Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1(rd8) mouse model

Author summaryInherited eye diseases affect roughly 1:1,000 individuals worldwide. Although these diseases are often linked to variants of a single gene, it is increasingly recognized that a second variant in other genes may modify disease characteristics, including age of onset, severity, and lesion appearance. Identifying such modifier genes in humans is difficult. In this study, two modifiers of a gene associated with retinal damage leading to childhood blindness in humans (CRB1) were identified in mice. Retinal damage caused by Crb1 mutation alone was less severe than in the presence of Arhgef12 or Prkci mutations. Furthermore, the modifier gene mutations caused retinal damage only in the presence of the Crb1 mutation. Our results point to a role of mouse Crb1 and the modifying effects of Arhgef12 and Prkci in a biological network that controls adhesive interactions between cells. The variation in disease severity, lesion appearance, and visual responses in these mice provide a dramatic example of modifier gene influence. This work may lead to an improved understanding of the molecular basis of CRB1-associated retinal disease, with possible relevance to diagnostic and therapeutic intervention in humans. Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1(rd8)/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1(rd8) allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Muller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1(rd8)/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1(rd8)/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.