Development of a High Throughput Assay

Abstract

Purpose : The goal of our study was to develop a high throughput splicing assay (HTSA) to study the effect of rare variants of unknown significance (VUS) on splicing.

Methods : The HTSA is an improved version of a previously published splicing assay approach by Cheung et al. 2019, which relies on a split-GFP construct, into which test sequence is cloned and a control mCherry fluorophore. We changed the original construct to enable the study of out-of-frame exons. Our assay was tested by a control IRD exon oligo library (363 reference oligos and splice site mutations) and a variant library (1099 VUSs). The libraries were cloned as a pool into HTSA construct, expressed in HEK293T landing pad cells and FACS sorted into GFP-ve cells (exon inclusion) and GFP+ve cells (exon skipping). Deep sequencing of the integrated constructs enabled counting of the studied sequences and calculation of the exon inclusion index (EII). The impact of variants on exon inclusion compared to reference was calculated by ΔEII = EII_Var - EII_Ref. HTSA transcript sequencing was performed to determine other than exon-skipping events. A minigene assay was performed on a subset of variants to test HTSA accuracy and sensitivity. Constructs containing the exon of interest and flanking exons containing reference, variant, and splice site mutations were subcloned into pCSGW2+ and individually transfected into HEK293T cells. Transcripts were sequenced, aligned to hg38 reference genome and visualized by Sashimi plots.

Results : The improved HTSA construct performed well regardless of the frame of the studied exon and showed high reproducibility. The assay depends on strong splicing signals of the reference sequences, where 282 exons had EII>0.5 (median EII = 0.85). EII was significantly reduced by the essential splice site (SS) mutations, where mutating donor SS had a higher impact on exon skipping than the acceptor SS. (donor: EII_Med=0.06, acceptor:EII_Med=0.39). Of the 1099 studied VUSs, 103 led to decreased exon inclusion (ΔEII ≤ −0.3). Further evaluation of HTSA transcripts will be shown to reveal the possible intron retention and partial exon skipping events in the control and VUS library. Our minigene assay confirmed these outcomes when employed as a second method of validation in a subset of variants.

Conclusions : HTSA is a robust method to study simultaneously thousands of variants that may alter splicing, which can lead to an increase in genetic diagnoses.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.