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.

日本語要旨:

Duchenne/Becker型筋ジストロフィー(DMD/BMD)の原因遺伝子であるジストロフィン遺伝子異常の診断に次世代シークエンス法(IonPGMによるシークエンス)を用いた。欠失は全例診断可能であり、一方重複は7割が評価可能であった。微小変異は連続した塩基の挿入変異の検出が困難であった。現行のMLPA法よりも診断率が高く、治療が期待されるナンセンス変異の検出も一度に可能であり、今後次世代シークエンス法がDMD/BMD診断の第一選択となることが期待される。