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Stefania Marsili, Jan Riley, Alan L Robin, John H Fingert, Genetics; TBK1 protein production in fibroblast cell cultures from NTG patients with TBK1-related glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2545.
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© ARVO (1962-2015); The Authors (2016-present)
Duplications and triplications of the TBK1 gene have been associated with normal tension glaucoma (NTG). Cultured fibroblast cells from patients with TBK1 gene duplications were previously shown to have increased transcription of TBK1 mRNA. The purpose of this study was to determine the effect of TBK1 gene duplications on TBK1 protein production in human fibroblast cells.
Fibroblast cell cultures from three NTG patients with TBK1 gene duplications and from two control subjects (with no TBK1 duplications) were established from skin biopsies. TBK1 protein production in these cell lines were compared using Western blot analysis with a TBK1 antibody. TBK1 levels were assessed using densitometry and were normalized to tubulin levels.
Densitometry of TBK1 Western blots detected no difference in TBK1 protein production between fibroblast cell cultures from NTG patients with a TBK1 gene duplication and fibroblast cell cultures from control subjects.
TBK1 copy number variations are an important cause of NTG. However, the mechanism by which these duplications lead to optic nerve damage and NTG is not yet known. Prior studies to investigate the pathogenesis of TBK1-related glaucoma showed that in fibroblast cells, TBK1 gene duplications cause an increase in TBK1 gene transcription, which suggested that the pathogenicity of TBK1 copy number variations might be mediated by increased production of TBK1 protein. However, in the current study of the same fibroblast cell culture system, TBK1 gene duplications did not appear to cause a detectable increase in TBK1 protein, suggesting that TBK1 is under post-transcriptional control and that TBK1 protein production might have tissue-specific regulation, i.e. copy number variations might only cause increased TBK1 protein production in cell types relevant to glaucoma pathogenesis. On-going studies are investigating this hypothesis by measuring TBK1 protein production in stem cell-derived retinal ganglion cells that carry TBK1 copy number variations.
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