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,

The authors have declared no competing interest.

This work was supported by grants from the National Eye Institute R01EY026904 (KMB/EAP), [R01EY012910 (EAP) and P30EY014104 (MEEI core support)], the Foundation Fighting Blindness [EGI-GE-1218-0753-UCSD, (KMB/EAP)] and the Research to Prevent Blindness International Research Collaborators Award (KMB). Exome sequencing and analysis were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and was funded by the National Human Genome Research Institute, the National Eye Institute, and the National Heart, Lung and Blood Institute grant UM1HG008900 and in part by National Human Genome Research Institute grant R01 HG009141.

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The study was approved by the Institutional Review Board at the Massachusetts Eye and Ear (Human Studies Committee MEE, Mass General Brigham, USA)

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The raw data from the splicing assay are provided in the supplementary files