A rare optineurin mutation in an Indian family with coexistence of JOAG and PCG

Purpose: This study focused on the genetic screening of Myocilin (MYOC), Cytochrome P450 family 1 subfamily B member 1 (CYP1B1), Optineurin (OPTN), and SIX homeobox 6 (SIX6) genes in a family with coexistence of primary congenital glaucoma (PCG) and juvenile open?angle glaucoma (JOAG). Methods: Sanger sequencing was used to examine the coding region of all four genes. Six different online available algorithms were used for the pathogenicity prediction of missense variant. Structural analysis was done using Garnier–Osguthorpe–Robson (GOR), PyMol, ChimeraX, and Molecular Dynamic (MD) Simulations (using Graphics Processing Unit (GPU)?enabled Desmond module of Schrödinger). Results: There were a total of three sequence variants within the family. All seven algorithms determined that a single mutation, G538E, in the OPTN gene is pathogenic. The loops connecting the strands became more flexible, as predicted structurally and functionally by pathogenic mutations. Mutations create perturbations and conformational rearrangements in proteins, hence impairing their functioning. Conclusion: In this study, we describe a North Indian family in which members were having JOAG and PCG due to a rare homozygous/ heterozygous mutation in OPTN. The coexistence of two types of glaucoma within a single pedigree suggests that certain OPTN mutations may be responsible for the onset of different glaucoma phenotypes.

Inhibitory effect of myocilin on fibronectin expression and adhesion in trabecular meshwork cells of glaucoma / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To investigate the effect of myocilin on the expression of fibronectin (FN) and the adhesion function in trabecular meshwork cells of primary open angle glaucoma (POAG) in vitro. ·METHODS: The concentration of recombinant myocilin protein was 0 (control group), 0. 5, 1, 1. 5, 2 and 2.5μ g/mL, respectively. The expression of FN in the cells and the cell culture medium were detected by Western blot and ELISA. The effect of myocilin protein on the adhesion of cultured POAG trabecular meshwork cells was detected by CCK-8 method. ·RESULTS: Western blot showed that the ratio of FN/β-actin was 34.8±0.6,33.4±1.0,28.9±0.8,21.6±0.9,15.9± 1.1 and 11. 9 ± 0. 8; that of 0. 5μ g/mL group was not significantly different compared with that of control group (P=0.092);that of 1.0,1.5,2 and 2.5μ g/mL group were significantly different compared with that of the control group (F=346.131, P<0.05). The concentration of FN in the cell culture medium was 0. 4654 ± 0. 0039, 0. 4596 ± 0.0032, 0.4216± 0.0037, 0.4214 ± 0.0039, 0.4043 ± 0.0039, 0.3806±0.0071μ g/mL by ELISA;difference between that of 0.5μ g/mL group and that of control group was not statistically significant (P=0.120);the difference between of 1,1.5,2,2.5μ g/mL group and the control group were statistically significant (F=176.054, P<0.05). The mean values of the absorbance values of each group were 1.9814 ± 0.1624, 1.8848 ± 0.0267, 1.4895 ± 0.0916, 1.4120 ± 0.1087,1.3392 ± 0.1391, 1.0310 ± 0.0639 through CCK-8 method; that of 0. 5μ g/mL group was not significantly different compared with that of control group(P=0.300);the difference between of 1,1.5,2,2.5μ g/mL group and the control group were statistically significant (F =177.818,P<0.05). · CONCLUSION: Myocilin protein can inhibit the fibronectin expression in POAG trabecular meshwork cells, showing a dose dependent manner. Myocilin protein can reduce the adhesion of POAG trabecular meshwork cells.

Co-expression of GRP78 and Myocilin in trabecular meshwork cells of primary open angle glaucoma / 中华实验眼科杂志

Background Misfolding of Myocilin protein plays an important role in the pathogenesis of primary open angle glaucoma (POAG).GRP78 can transfer the misfolded proteins out endocytoplasmic reticulum and keep the protein synthesis function of endoplasmic reticulum.However the relationship between GRP78 and Myocilin in the pathogenesis of POAG has not been elucidated.Objective This study was to investigate and analyze the coexpressions of GRP78 and Myolicin in trabecular meshwork cells (TMCs) of POAG.Methods The trabecular meshwork tissues were obtained during the surgery of POAG and healthy donor for the isolated and in vitro culture of TMCs,and the morphology of the cells was compared between POAG-derived TMCs and donor-derived TMCs.The expression of specific factors for TMCs were detected by immunochemistry.The cells were divided into normal control group,tunicamycin (Tm)-treated group and staurosporine (STS)-treated group,and the cells were cultured by regular medium,the 1 μ mol/L Tm medium or 0.1 μmol/L STS medium for 24 hours respectively.The co-expression of GRP78 and Myocilin in the cells were detected using immunofluorescence assay.This study was approved by Ethics Committee of Xi'an No.4 Hospital,and written informed consent was obtained from each patient before surgery.Results Primarily cultured cells showed the fiat star-like and irregular appearance with 3-5 processes,round nuclei,abundant cytoplasm and black engulf particles.The 3-7 generations of POAG-derived cells grew well,showing positive expressions of laminin (LN),fibronectin (FN),neuronal specific enolase (NSE) and Vimentin.Immunofluorescence assay showed positive expressions of GRP78 and Myocilin in the cells of the Tm group,STS group and normal control group,with the reddish and green fluorescence separately.Incomplete co-expression of GRP78 and Myocilin was seen in donor-derived TMCs,and complete co-expression of GRP78 and Myocilin was found in the POAG-derived TMCs in the normal control group.Complete co-expression of GRP78 and Myocilin was exhibited in both donor-and POAG-derived TMCs in the Tm group and STS group.In addition,accumulation of GRP78 protein around nucleus was seen in both donor-and POAG-derived TMCs in the Tm group.Conclusions GRP78 and Myocilin proteins are completely co-expressed in POAG-derived TMCs,inferring that GRP78 and Myocilin play an interaction role in the pathogenesis and development of POAG.GRP78 may play a cytoprotective role against the apoptosis of TMCs caused by endoplasmic reticulum stress.

