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Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform
  1. Byung Chan Lim1,
  2. Seungbok Lee2,3,
  3. Jong-Yeon Shin2,4,
  4. Jong-Il Kim2,3,4,5,
  5. Hee Hwang1,
  6. Ki Joong Kim1,
  7. Yong Seung Hwang1,
  8. Jeong-Sun Seo2,3,4,5,6,
  9. Jong Hee Chae1
  1. 1Department of Pediatrics, Seoul National University College of Medicine, Seoul National University Children's Hospital, Seoul, Korea
  2. 2Genomic Medicine Institute (GMI), Medical Research Center, Seoul National University, Seoul, Korea
  3. 3Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Korea
  4. 4Psoma Therapeutics Inc., Seoul, Korea
  5. 5Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Korea
  6. 6Macrogen Inc., Seoul, Korea
  1. Correspondence to Professor Jong Hee Chae, Department of Pediatrics, Seoul National University College of Medicine, Seoul National University Hospital, 101, 110-744 Daehak-ro, Jongno-gu, Seoul, Korea; chaeped1{at}snu.ac.kr Dr Jeong-Sun Seo, Genomic Medicine Institute, Medical Research Center, Seoul National University College of Medicine 101 Daehak-ro, Jongno-gu, 110-799, Seoul, Korea; jeongsun{at}snu.ac.kr

Abstract

Background Duchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large size of the dystrophin gene require two or more analytical methods and have a high cost. The authors tested whether large deletions/duplications or small mutations, such as point mutations or short insertions/deletions of the dystrophin gene, could be predicted accurately in a single platform using next-generation sequencing technology.

Methods A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular-dystrophy-related genes. A multiplexing strategy, wherein four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyser, was applied. The study subjects were 25 patients: 16 with deficient dystrophin expression without a large deletion/duplication and 9 with a known large deletion/duplication.

Results Nearly 100% of the exonic region of the dystrophin gene was covered by at least eight reads with a mean read depth of 107. Pathogenic small mutations were identified in 15 of the 16 patients without a large deletion/duplication. Using these 16 patients as the standard, the authors' method accurately predicted the deleted or duplicated exons in the 9 patients with known mutations. Inclusion of non-coding regions and paired-end sequence analysis enabled accurate identification by increasing the read depth and providing information about the breakpoint junction.

Conclusions The current method has an advantage for the genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy wherein a comprehensive mutational search may be feasible using a single platform.

  • Duchenne muscular dystrophy
  • Becker muscular dystrophy
  • genetic diagnosis
  • next-generation sequencing
  • genetics
  • neurology
  • epilepsy and seizures

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Footnotes

  • BCL and SL contributed equally to this work.

  • Funding This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (Grant No. A080588) and Seoul Broadcasting System Grant-in-Aid for Seoul National University Children's Hospital Research (Grant No.30-2010-0190).

  • Competing interests None.

  • Ethics approval Ethics approval was provided by the Institutional Review Board of the Seoul National University Hospital.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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