June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Quantification of exon 3 skipping in human L and M opsin genes
Author Affiliations & Notes
  • Candice Davidoff
    Ophthalmology, University of Washington, Seattle, WA
  • Jessica Rowlan
    Ophthalmology, University of Washington, Seattle, WA
  • Jay Neitz
    Ophthalmology, University of Washington, Seattle, WA
  • Maureen Neitz
    Ophthalmology, University of Washington, Seattle, WA
  • Footnotes
    Commercial Relationships Candice Davidoff, None; Jessica Rowlan, None; Jay Neitz, None; Maureen Neitz, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1260. doi:
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      Candice Davidoff, Jessica Rowlan, Jay Neitz, Maureen Neitz; Quantification of exon 3 skipping in human L and M opsin genes. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1260.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Many human diseases involve gene splicing errors, including cystic fibrosis, Duchenne muscular dystrophy, and retinitis pigmentosa. Recently, Ueyama et al (Biochem Biophys Res Commun, 424,152, 2012) found that variants of the cone opsin genes associated with red-green color vision deficiency led to splicing errors that resulted in the absence of exon 3 from the final mRNA. The human L and M opsin genes are highly variable and in this work, we designed an assay to survey opsin gene variants to identify those associated with exon 3 splicing defects. In addition, among variants associated with exon skipping we sought to quantify the fraction of mRNA that contains normal full-length message compared to message lacking exon 3.

Methods: The Sequenom Mass Array instrument performs single nucleotide level genetic analysis using allele specific primer extension followed by mass spectrometry. We designed an assay to quantify the fraction of L opsin mRNA that lacked exon 3. The assay was first tested with known mixtures of full length and exon skipped cDNA. We then used the assay to probe exon skipping across all 128 possible variants of L opsin exon 3 using a minigene test in which HEK293 cells were transfected with a plasmid containing the full length opsin sequence with introns removed except for the two flanking exon 3. mRNA was harvested and used to produce cDNA for testing on the Mass Array.

Results: The exon skipping assay was found to be accurate within 5%. Across all exon 3 opsin variants, a surprising number were associated with exon 3 splicing defects in which some amount of mRNA lacked exon 3. The median fraction of mRNA in which exon 3 was skipped was 9% and the mean 25%. LIAVA, MIAVA, and LVAVA encoding haplotypes were found to be among the highest skipping variants along with newly discovered LIVVA and MIVVA. Sequences encoding an alanine instead of serine at position 180 skipped more on average (38% vs 11%), as did sequences encoding valine instead of isoleucine at position 178 (44% vs 6%).

Conclusions: Splicing defects of the cone opsin genes leading to excision of exon 3 are common and the amount of message with exon 3 skipped varies widely depending on exon 3 haplotype. Exon 3 skipping may underlie the cone opsin gene contribution to myopia and other eye disorders. The Mass Array is a promising tool for studying exon skipping, especially as its multiplexed format would allow for testing multiple alternative splicings.


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