Abstract
Purpose :
Inconclusive interpretation of variant pathogenicity is a common problem in Mendelian disease diagnostics. Variants outside of the canonical splice sites can lead to splicing alteration and sometimes disease. The purpose of this study was to test if rare variants of unknown significance (VUS) in or in a proximity of an exon lead to aberrant pre-mRNA splicing.
Methods :
We developed a high throughput splicing assay, which relies on a split GFP mini gene. Reference, control and variant sequences (2296 oligos) from 380 exons of 89 inherited retinal degeneration (IRD) genes were cloned as a pool into the split GFP intron and expressed in landing pad RCA7 HEK293T cells. Exon inclusion led to GFP disruption and exon skipping led to GFP reconstitution, enabling to separate GFP+ve and GFP-ve cells by fluorescence activated cell sorting. NGS read-based counts of each studied oligo per GFP cell pool enabled calculation of the exon-inclusion ratio (% spliced in) and subsequent comparison of the reference and variant sequences.
Results :
In six recessive IRD cases, we were able to provide functional evidence of splicing defect for VUSs that were coupled with likely pathogenic variants in the same gene. Variants in ABCA4 (c.1928T>G, p.Val643Gly; c.5693G>A, p.Arg1898His, c.5680C>T p(=)) and USH2A (c.5298+3A>G ) led to exon skipping in 34-69% of transcripts. Variants in EYS (c.1056+3A>C) and FLVCR1 (c.1026T>C p.(=)) led to exon skipping in over 70% of transcripts. Three additional male cases carried mutations in RPGR (c.310G>A, p.Glu104Lys; c.310+7T>G) and CHM (c.940G>A, p.Gly314Arg) that led to exon skipping in over 60% of transcripts. One subject carried a c.2237G>A, p.Ser746Asn allele in JAG1leading to an in-frame exon 18 skipping in 30% of transcripts. It is unclear if this variant is the cause of disease in the studied subject, however given that JAG1 is mutation intolerant and dosage sensitive, it important to consider the effect on splicing of this allele.
Conclusions :
HTSA offers a robust method to study the effects of VUSs on splicing. However, interpretation of the pathogenicity of variants that lead to splicing alterations is a complex process that requires consideration of multiple criteria such as: the extent of exon skipping; the frame of the exon that is eliminated; protein domains encoded by the skipped in-frame exon; mode of inheritance and pathogenicity of the allele in trans for recessive genes; gene dosage sensitivity.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.