Deep-intronic variants in USH2A: the importance of model selection in variant interpretation and pathogenicity assessment

In the USH2A deep-intronic variants c.5573-834A>G and c.11047+345A>G, only one reproducibly drives pseudoexon inclusion, and residual wildtype transcripts preserve usherin expression in zebrafish.

• Why it matters: Splice-altering variants that affect only a subset of transcripts are hard to interpret for pathogenicity, risking overclassification based on alternative splicing alone.
• What they did: The authors tested both variants with a minigene splice assay and transcript analysis in patient-derived photoreceptor precursor cells and nasal epithelial cells, then modeled mixed transcript effects using the humanized ush2aPE40+M1 knock-in zebrafish.
• The result: c.5573-834A>G showed partial pseudoexon inclusion in patient-derived cells but did not reproduce in minigene, while c.11047+345A>G produced pseudoexon inclusion in minigene yet no detectable aberrant transcripts in patient-derived cells; zebrafish maintained detectable usherin expression, supporting likely benign for c.11047+345A>G and not supporting (likely) pathogenic for c.5573-834A>G.

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