Project description:To identify the specific genes in human trabecular meshwork (TM) related to POAG.Primary open-angle glaucoma TM specimens were obtained from routine trabeculectomy surgery. Nonglaucomatous control TM specimens were dissected from donor eyes using the same approach as a standard trabeculectomy. All cases were screened for myocilin (MYOC) mutations. Total RNA was extracted, labeled, and hybridized to Illumina HumanWG-6 BeadChips. Expression data were normalized and analyzed using the R package limma in Bioconductor. Pathway analyses were performed using DAVID Bioinformatics Resources.Our study included surgical TM specimens from 15 cases and 13 controls. One case was identified with a heterozygous Q368X MYOC mutation. If TMs were available from both eyes in an individual, the expression data were combined for analysis. The following three comparisons were performed for differential analyses: (1) MYOC POAG case versus 14 non-MYOC POAG cases, (2) MYOC POAG case versus 13 controls, and (3) 14 non-MYOC POAG cases versus 13 controls. Limited by one MYOC case in comparisons 1 and 2, expression changes were reported comparing the fold changes but without P values. Comparison 3 identified 483 genes, including 36 components of TM exosomes. Gene ontology analysis identified several enriched functional clusters, including cell adhesion, extracellular matrix, and secretion.This is the largest TM expression study of POAG cases and controls performed to date and represents the first report of TM expression in a patient having POAG with a Q368X MYOC mutation. Our data suggest the potential role of endocytic and exosome pathways in the pathogenesis of POAG.
Project description:Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wild-type. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma.
Project description:Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone ?B-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.
Project description:This study compared genome-wide expression profiles of individuals with and without Primary Open-Angle Glaucoma (POAG). One POAG case (case #6 with two replicates #10 and #11) carried a Q368X myocilin mutation. This study compared the genome-wide expression in human trabecular meshwork tissue between 13 controls and 15 POAG cases. Six controls and one POAG cases had the expression performed from both left and right eyes. One technical replicate was done between two cases. The average from the biological replicates for each inidividual was used for analysis.
Project description:Objective:Mutations in myocilin (MYOC) may cause either juvenile open angle glaucoma (JOAG) or adult-onset primary open angle glaucoma (POAG). MYOC encodes a glycoprotein that is normally secreted from trabecular meshwork cells that regulate intraocular pressure. Prior in vitro, transgenic rodent, and organ culture experiments have suggested that abnormal accumulation of MYOC protein within trabecular meshwork cells is a key step in glaucoma pathophysiology. We investigated the pathogenesis of MYOC glaucoma by examining a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation. Design:Case-control, immunohistochemical study of a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation and age-matched control donor eyes. Subjects:An eye from a 59-year-old male with JOAG caused by a Tyr437His MYOC mutation and eyes from five donors (ages 51-66) with no known ocular disease were examined. Methods:Frozen fixed sections of the iridocorneal angle were prepared from the donor eyes of the MYOC glaucoma patient and control eyes. We used antibodies directed against MYOC, collagen IV, and BiP/GRP78 as well as wheat germ agglutinin and concanavalin A lectins to localize MYOC protein in the trabecular meshwork. Main Outcome Measure:Qualitative comparison of MYOC protein labeling and localization in the trabecular meshwork of donor eyes from a glaucoma patient with a MYOC mutation and from control subjects. Results:Using immunohistochemistry, we detected more abundant MYOC protein within the trabecular meshwork of the MYOC glaucoma patient's eye than in control eyes. We further localized MYOC protein within the trabecular meshwork cells of the MYOC glaucoma patient's eye by co-labeling with the endoplasmic reticulum (ER) marker GRP78 (BiP). Little to no MYOC was identified within the trabecular meshwork cells of control eyes. Minimal extracellular MYOC was detected in both MYOC glaucoma eyes and control eyes. Conclusions:This is the first histopathological analysis of an eye from a glaucoma patient with a MYOC mutation. Furthermore, this analysis supports our model of MYOC-associated glaucoma, in which MYOC mutations cause abnormal intracellular retention of MYOC within the ER of trabecular meshwork cells as a key step towards development of glaucoma.
Project description:BACKGROUND/AIM To determine if overexpression of the glaucoma gene MYOC is involved in the development of open-angle glaucoma (OAG) and if its promoter variants are associated with glaucoma in the Korean population. METHODS Human trabecular meshwork cells were cultured in the presence of ophthalmic steroids such as fluorometholone, fluorometholone acetate, dexamethasone, prednisolone acetate and rimexolone. The cells were cultured at a hydrostatic pressure of 32 mm Hg above atmospheric pressure and induction of MYOC was evaluated by northern blot analysis. Genomic DNA was extracted from blood samples obtained from 74 normal controls and 168 unrelated Korean patients with OAG, including primary OAG, normal tension glaucoma and steroid-induced glaucoma. A 461 base pair (bp) DNA fragment of the MYOC promoter region was amplified using PCR and its genotype was analysed by directly sequencing the product. RESULTS The potencies of steroid eye drops in MYOC induction in vitro was the same regardless of their potential for elevating intraocular pressure in vivo. Hydrostatic pressure had no effect on MYOC induction. A dinucleotide repeat polymorphism and three single nucleotide polymorphisms were identified, but no obvious differences in the genotype distribution and allele frequency of the variants between the control group and any type of OAG were observed. CONCLUSION Our data suggest that MYOC overexpression is not a cause or an effect of intraocular pressure elevation and that MYOC itself is not associated with OAG.
Project description:PURPOSE: To analyze two candidate genes, trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) and human dioxin-inducible cytochrome P450 (CYP1B1), in a Chinese pedigree of primary open-angle glaucoma. METHODS: In a three-generation family containing 14 members, four of them were patients with primary open-angle glaucoma, one was a glaucoma suspect, and the rest were asymptomatic. All members of the family underwent complete ophthalmologic examinations. Exons of MYOC and CYP1B1 were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: Elevated intraocular pressure and impaired visual field were found in all patients. One MYOC heterozygous mutation G367R, in exon 3 was identified in four patients and the suspect, but not in the rest of the family members. Meanwhile, four single nucleotide polymorphisms in MYOC and CYP1B1 genes were found. CONCLUSIONS: Although the G367R mutation of MYOC, which causes primary open-angle glaucoma in the form of autosomal dominant inheritance, has been reported in some other ethnicities, it was found in Chinese pedigree for the first time.