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Jennifer J. Lentz, Francine M. Jokelka, William C. Gordon, Michelle L. Hastings, Nicolas G. Bazan; The Ush1c.216G>A Splice Site Mutation Results In The Accumulation Of Harmonin In The Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1364.
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© ARVO (1962-2015); The Authors (2016-present)
The molecular mechanisms associated with Usher syndrome, the leading cause of combined deaf-blindness, are unknown. Usher 1 patients have profound congenital deafness, vestibular areflexia, and adolescent-onset retinitis pigmentosa. Similarly, Ush1c.216AA knock-in mice (c.216AA) are born deaf, show circling and head-tossing behavior, and have progressive retinal degeneration. The USH1C gene encodes the protein harmonin, which has been found to play a critical role in hair cell bundle formation, structure and function. Its function in the retina, however, is unknown. The Ush1c.216G>A (c.216G>A) mutation is a single base pair substitution from a guanine (G) to an adenine (A) in the USH1C gene, which results in the loss of 35 base pairs at the end of exon 3 in the mRNA transcript. A truncated and out of frame mutant mRNA is detected in Acadian Usher 1 patients and in c.216AA mutant mice. The purpose of this study is to define the molecular mechanism(s) by which the c.216G>A mutation results in the accumulation of mutant Ush1c mRNA and mutant harmonin protein.
Targeted rt-PCR and western blot analysis were used to quantitate wild type (wt) and mutant Ush1c mRNA and harmonin protein in retina and cochlea of c.216AA mutant and wt littermates. Wt and mutant Ush1c minigenes were used in Hela nuclear extracts with antisense oligonucleotides (ASOs) to block the use of the mutant splice donor.
Targeted RT-PCR with wt-Ush1c specific primers shows mutant mice make wt transcript, but about half as much as wt mice. Mutant-Ush1c specific primers show that, at a cDNA dilution of 1:25, mutant mRNA is detectable in mutant retinas and cochleae, but not wt tissues. Western blot analysis shows both full length harmonin and an abundance of truncated mutant harmonin in the retina and cochlea from mutant mice. Cell-free splicing analysis of in vitro transcribed Ush1c216G and A RNA substrates with ASOs shows increased wt mRNA transcripts and decreased mutant transcripts in a dose-dependent manner.
The c.216G>A mutation changes the frequency with which the mutant and wt splice donors are used. Wt mice preferentially splice the Ush1c gene at the end of exon 3, whereas mutant mice preferentially use the 216A in exon 3 to splice, resulting in the accumulation of truncated mRNA and mutant harmonin protein. ASOs targeted to c.216G>A mutation in cell-free nuclear extracts suppress mutant splicing and increase wt splicing suggesting their consideration as a therapeutic option for Usher syndrome.
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