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

Area of Science:

• Genetics
• Molecular Biology
• Ophthalmology

Background:

• Genetic diagnosis is crucial for inherited retinal diseases (IRDs) due to emerging gene-specific therapies.
• Variants of uncertain significance (VUS) require further evidence for accurate classification and therapeutic eligibility.
• Predictive tools like SpliceAI can identify splicing alterations, but experimental validation is essential.

Purpose of the Study:

• To establish a functional assay pipeline to assess the splicing impact of VUS in the RPE65 gene.
• To reclassify RPE65 variants of uncertain significance (VUS) based on experimental splicing data.
• To determine patient eligibility for gene therapy by clarifying the pathogenicity of RPE65 variants.

Main Methods:

• Selected 73 RPE65 variants predicted to affect splicing by SpliceAI (delta score >0.1).
• Utilized minigene assays with 59 generated variant constructs to assess splicing effects.
• Quantified residual wild-type transcript levels to determine splicing aberration severity.

Main Results:

• Seven variants exhibited complete aberrant splicing (0% wild-type transcript).
• Eleven variants showed 5-20% residual wild-type transcript.
• Fifteen variants among 41 with >=20% residual transcript demonstrated no splicing effect.

Conclusions:

• Splice assay evidence enabled reclassification of seven RPE65 VUS to pathogenic (PVS1-very strong criterion).
• This functional validation aligns with ClinGen ACMG guidelines for RPE65 variant interpretation.
• Accurate variant classification improves diagnostic yield and guides gene therapy selection for IRDs.