Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: a multi-population diagnostic study

Abstract

Duchenne and Becker muscular dystrophies (DMD/BMD) are the most commonly inherited neuromuscular disease. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of the dystrophin gene and complex causative mutation spectrum. Such traditional methods as multiplex ligation-dependent probe amplification plus Sanger sequencing require multiple steps to fulfill the diagnosis of DMD/BMD. Here, we introduce a new single-step method for the genetic analysis of DMD patients and female carriers in real clinical settings and demonstrate the validation of its accuracy. A total of 89 patients, 18 female carriers and 245 non-DMD patients were evaluated using our targeted NGS approaches. Compared with traditional methods, our new method yielded 99.99% specificity and 98.96% sensitivity for copy number variations detection and 100% accuracy for the identification of single-nucleotide variation mutations. Additionally, this method is able to detect partial deletions/duplications, thus offering precise personal DMD gene information for gene therapy. We detected novel partial deletions of exons in nine samples for which the breakpoints were located within exonic regions. The results proved that our new method is suitable for routine clinical practice, with shorter turnaround time, higher accuracy, and better insight into comprehensive genetic information (detailed breakpoints) for ensuing gene therapy.

Noninvasive Prenatal Testing for Wilson Disease by Use of Circulating Single-Molecule Amplification and Resequencing Technology (cSMART)

“…Identification of the causative DMD mutations by conventional molecular diagnostic methods is complex, although recently, next generation sequencing methodologies have been developed to simplify the analysis (40 ). In the case of cSMART, deletion mutations would not be amenable to direct and equal targeting of the wild-type and mutant allele.…”

Clinical Utility Gene Card for: Becker muscular dystrophy

“…The DMD gene may be sequenced as part of an in-house gene panel, a commercially available sequencing gene panel such as TruSight One (Illumina), or a whole exome. Single-step methods able to detect exon copy variants and exonic point variants are also streamlining this process [23][24][25][26]. Point variants affecting splicing (including those deep within intronic regions of DMD) can affect RNA expression and/or processing.…”

“…MLPA has an analytical sensitivity of~71% and when combined with Sanger or massively parallel sequencing of the coding regions and splice sites following negative MLPA results, the analytical sensitivity becomes~97% [29]. Some mutational analyses using single platforms have achieved sensitivities from 92 to 99% [23,25,26]. Array CGH can identify complex rearrangements that go undetected by MLPA [36].…”

CUGC for Duchenne muscular dystrophy (DMD)

“…Then for cases where an exon copy number variant is not identified, or in positive cases where the patient is to be considered for exon-skipping therapy, sequencing of the entire coding region from genomic DNA should be performed [31]. Next generation sequencing (NGS) strategies facilitate this process and can include sub-exomic targeted gene panels or whole exome sequencing [32][33][34].…”