Abstract
Purpose :
Splice-altering variants account for approximately 9% of all pathogenic retinal disease variants. Synonymous variants (sSNV) have been often overlooked as potentially altering mRNA splicing. SpliceAI is a neural network based software with high predictive value of splice variant outcomes. To evaluate SpliceAI in detection of pathogenic synonymous variants, we reviewed inherited eye-related genes from a public variant database.
Methods :
SpliceAI scores were calculated for synonymous variants from the Illumina splice variant database in 20 genes associated with inherited ophthalmic disease (ABCA4, CDHR1, CHD7, CNGA1, CNGA3, CNGB1, CNGB3, EYS, PAX6, PDE6A, PDE6B, PDE6C, PDE6H, PNPLA6, PRPH2, RHO, RP1, RP2, RS1, USH2A) and filtered for a score ≥0.8 in a single category (donor gain [DG], donor loss [DL], acceptor gain [AG], acceptor loss [AL]) (88 variants). A subset of these variants (21) was validated by minigene functional assay. The RHCglo vector was used for site-directed mutagenesis with the NEB HiFi DNA Assembly kit. HEK293 cells were used for transfection, followed by RT-PCR and sequencing.
Results :
Synonymous variants with high SpliceAI scores included 88 variants in 14 of 20 ophthalmic disease genes. Sixty-four variants are predicted to have DG, 13 with AG, 2 with AL, 1 with DL, and 8 with both DG and DL. Minigene assay for a novel sSNV in CDHR1 (c.759C>G:p.Gly253Gly) confirmed the predicted cryptic donor site resulted in a frameshift due to 25 bp deletion (DL:0.70; DG:0.99). This variant was observed in an individual with adult-onset maculopathy in trans with a CDHR1 frameshift variant (c.1690dup;p.Ile564Asnfs*8), thus confirming the molecular diagnosis. Minigene assays for an additional 20 sSNVs were performed and correlated to SpliceAI scores and genotype-phenotype.
Conclusions :
SpliceAI is a reliable predictive tool confirmed by minigene functional studies that may improve molecular diagnostics. Synonymous variants should be reviewed when other causes have not been found. Understanding expected splice outcomes is helpful in developing potential therapies for inherited ophthalmic diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.