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J. Yang, X. Liu, M. Adamian, B. S. Pawlyk, X. Sun, D. R. McMillan, P. C. White, M. C. Liberman, T. Li; N-Terminal Mutation of Whirlin Disrupts the Usher Syndrome II (USH2) Protein Complex and Causes Retinal and Inner Ear Defects in Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4405.
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Mutant alleles of the whirlin gene cause different diseases in humans. A mutation near the C-terminus results in nonsyndromic deafness, while its N-terminal mutations are responsible for USH2D, a condition with both visual and hearing defects. In this study, we investigated the underlying mechanism for the variable disease expressions involving the whirlin gene.
Gene targeting was carried out by homologous recombination in embryonic stem cells followed by blastocyst injection of the targeted clones. Whirlin knockout mice were examined by light and scanning electron microscopy, electroretinography (ERG), and distortion product otoacoustic emissions (DPOAE) testing. Subcellular localization and expression levels of whirlin and other USH2 proteins were analyzed by immunofluorescence and Western blotting.
Whirlin colocalized with two known Ush2 proteins Ush2a and Vlgr1 at the periciliary ridge complex (PRC)-like structure in photoreceptors and at the stereocilia in hair cells. Disruption at the first exon of the whirlin gene in mice abolished the normal localization of Ush2a and Vlgr1 in photoreceptors and reduced their protein levels, indicative of destabilization. Photoreceptor outer segments were shortened and ERG amplitudes were reduced in whirlin mutant mice at 28 months of age. Localization of Ush2a and Vlgr1 in hair cells was also affected in the mutant. Stereocilia of inner ear hair cells in mutant mice were lost in the basal turn of the cochlea, and shortened or distorted in the middle turn. The threshold of hearing response was markedly elevated as demonstrated by DPOAE testing. The hearing loss appeared to be non-progressive.
Whirlin forms a multi-protein complex in vivo with the two other known USH2 proteins through its N-terminal domain. A mutation in this region causes both retinal degeneration and cochlear hair cell defects in mice as in humans. Subtle differences in functional requirements and transcript/splice variant expressions between photoreceptors and inner ear hair cells may explain the variable disease expressions caused by mutations in different regions of the whirlin gene.
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