A high throughput splicing assay to investigate the effect of variants of unknown significance on exon inclusion

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 r2 = 0.83). Reference exon sequences showed a high level of exon recognition (median EII = 0.88) which was significantly reduced by mutations to the essential splice sites (donor site variants: median EII=0.06; acceptor site variants: median EII=0.48). Of the 748 studied VUSs, 47 variants led to decreased exon inclusion (ΔEII ≤ −0.3) with 11 variants showing a strong effect (ΔEII ≤ −0,6). Using the HTSA data we were able to provide a likely genetic diagnosis to five 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement 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. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Institutional Review Board at the Massachusetts Eye and Ear (Human Studies Committee MEE, Mass General Brigham, USA) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The raw data from the splicing assay are provided in the supplementary files