Abstract
Purpose:
Late onset Fuchs endothelial corneal dystrophy (FECD), affecting 5% of the US population, is a major cause for corneal transplantation. Mutations of a variety of unrelated genes: SLC4A11, COL8A2, TCF8 and LOXHD1, are associated with FECD. Current pathalogical hypotheses include deficiency of the CE (corneal endothelium) pump function and/or induction of the unfolded protein response (UPR). This study aims to determine the contribution of the above two mechanisms by assessing the expression levels of: (1) Endothelial ion-transporters known to regulate stromal hydration and (2) UPR related genes, in CE obtained from FECD patients compared to that of normal controls.
Methods:
Fuchs Corneal Endothelium was collected immediately post- Keratoplastic surgery (at Midwest eye clinic, Indianapolis) and transferred to RNA stabilizing agent and refrigerated. Normal specimens were similarly collected at Lion’s eye bank, Indianapolis. Total RNA from six CE specimens from each category i.e. Fuchs and normal control were individually extracted using RNeasy Micro Kit (Qiagen). Expression levels of ion transporters and UPR genes were tested in each of the RNA samples using quantitative Real Time PCR and UPR specific PCR array, respectively. Primers specific to human SLC4A11, Na+/HCO3- co-transporter, Na+/K+/ATPase pump, Monocarboxylate Transporters (MCT -1, 2 and 4) and Na+/H+ exchanger 1 were designed and standardized for efficiency. β-actin was included as a normalizing control. First strand cDNA synthesis (Applied Biosystems) and RT2 amplification for qRT-PCR (SB Biosciences) were used to prepare samples for qRT-PCR and UPR PCR array (SB Biosciences), respectively; Stratgene Mx3000 was employed to run PCR.
Results:
The PCR array tested 84 UPR related genes. The PCR array data analysis showed upregulation of 41 genes and downregulation of 3 genes i.e, ~ 52% of the tested genes had their expression altered in FECD samples. 13 genes that were altered were significant with p-value < 0.05. These genes were validated by qRT-PCR. Among the ion-transporters tested, Na+/K+ ATPase and Mono Carboxylate Transporters 1 & 4 were significantly downregulated in FECD samples (p-value < 0.05).
Conclusions:
FECD samples had evident UPR with significant alteration of genes associated with the UPR pathways along with significant down regulation of ion transporters indicating simultaneous compromised CE pump function under dystrophic condition.
Keywords: 481 cornea: endothelium