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S. Gobeil, V. Raymond; Correlation Between Non–Secretion of Myocilin Mutant Proteins and Familial Primary Open–Angle Glaucoma (POAG) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):30.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Mutations in the myocilin (MYOC) gene, also known as the trabecular meshwork–inducible glucocorticoid response (TIGR) gene, are found in about 4 % of all patients affected by POAG, an optic neuropathy leading to a progressive loss of the visual fields. MYOC encodes a 504 aa polypeptide secreted in aqueous humor. More than 45 MYOC mutations have been reported. Several of these mutations have been studied in vitro and prevented secretion of the protein. To investigate if this observation was a general feature of mutant proteins, we analyzed the secretion status of 36 myocilin variants; 22 considered as mutations causing familial POAG, 3 as mutations for sporadic cases of glaucoma, 7 as polymorphisms and 4 as undefined. Methods: Myocilin variants were generated by site–directed mutagenesis of a vector encoding the human MYOC cDNA. COS–7 and immortalized human trabecular meshwork cells were transfected with wild–type or mutated MYOC constructs. Myocilin proteins were detected in cells and extracellular media by immunoblotting. Results: Of the 36 myocilin variants, 15 were secreted (5 mutations, 6 polymorphisms, 4 undefined) and 20 remained sequestered intracellularly (all mutations), while the truncated R46X protein was too small to be detected. Identical results were obtained for the two cell lines. Of the 26 variants located within the olfactomedin–homology (Olf) domain (aa 246–501), 19 remained sequestered intracellularly; 18 of these 19 variants were mutations causing familial POAG whereas the sequestered W286R mutation was associated with sporadic glaucoma. Of the 7 variants in the Olf domain that were secreted, 3 were mutations (2 familial, 1 sporadic), 1 was a polymorphism and 3 were considered as undefined. The 2 familial POAG mutations in the Olf domain that were secreted were the T377M and A427T mutants. The T377M was only partially secreted. The 20th sequestered mutation was the S502P variant located 1 aa outside the Olf domain and also causing familial POAG. The R126W mutant was the sole familial mutation outside the Olf domain secreted. Conclusions: Most variations, but not all, located near or within the Olf domain led to the intracellular sequestration of the myocilin polypeptide showing that this region may play an important role in the folding and/or oligomerization of the protein. Our data also demonstrated a high correlation between non–secretion of myocilin mutant polypeptides and familial POAG. Testing myocilin variants for secretion will help to decipher if these variations may be classified as polymorphisms or as disease–causing mutants.
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