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

Interpreting splice-altering USH2A variants is challenging when these result in the alteration of splicing in only a fraction of transcripts. We investigated two deep-intronic USH2A variants from a single case, the inherited variant c.5573-834A>G and the de novo variant c.11047+345A>G, using a minigene splice assay and transcript analysis in patient-derived photoreceptor precursor cells and nasal epithelial cells. The minigene assay revealed the inclusion of a pseudoexon in the presence of the c.11047+345A>G variant, but did not reproduce previously reported aberrant splicing for c.5573-834A>G. In patient-derived cells, c.5573-834A>G resulted in the inclusion of a pseudoexon in part of the transcripts, whereas c.11047+345A>G did not result in detectable levels of aberrantly spliced transcripts. To assess the impact of residual wildtype transcripts on retinal function, we used the previously developed humanized ush2aPE40+M1 knock-in zebrafish model which allows the modeling of the impact of mixed wildtype and pseudoexon-containing ush2a transcripts. This model demonstrated that detectable levels of usherin expression are maintained even when only a fraction of wildtype transcripts lacking pseudoexon inclusion remain. Collectively, these findings advocate for a more nuanced classification of partially splice-altering USH2A variants. The mere presence of alternative transcripts should not be considered sufficient evidence for pathogenicity. Within this framework, data on the c.5573-834A>G variant do not support a (likely) pathogenic classification, and findings related to the c.11047+345A>G variant are consistent with a likely benign classification.