Figure 3.

Strengthening 3′ splice sites rescues splice defects due to pathogenic 5′-splice-site variants in ABCA4 Exons 3 and 47. (A) The effect of the pathogenic variant c.302+4A>C as well as the combination of this variant with the rescuing variant c.161-3A>C in construct BA1. The Exon 3 skipping was fully corrected. (B) Schematic overview of the ABCA4 BA1 construct containing Exons 2–4 used in A, annotated with the Human Splicing Finder (HSF) scores [0–100] of both variants. (C) Semi-quantification of the ratio of correctly (light gray rectangles) and aberrantly spliced (dark gray rectangles) RT-PCR products of WT splicing, as well as Exon 3 skipping in the BA1 construct. (D) The effect of the pathogenic variant c.6478A>G, the rescuing variants c.6387C>T, and their combination in the BA29 construct. The partial Exon 47 skipping was fully rescued after the introduction of the 3′ splice-site variant. (E) Schematic overview of the BA29 construct containing Exons 46–48 used in D, annotated with the HSF scores of both variants. (F) Semi-quantification of the ratio of correctly and aberrantly spliced RT-PCR products of WT splicing as well as Exon 47 skipping in the BA29 construct. Red lettering indicates pathogenic variants. Green lettering indicates variants that rescued exon skipping. When multiple aberrant products were observed, their percentages were summed up.