Mechanisms Underlying Trabecular Meshwork Cell Death Caused by Mutant Myocilin Expression

PURPOSE: To determine whether the expression of mutant myocilin can lead to death of human trabecular meshwork (HTM) cells and to determine whether the mechanism by which this occurs is apoptosis. METHODS: HTM cells were transduced with a recombinant adenovirus expressing human mutant (Q368X) myocilin. The apoptotic death of HTM cells caused by expression of mutant myocilin was examined using a cell proliferation assay, flow cytometry, Western blot analysis, and immunocytochemistry. RESULTS: It appeared that the expression of mutant myocilin itself was not sufficient to cause HTM cell death. Furthermore, the expression of mutant myocilin did not lead to apoptosis of HTM cells although it did elicit a protein unfolding response. CONCLUSIONS: Our data suggest that the mechanism of myocilin glaucoma is not apoptotic death of HTM cells caused by mutant myocilin expression, and that the actual mechanism remains unknown.

Screening and Selection of Proteins Interacting with Myocilin

PURPOSE: To identify novel proteins which bind to myocilin, using a yeast two-hybrid screening system. METHODS: A bait plasmid of myocilin was constructed, and then transformed into yeast AH109. The transformed yeast cells were mated with yeast Y187 containing human skeletal muscle cDNA library plasmids in 2 X YPD medium. Mated diploid yeasts were plated on synthetic dropout medium (SD/-Trp-Leu-His and SD/-Trp-Leu-His-Ade). The positive clones grown on the selective medium were amplified, sequenced, and analyzed using the database of GenBank. The expression of selected genes in trabecular meshwork (TM) cells was detected by RT-PCR. RESULTS: The expression of bait protein in yeast was confirmed by Western blot analysis. Extensive screening of a cDNA library led to the selection of twenty-four positive clones which represent eight genes, including six of cytoskeleton associated proteins such as alpha-actin, myosin regulatory light chain 2A, dynactin, syntrophin alpha1, microtubule associated protein 1B, and myosin binding protein C. CONCLUSIONS: Myocilin may interact with cytoskeleton associated proteins in TM cells. Further studies on their interactions will provide functional clues of myocilin.

The Effect of Accumulation of Mutant Myocilins in Human Trabecular Meshwork Cells

PURPOSE: To investigate the cellular fate of the mutant myocilins expressed in human trabecular meshwork (TM) cells and their potential cytotoxicity. METHODS: Cultured human TM cells were transduced with adenoviral vectors that expressed green fluorescence protein (GFP) tagged mutant myocilins (G364V, K423E, Y437H, or I477N). The cytopathic effects of the mutant myocilins on the TM cells were verified by protein blot analysis, microscopic examinations, WST-1 cell prliferation assay and yeast two hybrid assay. RESULTS: All of the mutant myocilins examined in the present study accumulated in the endoplasmic reticulum (ER), which was thought to induce ER stress, severe malformed morphology and diminished cell proliferation. In addition, the expression of mutant myocilin could selectively inhibit the secretion of wild-type myocilin. CONCLUSIONS: The dysfunction or decreased number of trabecular meshwork cells due to the cytopathic effect of mutant myocilins may cause diminished turnover of the extracellular matrix of the trabecular meshwork, and thereby increase the resistance of the trabecular outflow, which may be associated with the mechanism of primary open-angle glaucoma.

The Cytotoxicity of Truncated (Q368X) Myocilin in Trabecular Meshwork (TM) Cells

PURPOSE: To investigate the cytotoxicities of wild type myocilin and trunctated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: GFP tagged truncated myocilin and DsRED1 tagged wild type myocilin were expressed in TM cells using adenoviral vectors and observed colocalization by confocal microscope. Cytopathic effects in the cells were examined by light microscope and WST-1 cell proliferation assay. RESULTS: Colocalization of wild type and truncated myocilin was observed in cells co-expressing the proteins. Truncated myocilin was found to be toxic to cells, leading to deformed cellular morphology and diminished cell proliferation. CONCLUSIONS: The intracellular accumulation of truncated myocilin exhibited cytotoxicity in trabecular meshwork cells, and eventually resulted in diminished number and dysfunction of trabecular meshwork cells, which might be involved in the pathogenesis of glaucoma.

Expression of Wild Type and Truncated Myocilins in Trabecular Meshwork Cells: their Subcellular Localization

PURPOSE: To investigate subcellular localizations of wild type myocilin and trunctated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: GFP and GFP tagged truncated myocilin, full-length myocilin, and stromelysin were expressed in TM cells using adenoviral vectors, and their secretory properties were examined by western blotting. To determine the subcellular localizations of myocilins, cellular organelles of the infected TM cells were stained with antibodies or orgenelle specific fluorescent indicators and examined under confocal microscope. RESULTS: Wild type myocilin was expressed as discrete fine vesicles in the perinuclear region of TM cells as well as in ER, but not in microtubules or mitochondria. Colocalization of wild type and truncated myocilin, indicative of in vivo interaction of the proteins, was also observed in cells co-expressing the proteins. Truncated myocilin was found to be accumulated as aggregates in ER, and inhibited the secretion of normal myocilin. CONCLUSIONS: Our result suggests that intracellular accumulation of truncated myocilin may cause a dysfunction of the cells, resulting in alterations in structural compartmentalization of trabecular ECM and obstruction of aqueous outflow.