Fig. 2

a A schematic map of the RT-PCR primer designed in relation to the exon–intron structure and mutations (G843E and S1653Kfs) in EYS and published transcript variants (Tv)²⁵. The locations of G843E (Exon 16) and S1653Kfs (Exon 26) are indicated by the arrows. Exon numbers are based on Tv1. Note, Tv5 was identified only in fibroblasts²⁵. b RT-PCR analysis. The regions for exons 5–6, exons 14–18, and exons 40–43 of EYS were amplified on cDNA generated from patient-derived lymphoblast cell lines with wild-type EYS (normal), homozygous S1653Kfs, and homozygous G843E. The Y79 retinoblastoma cell line was used as a positive control. Note C-terminal exons of the long isoform Tv1 were detected in LCLs with homozygous G843E but not in that with homozygous S1653Kfs. Sanger sequencing of the RT-PCR amplicon confirmed the expression of the G843E mutation using a primer pair targeting exons 14–18. Meanwhile, mRNA for exons 4–5 and 14–18 were detectable, possibly reflecting the differential expression of distinct EYS isoforms²⁵. c Chromatogram for RT-PCR amplicon (exons 14–18). Note, G843E variant is present in the patient’s mRNA. d RT-PCR analysis after mutation replacement genome-editing treatment (GE) or inhibition of nonsense-mediated mRNA decay (NMD) in LCL from an S1653Kfs homozygote, after which expression of exons 40–43 was detected.