The severe neovascular phenotype of a mouse model for Coats-like vasculopathy, rnv3, is dependent on Crb1^rd8

Abstract

Purpose : To identify the genetic basis of rnv3 (previously reported as JR5558 and Nrv2), a mutant with an early-onset retinal neovascular phenotype.

Methods : Longitudinal analysis by fundus examination, brightfield fundus imaging, fluorescein angiography and optical coherence tomography (OCT) was carried out on live animals, and histology was performed at various ages. The rnv3 mutation was mapped in a linkage cross using polymorphic markers. An exome capture library generated from a rnv3 mutant was subject to high-throughput sequencing and analysis. The mutation was corrected directly in zygotes, using TALEN and oligonucleotide homology directed repair.

Results : OCT indicated lesions in the subretinal space and fluorescein angiography indicated aberrant vessels with leakage. Histologically, retinal vessels span the outer retina and form lesions in the subretinal space as early as 3 weeks of age. Marker analysis indicated fragmentation of the external limiting membrane. The rnv3 phenotype mapped to Chr.1. Examination of sequences within the critical region indicated a mutation in the Crb1 gene; the same mutation reported for Crb1^rd8. TALEN mediated correction of the Crb1^rd8 allele, Crb1^corrrd8 in the homozygous rnv3 genetic background showed a 90% bilateral reduction in the number of neovascular lesions compared to uncorrected rnv3 mutant fundus images.

Conclusions : Genetic analysis has linked mutations in CRB1 with many retinal diseases including retinitis pigmentosa (RP) with para-arteriolar preservation of the RPE, RP with Coats-like exudative vasculopathy, Leber congenital amaurosis, pigmented paravenous retinochoroidal atrophy and macular dystrophy. A large degree of variation is also seen both in the age of onset and the severity of vision loss among patients with CRB1 variants. Our results indicate that Crb1^rd8 is necessary for the severe neovascular abnormalities in rnv3 mutants, as only a low incidence of neovascular lesions is observed among corrected mice. Since some neovascular lesions were still observed, and B6/J.Cg-Crb1^rd8/rd8 and B6/NJ-Crb1^rd8/rd8 mice normally do not present with neovascular lesions at three weeks, this suggests that additional gene(s) must interact with Crb1^rd8 to cause early, aberrant vessel formation. Hence, yet unidentified genes in the rnv3 genetic background result in a neovascular phenotype that is dramatically increased by the Crb1^rd8 mutation.

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