Genetic diagnosis of Duchenne muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform

Next generation sequencing technology(NGS) can be a useful tool for genetic diagnosis of certain diseases where laborious sequencing efforts are required because of the large gene size or the presence of multiple causative genes. We considered Duchenne/Becker muscular dystrophy as a suitable candidate disease for this purpose because the complex mutational spectrum and large size of the dystrophin gene require two or more different analytical metholdsand have a high cost. A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular dystrophy-related genes (SureSelect Target Enrichment System kit, Agilent Technology). A multiplexing strategy was applied in which four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyzer. Using these methods, whole spectrum of the dystrophin gene mutations from point mutations or short insertion/deletions to large deletion/duplication mutations were successfully identified. These methods would save the time to reach the genetic diagnosis of Duchenne/Becker muscular dystrophy compared with the current methods, combination of multiple ligation-dependent probe amplification (MLPA) and Sanger sequencing. Moreover, multiplexing and inclusion of other muscular dystrophy related genes might contribute to reducing the cost, which is one of the main obstacles for clinical application of NGS. (By Byung Chan Lim MD, )

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