Purpose: Inconclusive interpretation of pathogenicity of variants is a common problem in Mendelian disease diagnostics. We hypothesized that some variants of unknown significance (VUS) may lead to aberrant pre-mRNA splicing. To address this we have developed a high throughput splicing assay (HTSA) than can be utilized to test the effects of 1000s of variants on exon recognition. Methods: 2296 reference, control and variant sequences from 380 exons of 89 genes associated with inherited retinal degenerations (IRDs) were cloned as a pool into a split-GFP HTSA construct and expressed in landing pad RCA7 HEK293T cells. Exon inclusion led to disruption of GFP and exon skipping led to GFP reconstitution, enabling to separate GFP+ve and GFP-ve cells by fluorescence activated cell sorting. After deep sequencing-based quantification of studied sequences in each cell pool, exon inclusion index (EII) was determined, where EII = GFP-ve oligo count/total oligo count. Results: HTSA showed high reproducibility when compared between different biological replicates (tetrachoric correlation coefficient r = 0.91). Reference exon sequences showed a high level of exon recognition (median EII = 0.85) which was significantly reduced by mutations to the essential splice sites (donor site variants: median EII=0.06; acceptor site variants: median EII=0.39). Of the 1099 studied VUSs, 103 variants led to decreased exon inclusion (∆EII ≤ -0.3) with 23 variants showing a strong effect (∆EII ≤ -0,6). Using the HTSA data we were able to provide a likely genetic diagnosis to ten IRD cases. Conclusion: HTSA offers a robust method to study the effects of VUSs on exon recognition allowing to provide new diagnoses for patients with Mendelian disorders. Key Words: High throughput splicing assay, Mendelian trait, inherited retinal degeneration,
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