PAX6 and PAX6(5a) Transcript Levels in Conjunctival and Corneal Epithelial Samples and in Epithelial Cell Models of Congenital Aniridia

Abstract: PURPOSE: PAX6 haploinsufficiency plays a crucial role in the development of aniridia-associated keratopathy (AAK). Our purpose was to analyse PAX6 levels and the presence of different PAX6 isoforms in conjunctival and corneal epithelial samples and in epithelial cell models of congenital aniridia. Abstract: METHODS: PAX6 and PAX6(5a) mRNA levels were analysed in the following control and aniridia-derived samples: (a) primary limbal epithelial cell cultures from healthy donors (Ctrl-LECs) and aniridia patients (AN-LECs); (b) LECs transfected with control (siRNA Ctrl-LECs) or PAX6-targeting siRNA (siRNA PAX6-LECs); (c) immortalised limbal epithelial cells (t-LSCs) and genome-edited t-LSCs PAX6±; (d) conjunctival impression cytology from healthy individuals (Ctrl-CjECs-IC), aniridia patients (AN-CjECs-IC), and post-mortem donors (Pm-CjECs-IC); (e) corneal impression cytology from healthy living (Ctrl-CECs-IC) and post-mortem donors (Pm-CECs-IC); and (f) corneal epithelium (Ctrl-CECs) from healthy subjects. mRNA was extracted and reverse-transcribed to cDNA. PAX6 and PAX6(5a) were quantified via TaqMan qPCR with dual-labelled probes. Abstract: RESULTS: PAX6 mRNA was significantly reduced in AN-LECs, siRNA PAX6-LECs, t-LSCs, and t-LSCs PAX6± versus Ctrl-LECs (p = 0.022, 0.019, 0.006). In contrast, expression was significantly higher in Ctrl-CjECs-IC and Ctrl-CECs versus Ctrl-LECs (p < 0.001). Total PAX6 was significantly lower in AN-LECs, siRNA PAX6-LECs, t-LSCs PAX6±, and AN-CjECs-IC compared to their respective controls (p ≤ 0.039). PAX6(5a) levels were significantly lower than total PAX6 in certain examined groups (p ≤ 0.035). Abstract: CONCLUSIONS: PAX6 mRNA levels vary significantly across different epithelial cell types and models of congenital aniridia, with reduced expression observed in aniridia-derived samples and PAX6-deficient cell lines. The alternative isoform, PAX6(5a), is expressed at markedly lower levels than full-length PAX6 in most analysed tissues. This highlights the importance of both total PAX6 and PAX6(5a) expression in ocular surface epithelial homeostasis and provide further insight into the molecular alterations underlying AAK.