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Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin.

ABSTRACT: Heterozygous mutations in the myocilin gene (MYOC) cause glaucoma by an unknown mechanism. MYOC encodes an extracellular protein of unidentified function that undergoes intracellular endoproteolytic processing in the secretory pathway. It has been described that co-expression of wild-type/mutant myocilin reduces the secretion of the wild-type protein and that single expression of glaucoma myocilin mutants reduces its proteolytic processing. However, the effect of wild-type myocilin on mutant myocilin secretion and how mutant myocilin affects the proteolytic processing of wild-type myocilin have not been investigated. We herein analyze these two issues.We modeled the heterozygous state for 4 missense (E323K, R346T, P370L, D380A) and 1 nonsense (Q368X) myocilin mutants by transiently co-expressing each mutant with the wild-type protein in HEK-293T cells. Recombinant mutant and wild-type myocilin in both culture media and cellular fractions were quantified by western immunoblot and densitometry.A 24 h transient co-expression of each myocilin mutant with the wild-type protein elicited an augmented secretion of the mutant forms from 1.5 fold (D380A) to 5.4 fold (E323K). Under such conditions, extracellular mutant myocilin represented up to 20% of the total mutant protein. Other than this effect, secreted wild-type myocilin significantly decreased from 2.6 fold (E323K) to 36 fold (Q368X). When myocilin proteolytic processing was enhanced (96 hour co-expression) the extracellular amount of wild-type processed myocilin diminished from approximately 2.1 fold (E323K) to 6.3 fold (P370L). Nonreducing SDS-PAGE indicated that extracellular myocilin resulting from 24 h co-expression of wild-type myocilin and each of the 4 missense mutants forms hetero-oligomers and that glaucoma mutations do not increase the size of myocilin aggregates.Increased extracellular levels of mutant myocilin expressed in heterozygosis may play a relevant role in glaucoma pathogenesis. This effect is likely the result of intracellular mutant/wild-type myocilin hetero-oligomerization.

SUBMITTER: Aroca-Aguilar JD

PROVIDER: S-EPMC2585175 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin.

Aroca-Aguilar José-Daniel JD Sánchez-Sánchez Francisco F Martínez-Redondo Francisco F Coca-Prados Miguel M Escribano Julio J

Molecular vision 20081121

<h4>Purpose</h4>Heterozygous mutations in the myocilin gene (MYOC) cause glaucoma by an unknown mechanism. MYOC encodes an extracellular protein of unidentified function that undergoes intracellular endoproteolytic processing in the secretory pathway. It has been described that co-expression of wild-type/mutant myocilin reduces the secretion of the wild-type protein and that single expression of glaucoma myocilin mutants reduces its proteolytic processing. However, the effect of wild-type myocil ...[more]

PMID: 19023451

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Project description:Importance:Mutations in the myocilin (MYOC) gene are the most common molecularly defined cause of primary open-angle glaucoma that typically occurs in patients with high intraocular pressures (IOP). One MYOC mutation, p.Gln368Ter, has been associated with as many as 1.6% of primary open-angle glaucoma cases that had a mean maximum recorded IOP of 30 mm Hg. However, to our knowledge, the role of the p.Gln368Ter mutation in patients with normal-tension glaucoma (NTG) with an IOP of 21 mm Hg or lower has not been investigated. Objective:To evaluate the role of the p.Gln368Ter MYOC mutation in patients with NTG. Design, Setting, and Participants:In this case-control study of the prevalence of the p.Gln368Ter mutation in patients with NTG, cohort 1 was composed of 772 patients with NTG and 2152 controls from the United States (Iowa, Minnesota, and New York) and England and cohort 2 was composed of 561 patients with NTG and 2606 controls from the Massachusetts Eye and Ear Infirmary and the NEIGHBORHOOD consortium. Genotyping was conducted using real-time polymerase chain reaction that was confirmed with Sanger sequencing, the imputation of genome-wide association study data, or an analysis of whole-exome sequence data. Data analysis occurred between April 2007 and April 2018. Main Outcomes and Measures:Comparison of the frequency of the p.Gln368Ter MYOC mutation between NTG cases and controls with the Fisher exact test. Results:Of 6091 total participants, 3346 (54.9%) were women and 5799 (95.2%) were white. We detected the p.Gln368Ter mutation in 7 of 772 patients with NTG (0.91%) and 7 of 2152 controls (0.33%) in cohort 1 (P = .03). In cohort 2, we detected the p.Gln368Ter mutation in 4 of 561 patients with NTG (0.71%) and 10 of 2606 controls (0.38%; P = .15). When the cohorts were analyzed as a group, the p.Gln368Ter mutation was associated with NTG (odds ratio, 2.3; 95% CI, 0.98-5.3; P = .04). Conclusions and Relevance:In cohorts 1 and 2, the p.Gln368Ter mutation in MYOC was found in patients with IOPs that were 21 mm Hg or lower (NTG), although at a frequency that is lower than previously detected in patients with higher IOP. These data suggest that the p.Gln368Ter mutation may be associated with glaucoma in patients with normal IOPs as well as in patients with IOPs that are greater than 21 mm Hg.

Dataset Information

Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin.

Publications

Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin.

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