics, Hanoi, Vietnam. Electronic address: dungvu@nhp.org.vn.
authors.forename: Dung Chi
authors.initials: DC
authors.lastname: Vu
authors.affiliation: Department of Medical Genetics, Metabolism & Endocrinology and Clinical Research's Division in Research Institute for Child Health Vietnam, National Hospital of Pediatrics, Hanoi, Vietnam. Electronic address: khanhnn@nhp.org.vn.
authors.forename: Ngoc Khanh
authors.initials: NK
authors.lastname: Nguyen
authors.affiliation: Center for Gene and Protein Research, Hanoi Medical University, Hanoi, Vietnam. Electronic address: tathanhvan@hmu.edu.vn.
authors.forename: Van Thanh
authors.initials: VT
authors.lastname: Ta
authors.affiliation: Center for Gene and Protein Research, Hanoi Medical University, Hanoi, Vietnam. Electronic address: tranhuythinh@hmu.edu.vn.
authors.forename: Thinh Huy
authors.initials: TH
authors.lastname: Tran
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuo, Kobe, 650-0017, Japan. Electronic address: leetomo@med.kobe-u.ac.jp.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Lee
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuo, Kobe, 650-0017, Japan. Electronic address: takeshima@med.kobe-u.ac.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeshima
authors.affiliation: Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, 518 Arise, Ikawadani, Nishi, Kobe, 651-2180, Japan. Electronic address: mmatsuo@reha.kobegakuin.ac.jp.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Matsuo
mesh_terms: D000293:Adolescent; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis / Q000379:methods*; D016189:Dystrophin / Q000235:genetics*; D005260:Female; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007223:Infant; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D015203:Reproducibility of Results
chemical_list: D018389:Codon, Nonsense; C484258:DMD protein, human; D016189:Dystrophin
keywords: DMD; Duchenne muscular dystrophy; MLPA; Mutation