Improved understanding of the disease mechanism of steroid induced glaucoma using gene editing techniques

Abstract

Purpose : To have an improved understanding of the molecular and genetic mechanisms of steroid induced glaucoma (SIG), and to establish a functional assay to screen genetic risk variants.

Methods : The exact disease mechanism of steroid-induced glaucoma (SIG) is not understood, but decreased phagocytosis capability of the trabecular (TM) cells has been suspected. SIG shares clinical similarities with primary open-angle glaucoma (POAG), and almost all POAG patients are steroid responders. We generated HTM1 cells lines with either a heterozygous or homozygous POAG causing Y437H mutation in MYOC gene, as well as MYOC knockout HTM1 cell lines, using CRISPR/Cas gene editing approaches. By creating these MYOC mutant HTM1 cell lines with identical genetic backgrounds, we hope to isolate and identify the molecular and cellular changes caused by steroid treatment in the steroid responders.

Results : Characterization of Mutant HTM1 Cells
The effect of DEX treatment on the response of ER stress and the turnover time of myocilin were first evaluated. We observed that the expression levels of ER stress markers GRP78 and GRP94 detected by anti-KDEL antibody were similar between the wild type and MYOC KO lines, while it is significantly higher in the Y437H mutant lines, after 100 mM of DEX treatment for 15 days. The turnover time of myocilin in the HTM1 cells was significantly delayed in the Y437H mutant lines (Figure 1).
Phagocytosis Assay
We evaluated the effect of MYOC-Y437H mutation and DEX treatment on the phagocytosis ability of fluorescent labeled latex beads using flow cytometry. The results, as summarized in Figure 2, clearly indicate that 1) the phagocytosis ability of the TM cells decreases with the DEX treatment, and 2) the Y437H mutation causes significantly decreased phagocytosis capability, more so when it is homozygous, and even more so with the DEX treatment.

Conclusions : Using the CRISPR/Cas gene editing techniques, we a) created multiple isogenic HTM1 cell lines with known POAG disease-causing mutations; b) provided strong molecular evidences that decreased phagocytosis capability is indeed the most likely culprit for SIG; c) established a functional assay to screen for candidate SIG risk variants.

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

 

Figure 1. Myocilin turns over slower in MYOC Y437H mutant HTM1 cell lines.

 

Figure 2. Phagocytosis rate in wild-type and MYOC-Y437H mutant HTM1 cells before and after DEX treatment.