Genetic Identification of DMD Gene Pathogenic Mutations: Retrospective Genetic Analysis of 507 Patients Based on Next-Generation Sequencing

Background and purpose: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular diseases in children that resulted from mutations in the dystrophin gene. DMD stay in a high fatality rate, and currently, there is no specific medication or cure available. A comprehensive analysis of DMD gene mutations was conducted in patients with DMD/BMD to better understand the characteristics of the mutations and the intrinsic relationship between genotype and phenotype. The aim of this study is to improve the diagnosis and treatment efficacy of DMD/BMD by addressing the root cause of genetic mutations, thus providing a theoretical foundation for further development of exon skipping treatments. Methods: Between October 2018 and July 2021, a total of 507 DMD/BMD patients were collected from Hunan Provincial Children's Hospital. Initially, Multiplex Ligation-dependent Probe Amplification (MLPA) was employed to detect deletions and duplications in the 79 exons of the DMD gene. If negative, Next-Generation Sequencing (NGS) was used subsequently to identify point mutations. Finally, Sanger sequencing was utilized to confirm the identified point mutations. Results: Combined with MLPA, NGS and Sanger sequencing proved to be a cost-effective and high-efficient approach in providing gene diagnosis services for DMD/BMD patients. Among the 507 patients with DMD/BMD, the highest percentage of mutations observed was deletions in exons (64.9%), followed by point mutations (26.0%) and duplication mutations in exons (9.1%). The pathogenicity ratio of these mutations was P: LP: VUS=24.5:2.67:1. The hotspot regions of exon deletions in the DMD gene were primarily found in the distal exons in the range of 45-55 (79.64%), as well as in the proximal exons in the range of 2-20 (15.20%). The hotspot regions for exon duplications were concentrated in the proximal exons within the range of 3-9 (39.13%). Point mutations were distributed throughout the DMD gene, with the top four exons showing the highest mutation frequency being exon 22 with 9 mutations, exon 70 with 7 mutations, and exon 20 with 6 mutations. the top four exons with the highest mutation frequency are exon 22 with 9 mutations, exon 70 with 7 mutations, and exon 70 and exon 20 with 6 mutations, respectively. Additionally, 40 de novo mutations were identified in this study.