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

Area of Science:

• Genetics
• Molecular Biology
• Neuromuscular Disorders

Background:

• Aberrant pseudoexon (PE) inclusion in mRNA is a rare splicing defect implicated in Duchenne muscular dystrophy (DMD).
• Diagnosing intronic variants causing splicing defects in DMD can be challenging.

Purpose of the Study:

• To investigate the molecular mechanism of a deep intronic variant in the DMD gene identified in two Chinese males with Duchenne muscular dystrophy.
• To confirm the pathogenicity of the identified variant and its role in DMD pathogenesis.

Main Methods:

• Whole-genome sequencing and linkage-based filtering to identify genetic variants.
• Utilized patient-derived induced pluripotent stem cells (iPSCs) and differentiated them into myotubes for functional studies.
• Quantitative PCR (qPCR) to assess aberrant transcript levels and Western blotting (implied by dystrophin expression) to evaluate protein levels.

Main Results:

• Identified a deep intronic variant (c.6913-4037T>G) in intron 47 of the DMD gene.
• This variant activated a cryptic splice site, leading to the inclusion of a 72 bp pseudoexon (PE) between exons 47 and 48 in DMD mRNA.
• Aberrant transcripts containing the PE were highly abundant (~89% in myotubes, ~97% in muscle), resulting in near-complete loss of dystrophin expression.
• Patient-derived myotubes exhibited impaired fusion and altered calcium signaling.

Conclusions:

• The deep intronic variant c.6913-4037T>G is pathogenic and causes Duchenne muscular dystrophy through pseudoexon inclusion and subsequent loss of dystrophin.
• This finding highlights the diagnostic complexity associated with intronic DMD variants and expands the mutational spectrum of DMD.