Inter-Eye Molecular Discrepancies in the Corneal Epithelium Point to TFRC in the Keratoconus Severity Signature and Mechanism of Cone Formation

Purpose: There is no molecular evidence available confirming or contradicting inter-eye molecular variability in keratoconus (KTCN). The key question is whether the use of a model of more and less affected eyes of the same pair facilitates the identification of specific molecular features of KTCN severity.
Methods: This retrospective case–control study involved 21 KTCN patients (n = 132 experimental samples derived from 42 corneal epithelium samples separated into central, middle, and peripheral topographic regions) analyzed in the paired model. Transcriptomic (RNA-sequencing) and proteomic (MALDI-TOF/TOF MS/MS) profiling was performed. An additional non-paired model, including KTCN patients (n = 42) and controls (n = 14), strengthened the assessment. Then, key findings were validated in a rediscovery study with 20 patients using reverse-transcription quantitative polymerase chain reaction, immunofluorescence staining, and confocal microscopy.
Results: In the paired model, which included patients with ≥1 grade difference in topographic keratoconus classification, 48 differentially expressed genes were identified. Over-representation analysis highlighted the GO term “cell–cell adhesion” (adjusted P = 0.028), with key contributors including ACTN1, EPCAM, PCDH19, PVR, and TFRC. TFRC showed significantly higher expression in the middle topographic region of more severely affected eyes (P = 0.008, paired t-test), correlating with the average topographic region thickness (R = 0.53, P = 0.008) and flat keratometry (K1; R = 0.49, P = 0.016). Immunofluorescence confirmed intra-individual and regional differences in transferrin receptor (TFRC) protein expression, with increased expression in the middle topographic region of more advanced eyes, compared to eyes with forme fruste KTCN.
Conclusions: Inter-eye variability implicates TFRC as a component of the KTCN severity signature, thus adding the element to the mechanisms underlying KTCN cone formation.