Characterization of CRB1 splicing in retinal organoids derived from a patient with adult-onset rod-cone dystrophy caused by the c.1892A>G and c.2548G>A variants.

BACKGROUND Mutations in the human crumbs homologue 1 (CRB1) gene are associated with a spectrum of inherited retinal diseases. However, functional studies demonstrating the impact of individual CRB1 mutations on gene expression are lacking for most variants. Here, we investigated the effect of two CRB1 variants on pre-mRNA splicing using neural retinal organoids (NRO) derived from a patient with recessive rod-cone dystrophy caused by compound heterozygous mutations in CRB1 (c.1892A>G and c.2548G>A). METHODS The patient received ophthalmological examinations including multimodal imaging. NRO were differentiated from induced pluripotent stem cells (iPSCs) derived from the patient and a control subject. CRB1 transcripts were characterized by RT-PCR and Sanger sequencing. RESULTS The Patient displayed retinal thickening with disorganization of retinal layers and preservation of para-arteriolar retinal pigment epithelium. Both patient and control iPSC produced NRO containing photoreceptor progenitor cells expressing CRB1 mRNA. Patient NRO expressed a novel CRB1 transcript displaying skipping of exon 6. CRB1 transcripts containing the c.2548G>A substitution in exon 7 were expressed in patient NRO. CONCLUSIONS Together, these results confirm the pathogenicity of the c.1892A>G and c.2548G>A CRB1 variants in a family with recessive adult-onset rod-cone dystrophy and further demonstrate the effects of these variants on pre-mRNA splicing. This data provide important insights into the pathogenic mechanisms associated with these variants.