Figure 2.

Splicing defects caused by five missense ABCA4 variants creating new SASs. The RT-PCR of RNA derived from mutant midigenes and the corresponding WT plasmids were visualized with gel electrophoresis. The green triangles represent the splice site predictions for acceptor gain (AG) by SpliceAI. Acceptor losses (AL; red triangles) were also shown if the delta scores (DSs) were > 0.10. Fragments for which the sequence information suggested a PCR artifact are indicated by asterisks. (A) For the mutant c.1977G>A midigene, two splicing defects were detected (fragments 2 and 4) that corresponded to a skip of the first 41 nt of exon 14 and a complex splice defect resulting in the absence of the first 41 nt of exon 14 and the complete exon 15. Skipping of exon 15 was also observed in the WT construct as seen previously (20). (B) RT-PCR and sanger sequencing of the c.3703A>G BA17 midigene revealed an in-frame 96-bp deletion (fragment 2) at the 5′ end of exon 25, as opposed to the WT BA17 which did not show this defect. (C) Missense variants c.4454C>T and c.4457C>T led to the generation of fragments that were confirmed by Sanger sequencing to lack exon 29 (fragment 2) completely, and a combination of a deletion of exon 29 and the first 114 nt of exon 30 (fragment 3). The latter was absent in the RNA derived from WT BA20. (D) Variant 5088C>G led to a novel transcript with an in-frame deletion of 69 nt at the 5′ end of exon 36 (fragment 2). A complete deletion of exon 35 was observed in fragment 3.