Whole dystrophin gene analysis by next-generation sequencing: a comprehensive genetic diagnosis of Duchenne and Becker muscular dystrophy

Abstract

Duchenne/Becker muscular dystrophies are the most frequent inherited neuromuscular diseases caused by mutations of the dystrophin gene. However, approximately 30% of patients with the disease do not receive a molecular diagnosis because of the complex mutational spectrum and the large size of the gene. The introduction and use of next-generation sequencing have advanced clinical genetic research and might be a suitable method for the detection of various types of mutations in the dystrophin gene. To identify the mutational spectrum using a single platform, whole dystrophin gene sequencing was performed using next-generation sequencing. The entire dystrophin gene, including all exons, introns and promoter regions, was target enriched using a DMD whole gene enrichment kit. The enrichment libraries were sequenced on an Illumina HiSeq 2000 sequencer using paired read 100 bp sequencing. We studied 26 patients: 21 had known large deletion/duplications and 5 did not have detectable large deletion/duplications by multiplex ligation-dependent probe amplification technology (MLPA). We applied whole dystrophin gene analysis by next-generation sequencing to the five patients who did not have detectable large deletion/duplications and to five randomly chosen patients from the 21 who did have large deletion/duplications. The sequencing data covered almost 100% of the exonic region of the dystrophin gene by ≥10 reads with a mean read depth of 147. Five small mutations were identified in the first five patients, of which four variants were unreported in the dmd.nl database. The deleted or duplicated exons and the breakpoints in the five large deletion/duplication patients were precisely identified. Whole dystrophin gene sequencing by next-generation sequencing may be a useful tool for the genetic diagnosis of Duchenne and Becker muscular dystrophies.

Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and neuromuscular, rehabilitation, endocrine, and gastrointestinal and nutritional management

“…22 These mutations include point mutations (nonsense or missense), small deletions, and small duplications or insertions, which can be identified using next-generation sequencing. 23–25 Finally, if genetic testing does not confirm a clinical diagnosis of DMD, then a muscle biopsy sample should be tested for the presence of dystrophin protein by immunohistochemistry of tissue cryosections or by western blot of a muscle protein extract.…”

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.…”

Duchenne muscular dystrophy in the Western Cape, South Africa: Where do we come from and where are we going?

“…Because of its large size, testing for small/point mutations in the DMD gene is challenging and has not yet become part of the routine diagnostic repertoire of the NHLS laboratories. Highresolution melting curve analysis (HRM) for small/point mutation detection was successfully attempted locally and the results were published by Esterhuizen et al [11] However, HRM in B/DMD has been superseded by next-generation sequencing (NGS) [12] which is currently undergoing validation in the Division of Human Genetics at UCT and the NHLS in Cape Town. Fig.…”