Overexpression of extracellular matrix proteins, increased cell death and reduced cell proliferation contribute to the pathophysiology of microphthalmia across in vitro patient-derived models

Genetic perturbations influencing early eye development can result in microphthalmia, anophthalmia and coloboma (MAC). Over 100 genes are associated with MAC, but little is known about common disease mechanisms. In this study, we generated induced pluripotent stem cell (iPSC) derived optic vesicles (OVs) from two unrelated microphthalmia patients and healthy controls. At day 20, 35 and 50 microphthalmia patient OV diameters were significantly smaller, recapitulating the 'small eye' phenotype. RNA-seq analysis revealed upregulation of apoptosis-initiating and extracellular matrix (ECM) genes at day 20 and 35. Western blot and immunohistochemistry revealed increased expression of lumican, nidogen and collagen type IV, suggesting ECM overproduction. Increased apoptosis was observed in microphthalmia OVs with reduced pH3+ cells confirming decreased cell proliferation at day 35. Pharmacological inhibition of caspase-8 activity with Z-IETD-FMK decreased apoptosis in one patient model, highlighting a potential therapeutic approach. These data reveal shared pathophysiological mechanisms contributing to a microphthalmia phenotype. Overall design: To identify common developmental pathway aberrations in microphthalmia patients with similar phenotypes but varying genotypes, we generated patient-specific induced pluripotent stem cell-derived optic vesicles carrying pathogenic variants. These were differentiated to day 20 and day 35 and taken for RNA-seq analysis. From each patient line, 4 replicates from 2 clones each were included in the RNA-seq analysis. Pairwise comparisons between each patient and the healthy control optic vesicles were performed as well as groupwise patient v control comparisons. Differentially expressed genes and enriched gene ontology terms were analysed in each comparison.