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 individualCRB1mutations on gene expression are lacking for most variants. Here, we investigated the effect of twoCRB1variants on pre-mRNA splicing using neural retinal organoids (NRO) derived from a patient with recessive rod-cone dystrophy caused by compound heterozygous mutations inCRB1(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.CRB1transcripts 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 expressingCRB1mRNA. Patient NRO expressed a novelCRB1transcript displaying skipping of exon 6.CRB1transcripts 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>ACRB1variants 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.