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S.F. Geller, J. Isosomppi, H. Makela, E.M. Sankila, P.T. Johnson, J.G. Flannery; Vision Loss in Usher Syndrome Type III is Caused by Mutations in Clarin–1, an Inner Retinal Protein. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5123.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Clarin–1 is the gene product of USH3A, and in its mutant form is hypothesized to be responsible for Usher's Syndrome type III. We have further characterized the retinal localization, sub–cellular trafficking, and function of Clarin–1 in the retina. Methods: Clarin–1 mRNA transcripts were RTPCR amplified from wild–type (WT) c57BL/6 and mutant C3H mice, which exhibit advanced photoreceptor degeneration and loss. Transcripts were localized to specific nuclear layers by laser capture microdissection (LCM). LCM was performed on ethanol–fixed cryostat sections of adult WT c57BL/6 mouse eyes. Subcellular localization was examined by cloning human WT and Tyr176Stop mutant Clarin–1 into a hemagglutinin (HA)–tagged expression vector and transient transfection of BHK–21 cells, followed by anti–HA immunofluorescence microscopy. Results: Both quantitative and semi–quantitative PCR amplification of Clarin–1 retinal cDNA, from both c57BL/6 and C3H/HeJ mice, suggest that mice that lack [all] photoreceptors express the same amount of Ush3A mRNA in their retinas as control mice. In contrast, rod opsin cDNA levels were analyzed as a control for photoreceptor loss, and were reduced approximately 50,000–fold in rodless mice. These data suggest that Clarin–1 mRNA is not in photoreceptors. Corroborative studies with laser capture microdissection of retinal sections, followed by RNA isolation and RT–PCR shows Clarin–1 to be restricted to the retinal inner nuclear layer. These data further support the hypothesis that Clarin–1 mRNA is expressed in the inner retina and not expressed in photoreceptors. Transfection experiments show WT Clarin–1 to be synthesized and targeted through the perinuclear region to the plasma membrane. Mutant (truncated) Clarin–1, however, appears to be retained within the endoplasmic reticulum. Conclusions: In contrast to the rod cell death mechanisms demonstrated in RP, our data suggest that Ush3A mRNA is expressed in INL cells, and not in photoreceptors. Our data show that the Clarin–1 protein is present in bipolar or horizontal cells, and that mutant Clarin–1 may dysfunction due to aberrant intracellular processing and/or trafficking. We hypothesize that null mutations in Clarin–1 causes vision loss in Usher syndrome type III through a defect in synaptic transmission to the inner retina.
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