Pseudoexon activating by a deep intronic variant and phenotype variation in a Chinese family with dystrophinopathy

Aberrant inclusion of pseudoexons (PE) in mature mRNA is a rare splicing defect contributing to Duchenne muscular dystrophy (DMD) pathogenesis. In this study, we described two affected males from a Chinese family who presented with progressive muscle weakness, elevated creatine kinase (CK) levels, and dystrophic changes on muscle pathology. Whole-genome sequencing followed by linkage-based filtering identified a shared deep intronic variant in intron 47 of DMD gene (c.6913-4037T>G), which activated a cryptic splice site and resulted in the inclusion of a 72 bp PE between exons 47 and 48. Patient induced pluripotent stem cells (iPSCs)-derived myotubes from the patient confirmed the presence of this PE, with a significant reduction in dystrophin expression compared to controls. Quantitative PCR revealed that aberrant transcripts comprised ~89% of total DMD transcripts in myotubes and ~97% in muscle, correlating with near-complete loss of dystrophin. Functional assays further showed impaired myotube fusion and altered calcium signaling. This study underscores the diagnostic complexity of intronic DMD variants and provides evidence supporting the pathogenicity of c.6913-4037T>G.