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Courtney Danielle Linne, Philip Andrew Ruzycki, Anne Hennig, Shiming Chen; Crx-L253X mutation produces dominant photoreceptor defects in TVRM65 mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3588.
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© ARVO (1962-2015); The Authors (2016-present)
The Cone-Rod Homeobox (CRX) transcription factor is essential for photoreceptor gene expression, differentiation and survival. Human CRX mutations cause dominant retinopathies of varying onset and phenotype severity. Frameshift CRX mutations that introduce a premature termination codon (PTC) result in inactive truncated proteins that interfere with normal CRX function. Two animal models of early PTC showed mutant mRNA/protein overproduction in heterozygotes, resulting in severe photoreceptor defects. We hypothesize that the pathogenicity of frameshift mutations depends on the PTC position and the overabundance of mutant mRNA/protein. To test this hypothesis, we characterized the TVRM65 mutant mouse, from The Jackson Laboratory, which carries a late PTC mutation (Crx-L253X), initially reported to model recessive blindness. We predict that, like other CRX PTC mutants, L253X has a dominant phenotype.
Retinas of homozygous (L253X/X), heterozygous (L253X/+), and wild-type control C57BL/6J (WT) mice collected at various ages were assessed for: 1) retinal cell morphology (histology of H&E sections), 2) the relative abundance of mutant CRX protein/mRNA to WT (quantitative Western Blot and digital droplet PCR), 3) gene expression (q-RTPCR and IHC) and 4) retinal function differences (ERG).
L253X/X showed a lack of photoreceptor function at 1 month and greater reductions in retinal thickness, but similar changes in gene expression to Crx null (on the same C57BL/6J background), suggesting that the L253X/X phenotype is at least as severe as that of null animals. L253X/+ mutants showed normal retinal morphology at all ages tested. However, both rod and cone retinal responses to visual stimuli were reduced, beginning at 1 month. q-RTPCR analysis described a complex phenotype of developmental delay and an inability to maintain proper gene expression in mature photoreceptors. L253X mRNA/protein were overexpressed relative to normal Crx, suggesting a pathogenic mechanism similar to other PTC mutants. However, the overexpression is less pronounced, correlating with a relatively mild dominant phenotype.
Crx L253X causes a dominant phenotype of altered gene expression and photoreceptor malfunction. The pathogenicity of CRX frameshift mutations depends on the positon of PTC (early is more toxic than late), which determines the degree of mutant mRNA/protein overproduction and phenotype severity.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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