Human aniridia limbal epithelial cells lack expression of keratins K3 and K12

Abstract

Patients with aniridia often develop aniridia-related keratopathy (ARK), due to limbal stem cell insufficiency. This study aimed to determine the proliferative capacity and differentiation status of limbal epithelial cells (LECs) in patients with ARK. We also investigated the influences of PAX6 genotype on PAX6-transcript and protein level. Here two patients with aniridia underwent keratoplasty were examined. During the surgery, small limbal biopsies and pannus tissue were excised and processed for cell culture. Cells were analyzed with RT-PCR, qPCR and Western blot to evaluate expression of the developmental transcription factor, PAX6, potential stem cell markers, DeltaNp63alpha and ABCG2, and corneal differentiation markers, keratin 12 (K12) and K3. Conjunctival differentiation markers, keratin 13 (K13) and K19 were also investigated. Protein coding sequence of PAX6 from patient LEC-cDNA was cloned and sequenced. Cells were immunostained to evaluate K3, PAX6, and p63alpha protein expression. Epithelial cells isolated from the limbus region of two patients were successfully expanded in vitro. RT-PCR showed that K12 and K3 transcripts were absent from patient cells, but present in healthy control preparations. Transcription levels of PAX6, ABCG2, and p63alpha appeared to be unaffected in aniridia-LECs. One aniridia-patient showed elevated K13 levels indicating conjunctival contamination of this sample. This patient showed a loss of stop codon in half of the cloned transcripts. The mutation c.174C > T (Refseq. NM_000280) of this patient 2 could not be observed at cDNA level. However, Western blot showed reduced PAX6 protein levels in aniridia-LECs compared to control-LECs. Immunostaining also showed reduced PAX6 and K3 expression in aniridia-LECs compared to control-LECs. We found that LECs from patients with aniridia had unaltered proliferative potential. Aniridia-LECs expressed low K3 and K12 levels, most likely because aniridia-LECs could not differentiate into mature corneal epithelial cells. In patient 1 this could be additional explained by a conjunctival contamination of the sample. Mutation in patient 1 could be identified in cDNA. In patient 2 mRNA degradation through nonsense mediated decay seems to be very likely since we could not identify mutation, or misspliced transcripts in cDNA. Interestingly PAX6 transcription levels were not altered in qPCR. Western blot analysis implicates the degradation of the prolonged PAX6 protein of patient 1.