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

Abstract

Background Duchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large size of the dystrophin gene require two or more analytical methods and have a high cost. The authors tested whether large deletions/ duplications or small mutations, such as point mutations or short insertions/deletions of the dystrophin gene, could be predicted accurately in a single platform using next-generation sequencing technology. Methods A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular-dystrophy-related genes. A multiplexing strategy, wherein four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyser, was applied. The study subjects were 25 patients: 16 with deficient dystrophin expression without a large deletion/duplication and 9 with a known large deletion/duplication. Results Nearly 100% of the exonic region of the dystrophin gene was covered by at least eight reads with a mean read depth of 107. Pathogenic small mutations were identified in 15 of the 16 patients without a large deletion/duplication. Using these 16 patients as the standard, the authors' method accurately predicted the deleted or duplicated exons in the 9 patients with known mutations. Inclusion of non-coding regions and paired-end sequence analysis enabled accurate identification by increasing the read depth and providing information about the breakpoint junction. Conclusions The current method has an advantage for the genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy wherein a comprehensive mutational search may be feasible using a single platform.

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

“…19 Recent studies applying NGS have proven its ability in detecting SNVs and large deletions/ duplications in the dystrophin gene in males. 15,20 However, the detection of CNV in female carriers by targeted NGS approaches had not been reported to date. Compared with haploid X chromosomes in male, the CNV detection in females with diploid X chromosomes would be more complex.…”

“…13,14 Lim et al 15 reported a mutational search platform for the genetic diagnosis of DMD/BMD, but only a small number of samples was included; these authors only reported the detection of large deletion/duplication mutations in male patients. In the present study, we developed a novel computational framework and applied this platform to identify copy number variations (CNVs) and single-nucleotide variations (SNVs) in a large cohort at the same time.…”

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].…”

Genetic diagnosis of Duchenne/Becker muscular dystrophy using next-generation sequencing: Validation analysis of DMD mutations

“…Lim et al 13 reported a mutational search platform for the genetic diagnosis of DMD/BMD. Wei et al 14 also reported a mutational search for DMD/BMD and female carriers using targeted NGS on HiSeq2000 (Illumina, San Diego, CA, USA).…”