Objective. Inherited retinal diseases (IRDs) are a group of disorders that cause progressive visual impairment and affect approximately 1 in 2000 people. They are associated with mutations in over 300 genes, making them genetically and clinically heterogeneous. Among these genes is CRX, which encodes a cone-rod homeobox transcription factor essential for photoreceptor development. Pathogenic variants in CRX are mainly associated with autosomal dominant (AD) IRDs forms, such as Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa and Leber Congenital Amaurosis. Here, we study a CRD patient and an asymptomatic (AS) sibling, both carrying the c.121C>T (R41W) variant located in CRX exon 3. To investigate the underlying phenotypic differences, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from both individuals.

Methods. Three iPSC lines (Control, CRX-R41W-CRD, CRX-R41W-AS) were differentiated into retinal organoids to analyse temporo-spatial gene and protein expression of retinal markers throughout differentiation. Additionally, minigene assays were conducted on CRX variants similar to R41W to study exon 3 splicing.

Results. We observed an altered expression profile in the organoids of the R41W individuals compared to control. In general, qPCR and immunofluorescence analyses identified anomalies in photoreceptor-specific gene expression. Early in the differentiation period, we observed a down-regulation of genes/proteins known to interact with CRX, such as NR2E3 and NRL. At later stages, the expression of mature photoreceptor markers, such as the opsins, was impacted. Interestingly, although clinically unaffected, the CRX-R41W-AS organoids showed mildly deregulated expression profiles. Moreover, the R41W variant appeared to affect exon 3 splicing, with differing frequencies between the CRD and AS individuals. Similar splicing effects occurred at varying extents relative to control for other missense variants in the same exon.

Conclusion. Comparing the retinal organoids from a CRD patient and an AS sibling carrying the same variant will enhance our understanding of CRD pathophysiology and help elucidate the mechanisms underlying the emergence of clinical signs. Furthermore, the results of minigene assays investigating similar variants may contribute to unravelling the genotype-phenotype correlation in the heterogeneous group of CRX-related disorders and could support the development of targeted therapeutic strategies.