Classification of noncanonical splice site variants according to the splice defects The percentage of correctly spliced ABCA4 RNA product for NCSS variants was calculated by using a capillary electrophoresis system (Fig. 4; Supplemental Table S3; Supplemental Fig. S5). Three variants, c.4634G>A, c.5196+3_5196+8del, and c.5585−10T>C, showed 100% correctly spliced ABCA4 mRNAs. We could rule out pathogenicity for the last two variants but not for c.4634G>A p.(Ser1545Asn), as this variant is significantly enriched in >3000 Caucasian STGD1 patients compared to the non-Finnish ExAC population (Cornelis et al. 2017). Sixteen variants showed between 4.3% and 79.6% normal RNA and were tentatively classified as severe, moderately severe, and mild, while the remaining 26 NCSS variants did not show any normal RNA and were thus deemed severe variants. For c.2382+5G>C and c.2588G>C, no quantification was performed as the wild-type BA12 clone also showed exon skipping. Based on the Sanger sequence validation, we were able to annotate all the variants at the RNA level and predict their effect at the protein level (Table 1). Figure 4. Open in a new tab Percentages of remaining normal ABCA4 transcripts due to noncanonical splice site variants based on capillary electrophoresis system analyses. The percentages of normal ABCA4 transcript for 45 noncanonical splice site variants are represented by black bars. Nineteen variants showed varying fractions (4.3%–100%) of correct ABCA4 mRNA. Four of them also result in missense changes as depicted, but only p.(Ser1545Asn) and p.(Lys2160Glu) are likely to have an effect on protein function, as significant amounts of ABCA4 protein will be produced. For the remaining 26 variants (in the square box), no residual RNA was observed. (#) For this variant, densitometric scanning was performed.Table 1. In vitro tested noncanonical splice site variants and their observed RNA and predicted protein effects Open in a new tabWe correlated the predicted effect of the NCSS variants with the phenotypes in reported cases and, when the variants were not present in a homozygous state, with the severity of the second allele. For 25 of 47 variants, sufficient clinical data were available, and in all these cases, our predicted effect of the NCSS variants correlated with the observed phenotypes (Supplemental Table S4).