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Maureen Neitz, Jay Neitz; Identification of rhodopsin exon mutations underlying retinitis pigmentosa that alter splicing. Invest. Ophthalmol. Vis. Sci. 2017;58(8):584.
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Mutations in the rhodopsin gene (RHO) are associated with autosomal dominant retinitis pigmentosa (ADRP), with >120 disease-causing mutations identified. The disease mechanism for individual mutations is unclear but important for understanding and developing therapeutic strategies. Individual nucleotide changes in exons have been presumed to exert pathology at the level of protein structure/function, overlooking the potential for effects on splicing. We tested the hypothesis that some exon mutations in rhodopsin alter splicing.
We tested 3 rhodopsin mutations based on bioinformatics predictions that they may alter splicing, and 2 randomly chosen mutations not predicted to alter splicing (Table 1). We used wild-type RHO as the control. Splicing assays were performed using full length rhodopsin genes driven by the ubiquitous CMV promoter. Plasmids carrying the rhodopsin genes were sequence-verified, then transfected into duplicate cultures of HEK293T cells. mRNA was isolated 48 hours later and subjected to reverse transcription (RT) and the polymerase chain reaction (PCR). Assays were performed in triplicate for each mutant. Gel purified RT-PCR products were directly sequenced by fluorescent sequencing on an ABI 3500 capillary sequencer.
The wild-type RHO gene, the p.A164Q and p.W184S mutants only gave rise to correctly spliced, full-length mRNA (confirmed by sequencing); however the other three mutations altered splicing. Both the p.Y136X and the p.C167W produced only mRNA in which exon 2 was skipped. The p.Q312X mutant used a cryptic splice site in exon 4, deleting the last 4 bases of exon 4.
If spliced correctly, the p.Y136X and p.C167W mutants are predicted to have quite different disease mechanisms. The p.Y136X mRNA is likely targeted to nonsense mediated decay (NMD) due to the premature translation termination codon, whereas the p.C167W mRNA is predicted to be translated into a mutant opsin protein. However, both mutations cause exon 2-skipping, which introduces premature stop codons for both that likely target the mRNAs to NMD, and thus they share the same disease mechanism. Rather than producing a truncated protein, the altered splicing observed for the p.Q312X mutation shifts the reading frame and encodes a protein that is 10 amino acids longer than RHO.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Rhodopsin mutations investigated.
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