Proteomic comparison of keratoconus with and without vernal keratoconjunctivitis in individuals from Saudi Arabia.

Abstract

Purpose : Keratoconus (KC) is a vision-debilitating disease where the cornea undergoes progressive, degenerative thinning and steepening, with total loss of vision in 20% of severe cases. In Saudi Arabia there is a high incidence of keratoconus (KC), often associated with vernal keratoconjunctivitis (VKC). To elucidate pathogenic events that distinguish VKC from KC alone we performed highly sensitive multiplex mass spectrometry on individual cornea tissues from these two groups.

Methods : Three DN samples deemed unsuitable for transplantation (Tissue Bank International, Baltimore) and patient corneas from 3KC and 3 VKC (consented under an approved IRB, King Khalid Eye Specialist Hospital) were extracted using a Barocycler. Equal amounts of protein were reduced, alkylated and subjected to trypsin digestion. The resulting peptides were labeled with Tandem Mass Tag™ reagents as per the manufacturer's instructions. The peptides were resolved by basic pH reverse phase chromatography into 24 fractions. These fractions were analyzed on an Orbitrap Fusion Lumos mass spectrometer coupled with Proxeon EASY-nLC 1000 liquid chromatography. The Proteome Discoverer software suite was used for MS/MS searches and protein quantification.

Results : A total of 3624 proteins were identified at high protein FDR confidence. We detected 33 different collagen polypeptides; basement membrane Col4A2-A6 chains were present at similar levels in DN and both KC types. Epithelial KRT3 was similar in DN and VKC, but increased in KC, while KRT12 was similar across all samples. Fibrillar collagen types, COL1A1, COL1A2, COL3A1 COL5A1, COL5A2 and COL5A3 were all elevated in KC, but not in VKC compared to DN samples. Whereas, other collagens like COL6A1-3 and COL8A1 were present at similar levels in all samples. The collagen associated proteoglycans; KERA and DCN were lower in the VKC samples.

Conclusions : The epithelial ECM proteins show fewer differences between KC and VKC. However, changes in stromal collagens and proteoglycans speak of loss in stromal ECM functions. Additional analyses of the entire dataset are underway to understand cellular and matrix pathogenesis underlying keratoconus and those associated with vernal keratoconjunctivitis.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.