Variant-specific disruption to SOX2-mediated Notch signalling in PAX6 patient hiPSC optic vesicles

Abstract

Homeobox-containing transcription factor PAX6 is a key regulator of eye development. Pathogenic heterozygous PAX6 variants lead to variable ocular phenotypes, notably aniridia, typically resulting from haploinsufficiency. Missense variants are often associated with milder phenotypes, however mutations in the DNA-binding paired domain lead to severe ocular phenotypes including microphthalmia, but precise molecular targets are poorly understood. hiPSC derived 3D optic vesicles were generated from patients with variants (i) PAX6^N124K displaying combined microphthalmia, aniridia and optic nerve coloboma, and (ii) PAX6^R261X exhibiting typical aniridia. Total RNA sequencing revealed significant transcriptomic differences between samples, including downregulation of Notch signalling components in missense PAX6^N124K vesicles. Significant reduction in expression of Notch regulator SOX2 in retinal progenitor cells suggests altered DNA-binding by PAX6^N124K. This misregulation of SOX2-mediated Notch signalling perturbs retinal progenitor cell proliferation and differentiation. Our molecular analysis of in vitro models of PAX6-related oculopathies provides novel insights into PAX6 regulation of eye development and new potential disease mechanisms which elucidate genotype-phenotype correlations.