High-performance proteomics reveals immune, epithelial, and vascular dysregulation underlying lacrimal fluid defects in patients with aniridia

BACKGROUND: Congenital aniridia is a rare disorder presenting as a panocular malformation with variable severity, often complicated by progressive keratopathy. The purpose of this study was to characterise the tear-film proteome in adults with PAX6-related congenital aniridia and to identify dysregulated pathways linked to aniridia associated keratopathy (AAK). METHODS: Tears were obtained with Schirmer strips from four genetically confirmed patients and four age- and sex-matched healthy volunteers. Peptides prepared with the single-pot, solid-phase-enhanced (SP3) protocol were analysed by data-independent nanoLC-MS/MS. Proteins were identified with a false discovery rate (FDR) <1% during DIA data processing. Differential abundance between controls and patients samples was assessed using an adjusted p-value < 0.05. Proteins with |log₂-fold change| ≥1 were considered significantly expressed. Functional enrichment was evaluated with Enrichr (Gene Ontology, Reactome, JensenExp, Orphanet, TissueExp). RESULTS: A total of 3 162 proteins were detected; 2 633 showed a valid intensity in every sample of at least one group and were retained for statistical testing. Seventy-three (2.8%) were differentially expressed: 33 were over-expressed and 40 under-expressed in aniridia tears. Down-regulated proteins clustered in lipid homeostasis, epithelial junction integrity and wound-healing modules and included lacritin, secretoglobins and cytoskeletal adaptors, indicating a fragile, poorly repaired surface. Up-regulated species were dominated by neutrophil effectors (CD177 ≈ 50-fold) and reflected heightened innate immunity and abnormal epithelial maturation. Anti-angiogenic processes were significantly over-represented in both under and over-expressed protein sets. CONCLUSION: Our workflow proved highly sensitive, capturing more than 3 000 tear proteins and thus underscoring the robustness of our proteomic approach. The tear film in aniridia reflects dysregulation of various processes, including immunity, lipid and epithelial homeostasis, and vascular remodelling. Our approach highlights novel biomarkers critical for developing targeted therapeutic strategies. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05562115. Registered on 29 September 2022.