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A. Sigamani, M. A. Carbone, G. Li, R. R. H. Anholt, T. Borras; Elevated Concentrations of Q368X Myocilin Mutant Elicit Specific Changes in the Transcriptome of the Human Trabecular Meshwork (TM). Invest. Ophthalmol. Vis. Sci. 2008;49(13):1602. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The myocilin mutation Q368X is associated with POAG and elevated IOP. Our goal was to investigate mechanisms responsible for the onset of glaucoma caused by this mutant. Since myocilin is induced by stress conditions, we hypothesized that development of the disease is influenced by an increased concentration of the mutated protein in the TM.
The mutated cDNA was obtained by inserting a point mutation (C to T at nt 1123/accs# AF001620) in wild-type clone pMC2. A recombinant adenoviral (Ad) vector was generated by inserting the mutated cDNA into pCMVShuttle, and recombination with AdEasy1 (AdhTIG5). Paired anterior segments from postmortem human donors (n=2) were perfused at constant flow (3 µl/min). Eyes from each pair were injected with 2-3X1010 viral particles (VP) of AdhTIG5 and control AdCMVNull. TM tissues were harvested for RNA at 3-5d post infection. Primary HTM cells were infected with the mutant and control Ad vectors (moi = 10000 VP/cell) and harvested 3d later. RNAs were processed, hybridized to Affymetrix Human Genome U133 plus 2.0 (n=6) and analyzed by standard procedures.
Validation of the experiments was confirmed by upregulation of myocilin mRNA and truncated protein. Out of 54,676 genes, overexpression of the myocilin mutant significantly altered more than two-fold 1,697±231 (up-regulated) and 1,450±197 (down-regulated) genes. The most upregulated gene in all comparisons (197±54 fold) was Cadherin 15 (M-cadherin), a calcium-dependent intercellular adhesion protein whose upregulation causes fusion enhancement of myotubules. Interestingly, genes that appeared most upregulated by wild-type myocilin overexpression (EER82,1002,2006) such as angiopoietin2, thrombomodulin and MMP1 were highly downregulated (-23, -7.6 & -2.3 folds) by overexpression of the truncated mutant. Angiopoietin2 is involved in loosening cell-matrix contacts; thrombomodulin is the receptor for thrombin and MMP1 breaks down collagens.
The strong induction of M-cadherin together with the suppression of angiopoietin2 points towards an abnormal assembly of TM cell-cell and cell-matrix contacts upon overexpression of the Q368X myocilin mutant. These results suggest that such mechanism, which is likely to affect outflow facility, could be part of the cause by which the mutated myocilin triggers elevated IOP in humans.
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