Minigene Splice Assays Allow Pathogenicity Reclassification of

BACKGROUND/

objectivesObtaining a genetic diagnosis for patients with inherited retinal diseases has become even more important since gene-specific therapies have become available. When genetic screening reveals variants of uncertain significance (VUS), additional evidence is required to determine genetic eligibility for therapy. Confirming the effect on splicing that is predicted by SpliceAI could change their classification to either likely pathogenic or pathogenic and would therefore be of great importance when interpreting these variants when geneticists worldwide are trying to reach a diagnosis.

methodsUsing minigene assays, we established a pipeline to assess the effect on splicing for all variants. We selected 73

resultsUsing four wild-type vectors, we generated 59 constructs containing the variants of interest. Through these minigene assays, we assessed the effect on splicing of these VUS to enable reclassification. Upon quantification, we identified seven variants with a full, aberrant splicing effect without residual wild-type transcript. Eleven variants had between 5% and 20% remaining wild-type transcript. Forty-one variants had ≥20% residual wild-type transcript, among which fifteen variants showed no effect on splicing.

conclusionsFollowing the 2023 established ClinGen specific ACMG guidelines for