Dystrophinopathies: A NGS approach for the molecular analysis of DMD gene

The DMD gene, located on Xp21, comprises 79 exons along 2400 kb, whose mutations originate Duchenne/Becker muscular dystrophies (DMD/BMD). 70% of DMD/BMD patients show deletions/duplications of one or more exons. The remaining 30% present a nonsense, missense, frameshift or splicing mutations, distributed along the entire gene. Detection of point mutations is an expensive task in terms of technical and economic issues. The introduction of next-generation sequencing technology (NGS) in our laboratory has allowed exponentially increased sequencing throughput. We studied DNA samples from 80 patients with clinical suspicion of DMD/BMD who had previously ruled out the presence of an exon deletion or duplication in the DMD gene. The generation of amplicon libraries was performed using the kit DMD MASTR of Multiplicom and they were sequenced using the MiSeq platform from Illumina. Sequencing data were analysed using Variant Studio and DNA Nexus softwares. The pathogenic changes were confirmed by Sanger sequencing and were compared with the LOVD database and Alamut and Polyphen-2 softwares. We have identified 38 mutations, which have been confirmed as the cause of the pathology. The analysis of point mutations in the DMD gene by NGS is a breakthrough in molecular analysis time and cost, compared to the Sanger technique. Thus, targeted NGS can contribute to alleviate the diagnosis delay and to overtake the genetic counselling. In those patients in whom no mutation was identified in gDNA, the DMD/BMD clinical diagnosis should be confirmed by immunohistochemistry in muscular biopsies and afterwards the mRNA sequencing.