Minigene Splice Assays Allow Pathogenicity Reclassification of RPE65 Variants of Uncertain Significance

Background/objectives: Obtaining 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. Methods: Using minigene assays, we established a pipeline to assess the effect on splicing for all variants. We selected 73 RPE65 variants that were classified as either VUS or likely benign in the RPE65 Leiden Open Variant Database (LOVD) or ClinVar and were predicted to affect splicing by SpliceAI with a delta score of >0.1 and by using an analysis window of 5000 bp up- and downstream of the variant. Results: Using 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. Conclusions: Following the 2023 established ClinGen specific ACMG guidelines for RPE65 (Criteria Specification Registry), evidence from splice assays enabled reclassification of seven RPE65 variants from VUS to pathogenic through an assigned PVS1-very-strong criterium, as less than 5% of wild-type transcript was present. These findings contribute to the interpretation of variants observed in patients, which will in turn dictate their eligibility for gene therapy.