Objective To investigate the diagnostic value of implementing a stepwise genetic testing strategy (SGTS) in genetically unsolved cases with dystrophinopathies.
Methods After routine genetic testing in 872 male patients with highly suspected dystrophinopathies, we identified 715 patients with a pathogenic DMD variant. Of the 157 patients who had no pathogenic DMD variants and underwent a muscle biopsy, 142 patients were confirmed to have other myopathies, and 15 suspected dystrophinopathies remained genetically undiagnosed. These 15 patients underwent a more comprehensive evaluation as part of the SGTS pipeline, which included the stepwise analysis of dystrophin mRNA, short-read whole-gene DMD sequencing, long-read whole-gene DMD sequencing and in silico bioinformatic analyses.
Results SGTS successfully yielded a molecular diagnosis of dystrophinopathy in 11 of the 15 genetically unsolved cases. We identified 8 intronic and 2 complex structural variants (SVs) leading to aberrant splicing in 10 of 11 patients, of which 9 variants were novel. In one case, a molecular defect was detected on mRNA and protein level only. Aberrant splicing mechanisms included 6 pseudoexon inclusions and 4 alterations of splice sites and splicing regulatory elements. We showed for the first time the exonisation of a MER48 element as a novel pathogenic mechanism in dystrophinopathies.
Conclusion Our study highlights the high diagnostic utility of implementing a SGTS pipeline in dystrophinopathies with intronic variants and complex SVs.
- neuromuscular diseases
- RNA cleavage
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Contributors ZX, ZW and YY conceived and planned this study and take full responsibility for the paper. ZX, IS and ZW contributed to the methodology of the study. ZX, CS, YL, MY, YZ and LM conducted clinical and genetic research. JY and LZ conducted the experiments of dystrophin cDNA analysis. IS checked all the genetic analyses. ZX, WZ, ZW and YY contributed to the analysis and interpretation of the clinical and muscle MRI data. ZX took the lead in writing the manuscript. WZ, ZW and YY supervised this study. ZX, GW, DMC-S, TB, NP-T, SML, IS, ZW and YY contributed to the revisions of the manuscript. All authors read and approved the final manuscript.
Funding This study was supported by a grant from the Ministry of Science and Technology of China (grant number 2016YFC1300605) and the Beijing Municipal Science and Technology Commission (grant number Z191100006619034). ZX is supported by a grant from the China Scholarship Council (student ID 201906010371).
Competing interests JY and LZ are employees of Beijing Epigen Medical Technology Inc. Both of them conducted the experiments of dystrophin cDNA analysis.
Patient consent for publication Not required.
Ethics approval This study and related protocols were approved by the Ethics Committee at Peking University First Hospital (2019181) and performed in accordance with the tenets of the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from all the study participants and/or their parents.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request.
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