Analysis of protein composition and protein expression in the tear fluid of patients with congenital aniridia

Abstract

Aniridia is a rare congenital genetic disorder caused by haploinsuffiency of the PAX6 gene, the master gene for development of the eye. The expression of tear proteins in aniridia is unknown. To screen for proteins involved in the aniridia pathophysiology, the tear fluid of patients with diagnosed congenital aniridia was examined using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two-dimensional map of tear proteins in aniridia has been established and 7 proteins were differentially expressed with P less than 0.01 between aniridia patients and control subjects. Five of them were more abundant in healthy subjects, particularly alpha-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1 and two other proteins, zinc-alpha 2-glycoprotein and lactoferrin were more expressed in the tears of aniridia patients. Moreover, immunoblot analysis revealed elevated levels of vascular endothelial growth factor (VEGF) in aniridia tears which is in concordance with clinical finding of pathological blood and lymph vessels in the central and peripheral cornea of aniridia patients. The proteins with different expression in patients tears may be new candidate molecules involved in the pathophysiology of aniridia and thus may be helpful for development of novel treatment strategies for the symptomatic therapy of this vision threatening condition. Biological significance This study is first to demonstrate protein composition and protein expression in aniridic tears and identifies proteins with different abundance in tear fluid from patients with congenital aniridia vs. healthy tears

Tear fluid biomarkers in ocular and systemic disease: potential use for predictive, preventive and personalised medicine

“…To date, very few studies have profiled tear fluid proteins in these patients. Recent work by Ihnatko et al [ 134 ] used 2D electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare tear proteins in aniridia and control subjects. The authors noted seven differentially expressed proteins in aniridia patients and control subjects, including α-enolase, peroxiredoxin 6, CST4, gelsolin, apolipoprotein A-1, zinc-α2-glycoprotein and lactoferrin.…”

“…The authors noted seven differentially expressed proteins in aniridia patients and control subjects, including α-enolase, peroxiredoxin 6, CST4, gelsolin, apolipoprotein A-1, zinc-α2-glycoprotein and lactoferrin. Of these, the former five proteins were more highly expressed in healthy subjects, while the latter two proteins were higher in tears of aniridia patients, and western blot data showed increased tear vascular endothelial growth factor (VEGF) levels in those with aniridia [ 134 ]. Further, a recent study by Peral et al [ 135 ] sought to ascertain the tear levels of diadenosine polyphosphates (Ap4A and Ap5A), which have been identified previously as potential dry eye biomarkers, in subjects with aniridia (who also have a propensity to dry eye).…”

Recent applications of CE‐ and HPLC‐MS in the analysis of human fluids

“…The same approach was also used by Ihnatko et al. to analyze the protein composition and the protein expression in the tear fluid of patients with congenital aniridia. Seven proteins, among which α‐enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A‐1, were differentially expressed between aniridia patients and controls.…”

“…Among differences observed in the tear protein profile between KC and controls, a significant increase in the level of the cystatin family members and in lipocalin-1 was evident for KC patients. The same approach was also used by Ihnatko et al [138] to analyze the protein composition and the protein expression in the tear fluid of patients with congenital aniridia. Seven proteins, among which ␣-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1, were differentially expressed between aniridia patients and controls.…”

Diagnostic impact of anterior segment angiography of limbal stem cell insufficiency in PAX6‐related aniridia

“…11 p. 13). PAX6 syndrome (OMIM 106210, ORPHA77) has panocular and progressive manifestations (macular and optic hypoplasia, corneal opacifications, cataract, glaucoma, progressive scar formation) and often systemic involvement (early onset diabetes, disturbance of diurnal rhythms, cerebral malformations, auditory abnormalities, renal insufficiency) (Bamiou et al 2007;Ihnatko et al 2013Ihnatko et al , 2016. There is congenital visual impairment with progressive loss of vision mainly due to the negative effects of limbal stem cell insufficiency (LSCI) (Gregory-Evans et al 2011;Khan et al 2014;Lagali et al 2013;Le et al 2013;Netland et al 2011).…”