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Maureen Neitz, Jay Neitz; Role of nucleotide polymorphisms in exon 3 of the L and M cone opsin genes in splicing and disease.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4491.
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© ARVO (1962-2015); The Authors (2016-present)
Nucleotide polymorphisms (NPs) in exon3 of the L/M cone opsin genes are individually benign but occur in combinations that prevent inlcudsion of exon3 in the messenger RNA (mRNA). Exon3-skipping variants are associated with vision disorders including blue cone monochromacy, high grade myopia, cone dystrophy, and color blindness. Little is known about what amount of "exon3-skipping" is normal versus disease causing, or about the mechanism by which combinations of NPs cause exon3 skipping. We report results of experiments to measure exon3 skipping in the 128 possible combinations of exon3 NPs, and bioinformatics analyses to investigate the mechanism of exon3 skipping.
L/M opsin minigenes were made by removing all introns exons except those flanking exon3. Minigenes were transfected into HEK293T cells, mRNA was collected ~48 hours later, and converted to cDNA and amplified in a reverse-transcriptase polymerase chain reaction. cDNA was used in a MassArray® assay to measure the relative amount of mRNA that included versus excluded exon 3. Exon splicing regulatory elements that are altered by exon3 NPs were identified with Human Splicing Finder, and a Bonferroni-corrected t test was performed to estimate the effect of each polymorphism on exon 3 splicing. We used the ExonScan splicing simulation algorithm to determine whether combinations of exon 3 NPs result in a failure of the algorithm to correctly predict exon 3.
Fig. 1 summarizes the results. Minigene assay results were used to calculate the mean percent of mRNA lacking exon 3 for individual NPs. Pairwise comparisons of the mean % exon3 skipping for individual polymorphic codons was analyzed with a t test. The Bonferroni corrected p values show NPs in codons 178, 180, and 171 significantly alter exon3 inclusion. No combination of NPs resulted in a failure of ExonScan to correctly predict exon 3.
Data from these experiments allow the formulation of detailed, testable hypotheses about the role of exonic splicing regulators in exon 3 splicing. Splicing silencers created by G532 in codon 178 and G538 in codon 180 appear to be the major culprits for exon 3 skipping, likely affecting both 3' and 5' splice site selection.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Polymorphisms in L/M opsin exon 3 codons 178 and 180 likely play a major role in splice site selection.
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