Fig. 3: Splice defects due to variants in ABCA4 introns 13 and 44.

a Genomic structure of intron 13 containing three pseudoexons (PEs) due to four deep-intronic variants. PE2 and PE3 share a splice donor site (SDS) (for PE2) and splice acceptor site (for PE3). Variants c.1938-621G>A and c.1938-619A>G strengthen the same cryptic SDS of PE2 slightly or strongly, respectively, as based on the Human Splicing Finder (HSF). Variant c.1938-514A>G creates a new strong SDS of PE3. The canonical and putative canonical splice sequences are given in bold lettering. The first and last positions of the PEs are provided. b Agarose gel analysis of reverse-transcription polymerase chain reaction (RT-PCR) products for intron 13 variants upon HEK293T cell splice assays. PE2 and PE3 were observed as single insertions, but also in combination with PE1. ^#Heteroduplex fragments of the lower bands. c Schematic representation of all mutant transcripts identified upon RT-PCR in HEK293T cell splice assays and of PE1 and PE1/PE2 observed as naturally occurring PEs when analyzing photoreceptor progenitor cells (PPCs) derived from a healthy individual. Interestingly, PE1 was previously shown to be induced by variant c.1937+435C>G (*Sangermano et al.)⁹ and also can be part of mutant transcripts, together with PE2 or PE3. This is surprising as it is located far upstream of the other causal variants. **Reported by Fadaie et al.¹³ d Variant c.6148-84A>T strengthens a SDS and results in PE1a or PE1b by employing upstream or downstream splice acceptor sites, respectively. These splice acceptor sites are comparable in predicted strength based on HSF. The canonical splice sequences are given in bold. e Agarose gel analysis of RT-PCR products due to c.6148-84A>T. f The largest fragment shows a 173-nt PE insertion between exons 44 and 45. The second largest band contains a 221-nt PE insertion (PE1a) and skipping of exon 44. The third-largest fragment represents the WT messenger RNA (mRNA) and the smallest fragment misses exon 45. The relative amounts of the products are listed at the right side.