Abstract
Purpose :
Blue cone monochromacy (BCM) is an inherited X-linked disease which is caused by pathogenic variants in OPN1LW and OPN1MW leading to non-functional long wave sensitive opsin (L-Opsin) and medium wave sensitive opsin (M-Opsin). The combination of certain variants in BCM patients, termed interchange haplotypes, has been shown to induce exon 3 skipping in vitro. Here we investigated the effects of different interchange haplotypes on the amount of exon skipping in vitro. The potential of induced pluripotent stem cell (iPSC) derived retinal organoids (ROs) from BCM patients to model disease was also investigated.
Methods :
An in vitro mini-gene assay was established to investigate the effects of interchange haplotypes on exon skipping. HEK293 cells were transfected with mini-genes containing BCM interchange haplotype variants, including ‘LIAVA’, ‘LVAVA’, and ‘LVVVA’. The degree of exon 3 skipping was assessed by RT-PCR and qPCR. Fibroblast cells were isolated from skin biopsies from patients with BCM and reprogrammed to iPSCs. iPSCs were characterized for pluripotency markers using immunohistochemistry (IHC). iPSCs were differentiated to ROs using several published protocols and were analyzed for retinal marker expression using RT-PCR and IHC.
Results :
The in vitro mini-gene assay revealed different amounts of exon 3 skipping for the disease associated interchange haplotypes tested. For example, qPCR of ‘LVAVA’ showed that only 14% of transcripts expressed contained exon 3; whereas, for 'LVVVA' 33% of transcripts contained exon 3. Patient-derived iPSC RO development was comparable to controls, with neuroepithelial lamination observed at day 30, and outer segment formation observed from day 150. RT-PCR and IHC was used to characterize ROs for photoreceptor markers. In ROs differentiated from a patient harbouring ‘LVAVA’, cone arrestin and phosphodiesterase 6H were detectable in cone cell bodies, inner segments, and outer segments, whereas L/M-Opsin was undetectable by IHC, suggesting cone outer segment formation in the absence of L/M-Opsin.
Conclusions :
Interchange haplotypes in OPN1LW/MW lead to exon 3 skipping in vitro. BCM patient-derived ROs represent a potentially important experimental paradigm to assess how interchange haplotypes affect OPN1MW and OPN1LW expression in human photoreceptors and the pathobiology of BCM.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.