Comprehensive Molecular Analysis of DMD Gene Increases the Diagnostic Value of Dystrophinopathies: A Pilot Study in a Southern Italy Cohort of Patients

Abstract

Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked neuromuscular disease due to pathogenic sequence variations in the dystrophin (DMD) gene, one of the largest human genes. More than 70% of DMD gene defects result from genomic rearrangements principally leading to large deletions, while the remaining are small nucleotide variants, including nonsense and missense variants, small insertions/deletions or splicing alterations. Considering the large size of the gene and the wide mutational spectrum, the comprehensive molecular diagnosis of DMD/BMD is complex and may require several laboratory methods, thus increasing the time and costs of the analysis. In an attempt to simplify DMD/BMD molecular diagnosis workflow, we tested an NGS method suitable for the detection of all the different types of genomic variations that may affect the DMD gene. Forty previously analyzed patients were enrolled in this study and re-analyzed using the next generation sequencing (NGS)-based single-step procedure. The NGS results were compared with those from multiplex ligation-dependent probe amplification (MLPA)/multiplex PCR and/or Sanger sequencing. Most of the previously identified deleted/duplicated exons and point mutations were confirmed by NGS and 1 more pathogenic point mutation (a nonsense variant) was identified. Our results show that this NGS-based strategy overcomes limitations of traditionally used methods and is easily transferable to routine diagnostic procedures, thereby increasing the diagnostic power of DMD molecular analysis.

Age-Related Blood Levels of Creatine Kinase-MM in Newborns and Patients with Duchenne Muscular Dystrophy: Considerations for the Development of Newborn Screening Algorithms

“…However, it has yet to be determined how feasible and time-effective NBS algorithms that involve or do not involve sequencing and repeat CK-MM will be. Additionally, residual clinical sensitivity limitations remain with respect to DMD sequencing; therefore, a subset of DMD cases may test positive with CK-MM screening but will not be detected by current targeted next-generation sequencing tests [ 33 , 34 , 35 ]. Repeat CK-MM testing may reduce the need for sequencing; however, a new specimen procurement for repeat CK-MM testing will incur a separate cost and may delay diagnosis.…”

A manifesting female carrier of Duchenne muscular dystrophy: Importance of genetics for the dystrophinopathies

“…Next-generation sequencing (NGS) technologies are increasingly considered more efficient and cost-effective compared to the traditional approach of MPLA/CGH and Sanger sequencing. [ 22 ] NGS allows for multiple genes to be sequenced and analysed in parallel, providing high throughput and ability to detect both large deletions/duplications and small point mutations in coding and non-coding regions, leading to the potential of improved molecular diagnosis of DMD. [ 23 ] Targeted NGS for dystrophinopathies is available as panels of neuromuscular genes, providing additional coverage for the diagnosis of related neuromuscular conditions or mimics.…”

NGS-based targeted sequencing identified six novel variants in patients with Duchenne/Becker muscular dystrophy from southwestern China

“…Therefore, if ES and the other capture-based NGS had been performed, the variant might not have been detected. According to De Palma FDE et al [ 16 ], the authors compared capture-based NGS with traditional methods (MLPA/mPCR/Sanger sequencing) and concluded that capture-based NGS overcomes the limitations of MLPA and Sanger sequences. The capture-based NGS is easy to apply in clinical practice and should be the main strategy for the diagnosis of DMD.…”