Autosomal dominant vitreoretinochoroidopathy (ADVIRC), a retinal dystrophy often associated with glaucoma and cataract, forms part of a phenotypic spectrum of 'bestrophinopathies'. We have previously shown that ADVIRC results from BEST1 mutations that cause exon skipping and lead to the production of shortened and internally deleted isoforms. In this study we describe a novel ADVIRC mutation, demonstrate that it disrupts an exonic splice enhancer (ESE) site and that this alters the binding of a splicing-associated SR protein. As with previous ADVIRC mutations the novel c.704t>c mutation in exon 6 altered normal splicing in an ex vivo splicing assay. Both this and another exon 6 ADVIRC-causing mutation (c.707g>a) either weakened or abolished splicing in an ESE-dependent splice assay when compared to a nearby exon 6 mutation associated with Best disease (c.703g>c). Gel shift assays were undertaken with RNA oligonucleotides encompassing the ADVIRC and Best disease mutations with four of the most commonly investigated SR proteins. While SC35, SRp40 and SRp55 proteins all bound to the wildtype and mutated sequences with similar intensities, ASF/SF2 showed increased binding to the two ADVIRC-mutated sequences compared to the wildtype or Best disease-mutated sequences. The exon skipping observed for these two exon 6 ADVIRC mutations, and their affinity for ASF/SF2, suggests that the region encompassing these mutations may form part of a CERES (composite exonic regulatory elements of splicing) site.
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