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Jessica Clare Gardner, Gerald Liew, Yinghua Quan, Hisao Ueyama, Jan Liebelt, Jonathan B Ruddle, Anthony T Moore, Michel Michaelides, Alison J Hardcastle; Genotype-Phenotype Comparison and Functional Investigation of Cone Opsin Variants. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4543.
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© ARVO (1962-2015); The Authors (2016-present)
To undertake an extensive clinical and genetic analysis of 22 families with a range of diagnoses from Blue Cone Monochromatism to progressive XL-Cone Dystrophy in order to evaluate possible genotype/phenotype relationships.
Clinical evaluation included retinal imaging, colour vision testing and electrophysiological assessment. The sequence and structure of the OPN1LW (L-) and OPN1MW (M-) gene array including the Locus Control Region (LCR), gene promoters and coding sequence was determined using PCR and Sanger sequencing. The 5’ opsin gene and composition of the array was determined by long range PCR. Splicing of opsin genes was investigated in-vitro by transfection of HEK293 cells with five different expression constructs containing a variety of exon 3 SNP haplotypes, followed by extraction of total RNA, RT-PCR and sequencing of opsin transcripts.
The opsin array genotype of the 22 families was established and grouped into one of three classes: deletions of the LCR (5 families), missense mutation (p.C203R) in a single L/M hybrid gene (8 families), or exon 3 SNP interchange haplotypes in an otherwise normal single or multiple gene array (9 families). Three SNP interchange haplotypes, encoding LIAVA, LVAVA or MIAVA, were identified. Previously it has been demonstrated that an LIAVA haplotype results in exon 3 skipping. We show that SNP haplotypes encoding MIAVA and LVAVA also result in aberrant splicing events, with a residual low level of correctly spliced cone opsin.
All 22 families had mutations in the OPN1LW/MW array that were consistent with loss of fully functional L- and M- opsin. High myopia and macular atrophy were present across the genotype spectrum and showed both inter and intra familial variability. A functional investigation of ancestral interchange haplotypes showed significant differences in the splicing of LIAVA compared to LVAVA and MIAVA, which may play a role in the observed phenotypic differences.
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