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H.M. Vastinsalo, J. Isosomppi, S. Geller, J. Flannery, E. Heon, E.–M. Sankila; Functional Evaluation of Two Novel Ush3a Mutations by in vitro Expression . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2062.
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© ARVO (1962-2015); The Authors (2016-present)
We screened a cohort of Usher syndrome patients for novel USH3A mutations and evaluated the pathogenic role of these mutations by in vitro expression of the altered gene products in cell cultures.
USH3A coding region was sequenced from DNA samples of approximately 40 Canadian USH patients with no previously known USH mutations. We identified and introduced these sequence alterations into the primary USH3A transcript (NM_174878), previously cloned into a hemagglutin (HA)–tagged expression vector using the QuickChange site–directed mutagenesis kit. Cultured BHK–21 cells were transfected with these constructs and subcellular localization of both wild type and mutant Clarin–1 proteins were visualized using anti–HA antibodies and confocal immunofluorescent microscopy. A time–course analysis was used to assess the stability of the proteins following cycloheximide treatment.
We detected two potentially pathogenic novel USH3A sequence alterations resulting in predicted amino acid changes L54P and A123D. In previous studies, we showed that in vitro expressed wild type Clarin–1 is properly trafficked and inserted into the plasma membrane whereas mutant proteins representing several known USH3A mutations were retained in the endoplasmic reticulum. The staining pattern of the expressed A123D mutant suggests retention of the misfolded protein in the endoplasmic reticulum; supporting the pathogenic role of the A123D mutation. When protein synthesis was interrupted by cycloheximide treatment, wild type Clarin–1 remained stable in transfected BHK–21 cells, while the A123D mutant protein was markedly reduced after 1 hour of treatment and was almost completely absent by 4 hours. The study of L54P alteration is ongoing.
The abnormal cellular localization and instability of A123D mutated Clarin–1 indicates that this amino acid change, found in a homozygous Usher syndrome patient compatible with the USH3 phenotype, is the causative mutation. Site–directed mutagenesis and in vitro expression of mutant proteins is a feasible method of evaluating the possible pathogenicity of USH3A mutations.
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