Post-GWAS screening of candidate genes for refractive error in mutant zebrafish.

Abstract

Purpose : In the last decade, association studies have focused on dissecting the genetic factors underlying refractive error (RE). These studies resulted in the identification of almost 500 associated loci and potential candidate genes. To explore the genetic mechanisms driving RE development, we made a prioritization of 11 candidate genes based on biological and statistical evidence (i.e., PRSS56, FBN1, TJP2, PDE11A, SHISA6, LAMA2, LRRC4C, KCNQ5, GNB3, RBFOX1, and GRIA4), and generated mutant zebrafish models. In these models, we screened for abnormalities in axial length (AL) and refractive status.

Methods : Published transcriptome databases were assessed to examine whether candidate genes were expressed in human ocular tissues; expression in zebrafish ocular tissue was confirmed by RT-PCR. CRISPR-cas9 was used to generate 11 mutant zebrafish lines. Ocular biometry assessment was performed in lines without large morphological abnormalities and normal breeding rates (n=8). AL was measured at 2 and 4 months (n=20 eyes per group) using Spectral Domain Optical Coherence Tomography which visualized the entire eye; refractive status was measured using a custom eccentric photorefraction setup.

Results : Our database search and expression study in the zebrafish eyes confirmed that all genes were expressed throughout human and zebrafish eyes. Three of the eight studied mutant lines (lama2^-/-, lrrc4c^-/-, kcnq5^-/-) showed a significant (p<0.01) increase in AL and corresponding myopic shift in refractive status (p<0.01). Two of the three mutants showed the largest increase in AL at 2 months (lrrc4c^-/-: 140 µm, kcnq5^-/-: 151 µm) which stabilized at 4 months (lama2^-/-: 69 µm, lrrc4c^-/-: 114 µm, kcnq5^-/-: 92 µm). In line with previous reports in other animal models, the prss56^-/- mutant showed a significant reduction in AL (-157 µm, p<0.001) and a nanophthalmos-like phenotype at 2 and 4 months. In the other four studied lines (fbn1^-/-, tjp2^-/-, pde11a^-/-, shisa6^-/-) no significant changes in AL were observed.

Conclusions : Of the eight mutants lines, four showed a change in refractive status. Our study provides functional evidence that these candidate genes selected from GWAS studies induce changes in eye growth and presents new zebrafish eye models for the study of refractive error.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.