Zebrafish and Mouse Models with Mutations in the Novel Gene ASRGL1 Develop Retinal Degeneration (RD) Recapitulating the Human Phenotype.

Abstract

Purpose : We identified a homozygous G178R mutation in the human ASRGL1 (hASRGL1) segregating with early-onset recessive RD in a pedigree with predominant cone abnormalities. The purpose of this study was to evaluate the involvement of ASRGL1 mutations in retinal pathology.

Methods : Zebrafish overexpressing hASRGL1 was generated by injecting 1ng-5ng of wild type (wt) or G178R-hASRGL1 (mut) mRNA into zero day post fertilization (dpf0) embryos. The ocular phenotype in 6dpf zebrafish expressing wt or mut hASRGL1 was studied by evaluating gross morphology, axial length measurements and immunohistochemistry with photoreceptor marker antibodies. The Asrgl1 gene ablated mouse model (Asrgl1^mut/mut) was generated using CRISPR/Cas9 methodology. Retinal morphology of these mice was evaluated by imaging, optical coherence tomography, immuno staining with photoreceptor marker and ASRGL1 antibodies. Expression of Asrgl1 and photoreceptor marker genes was studied by qRT-PCR.

Results : The zebrafish overexpressing the G178R-hASRGL1 showed decreased axial length and increased mortality compared to wt-hASRGL1 and dye injected fish. Immunohistochemistry with rod and cone opsin antibodies showed no difference between dye-injected and wt-hASRGL1 mRNA injected fish. Whereas, fish injected with 4ng G178R-hASRGL1 mRNA showed smaller eye phenotype (axial length) and loss of blue cone opsins. The fish injected with >5ng of mutant mRNA developed severe pathology and mortality at 1dpf. However, fish injected with wt-hASRGL1 mRNA at concentrations over 200ng had normal eye morphology. The Asrgl1^mut/mut mice displayed significant decrease in the levels of expression of Asrgl1 (p<0.0001), Opn1mw (p=0.003), Opn1sw (p=0.02) and Rho (p=0.01) transcripts compared to those of age matched control mice. Immunostaining of the retinal sections of Asrgl1^mut/mut mice showed a significant loss of M-opsin expressing cones compared to age-matched control mice and near total loss of the ASRGL1 protein. The electroretinography recordings exhibited reduced cone response in six months old Asrgl1^mut/mut mice compared to the age-matched controls.

Conclusions : ASRGL1 gene ablated zebrafish and mouse models developed loss of cone photoreceptors, which is consistent with the phenotype observed in patients harboring mutations in ASRGL1, suggesting the involvement of ASRGL1 mutations in causing RD phenotype.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.