Genetic Modifiers of Crb1^rd8 Retinal Dysplasia Identified in C57BL/6N Single-Gene Knockout Mice

Abstract

Purpose : Mutations in the cell polarity protein CRB1 in humans and mice are linked to variable phenotypes, including retinal dysplasia, photoreceptor degeneration, and other pathological changes. This variability may be partly due to genetic modifiers. We sought to expand the CRB1 modifier gene network by screening for altered disease phenotypes in knockout (KO) mice that are also homozygous for Crb1^rd8.

Methods : Ocular phenotypes of 857 KO lines derived from C57BL/6NJ (B6N) mice at The Jackson Laboratory as part of the International Mouse Phenotyping Consortium (IMPC) were reviewed to identify candidate modifiers. Selected lines were rederived and retinal phenotypes were examined by fundus imaging and histological analysis. Expression of mRNA and protein encoded by the KO target gene was assessed by qRT-PCR and western analysis. KO lines were crossed with a strain carrying a corrected Crb1 allele to test for epistasis. Photoreceptor degeneration was assessed by counting outer nuclear layer nuclei at 1–3 and 10–12 months of age. Ocular distribution of the target proteins was determined by immunohistochemistry. Gene interactions were explored by literature review and pathway analysis.

Results : Ten IMPC strains were selected based on an abnormal retinal phenotype by indirect ophthalmoscopy and Crb1^rd8-like lesions in fundus images. On rederivation, an increase in light spots or patches was recapitulated in fundus images of Foxo3, Jam2, Limch1, Mpdz, Ncoa6, Rab11fip4, and Sorbs2 KO mice. Histological analysis revealed enhanced outer retinal dysplasia and photoreceptor degeneration in these strains compared to B6N controls. Loss of mRNA and/or protein was observed in all homozygous KO strains except for Ncoa6 KO mice, which were not tested. The KO allele in all strains was epistatic to Crb1^rd8. LIMCH1, MPDZ, and SORBS2 were localized to the retinal external limiting membrane. Bioinformatic analysis of these and previously reported Crb1 modifier genes suggested an influence of apicobasal polarity, cytoskeletal remodeling, transcriptional regulation, and inflammatory pathways on Crb1^rd8 disease.

Conclusions : We identified KO alleles of genes that modify retinal dysplasia and degeneration associated with the Crb1^rd8 allele in mice. Our findings link retinal dysplasia to defects in pathways that establish and fortify cell-cell adhesion, and they expand the gene network that may influence CRB1 disease variability.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.