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Shi Song Rong, Anna Larson, John Simmons Borchert, Janey L Wiggs; Rare ATXN2 Variants in Cases with Primary Open Angle Glaucoma Cause Visual Impairment in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2833. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Genome-wide association studies have identified significant association of common and rare ATXN2 variants with primary open angle glaucoma (POAG). To understand functional consequences of rare coding ATXN2 variants, we tested mutant human ATXN2 mRNA compared with wild type human ATXN2 mRNA in a zebrafish model with reduced atxn2 expression.
Five potentially pathogenic ATXN2 coding variants found in POAG cases and not in controls were introduced into human ATXN2 cDNA vector via mutagenesis and were transcribed into mRNA. A translation-blocking (TB) morpholino was injected into ABTL strain zebrafish embryos at the 1- to 4-cell stage to reduce atxn2 expression. Human wild type mRNA (wtRNA) and the 5 mutant ATXN2 mRNAs (mutantRNA) were tested by co-injection with the morpholino, respectively. External phenotype, eye size, and visual motor response (VMR) were compared between wtRNA, mutantRNA, TB morphants, and negative controls (NC) at 5 days post fertilization (5dpf). These phenotypes were also tested at 5dpf in zebrafish injected with CRISPR-Cas9 system to knock down atxn2. The expression of atxn2 mRNA in zebrafish retina was tested using in situ hybridization.
All 5 mutantRNA groups had reduced vision as measured by lower activities during the first second following light change in the VMR assay (P<0.05). Decreased eye size in contrast to body length was observed in 4 of the 5 mutantRNA groups (P<0.05). wtRNA successfully rescued the wild type external phenotype, eye size, and VMR in comparison with TB morphants (P<0.001), while none of the mutant mRNAs fully rescued any phenotype including VMR. Reduction of VMR was also observed in fish with atxn2 knocked down by CRISPR-Cas9 system (P<0.01) in comparison with controls. In situ hypbridization confirmed expression of atxn2 mRNA in zebrafish retina, particularly in the retinal ganglion cell (RGC) layer.
Rare ATXN2 coding variants identified in cases with POAG cause visual impairment in zebrafish. Reduced eye size was also apparent for 4 of the 5 mutations. Expression of atxn2 in RGC suggests that these mutations impact normal retinal function.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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