Abstract
Purpose: :
Myocilin, a protein linked to POAG, is induced by stress conditions. Overexpression of mutant myocilins by stress might be involved in the development of glaucoma. Our goal was to compare gene expression patterns induced by overexpression of different myocilin mutants in human trabecular meshwork cells (HTM) cells originating from a single individual.
Methods: :
Five recombinant adenoviruses, mutants Adh.Q368X, Adh.R342K, Adh.D380N, Adh.K423E, and wild-type (WT) Adh.WT were constructed by inserting the WT and mutated cDNAs into pCMVShuttle using the AdEasy system. All cDNA mutations were generated on the myocilin WT clone pMC2 which contains 1522 bp full coding cDNA. An AdNull carrying no gene was used as a control. Primary HTM-72 cells, generated from a 43 year old Caucasian male, were infected with 2.8x103 VP/cell. RNA was harvested at 50 h post-infection and hybridized to Affymetrix U133plus2.0 chips (n=17, 2 replicates per recombinant and 7 per control). Bioinformatics analysis was conducted using GS7.3 and GS10X.
Results: :
Validation of overexpression of each RNA was confirmed by upregulation of myocilin mRNA (TaqMan PCR) and protein (WB). Analyses were performed on the 1.5-fold altered gene lists. Paired comparisons of the mutants to the WT showed that Q386X had the highest number of genes differently regulated (6,175) versus 4,588 (R342K), 1,279 (D380N) and 1,309 (K423E).Overlapping Venn maps showed that there were 58 genes altered simultaneously in all four mutants; of these, 7 genes were not altered in the WT. One of the genes, Cystatin A (stefin) was upregulated in all mutants and not changed in the WT. The 20 most-altered genes were very similar between mutants Q368X and R342K. Changes elicited by D380N and K423E were more distinct. Potential relevant genes in lists also include MMP1, PDIA4, PTHLH, CALR, CDH15, and THBD.
Conclusions: :
Compared to WT, the Q368X myocilin mutant associated with highest IOP induces the most changes in the TM transcriptome. Identification of mutant-specific responder genes would provide key information on cellular mechanisms leading to myocilin-caused POAG.
Keywords: trabecular meshwork • gene microarray • mutations