Molecular Diagnosis of Duchenne Muscular Dystrophy Using Single NGS‐Based Assay

Duchenne Muscular Dystrophy (DMD) is an X-linked inherited neuromuscular disorder caused by pathogenic variants in the dystrophin gene (DMD; locus Xp21.2). The variant spectrum of DMD is unique in that 65% of causative mutations are intragenic deletions, with intragenic duplications and point mutations (along with other sequence variants) accounting for 6% to 10% and 30% to 35%, respectively. The traditional strategy for molecular diagnostic testing for DMD involves initial screening for deletions/duplications using microarray-based comparative genomic hybridization followed by a full-sequence analysis of DMD for sequence variants. This traditional strategy is expensive and time-consuming due to the involvement of two separate tests to detect all types of variants in the DMD gene. Recent advancements in next-generation sequencing (NGS) technology and improvements in analysis algorithms related to copy number variant detection ultimately resulted in the development of a single NGS-based assay to detect all variant types, including deletions/duplications and sequence variants. This article initially discusses the strategic algorithm for establishing a molecular diagnosis of DMD and later provides detailed molecular diagnostic protocols for DMD, including an NGS-based sequencing assay with sequence and copy number variant analysis.