A Simple Secretion Assay for Assessing New and Existing Myocilin Variants

Purpose A lack of sufficient functional information exists for appropriately categorizing a large number of myocilin (MYOC) variants and their involvement in primary open angle glaucoma, hindering their clinical significance classification. Most glaucoma-causing MYOC mutations result in protein non-secretion and intracellular insoluble aggregate formation in cultured cells. Herein, we generated a Gaussia luciferase-based MYOC fusion protein to quickly and sensitively track the secretion of MYOC variants and compared these results to the better-established western blotting assay for MYOC. Methods Fourteen clinically-derived MYOC variants with varying degrees of predicted pathogenicity were transfected into HEK-293A cells and analyzed by either a luciferase assay or western blotting. Results Eight of the variants (G12R, V53A, T204T, P254L, T325T, D380H, D395_E396insDP, and P481S) had not been biochemically assessed previously. Of these, P254L and D395_E396insDP demonstrated significant secretion defects reminiscent of glaucoma-causing mutations. The luciferase assay results agreed with western blotting for thirteen of the fourteen variants (93%), suggesting a strong concordance. Conclusions These results suggest that the Gaussia luciferase assay may be used as a complementary or standalone assay for quickly assessing MYOC variant behavior and we anticipate that these results will be useful in MYOC variant curation and reclassification.

Automated summary

By using the Gaussia luciferase assay, researchers were able to successfully track the secretion of MYOC variants, indicating that this method could be an effective way to quickly assess the behavior of MYOC variants.