The genetic background determines the severity of retinal pathology in homozygous Crb1^rd8/rd8 mice

Abstract

Purpose: Understanding phenotype-genotype correlations in monogenetic and multifactorial inherited retinal degenerations is still a major challenge. Different and even identical mutations within the same gene can lead to variable clinical manifestations, suggesting additional modifying factors. To understand the contribution of different genetic backgrounds to such phenotypic variability, we compared the retinal pathology of three homozygous Crb1^rd8/rd8 mouse lines with C57Bl/6J Ola Hsd (Crb1^wt/wt) mice between 2-10 months of age.

Methods: For in vivo assessment of the retinal phenotype, autofluorescent scanning laser ophthalmoscopy and optical coherence tomography (OCT) were used. Semithin histology and immunohistochemistry on retinal flat-mounts for vasculature (lectin B4) and microglia (Iba1) were used to further assess retinal lesions. Whole genome SNP screens provided information about the genetic background of the Crb1^rd8/rd8 lines, which were derived from previous backcross experiments with C57Bl/6J Ola Hsd (C57Bl/6J Crb1^rd8/rd8 (line 1 & 2)) or from the Jackson lab (Crb1^rd8/rd8).

Results: At 2 months of age, fundus images from all homozygous Crb1^rd8/rd8 lines revealed significantly increased numbers of autofluorescent signals in the inferior retina when compared to wildtype mice confirming the rd8 mutation as the cause for the retinal phenotype. However, a striking variability in this phenotype was observed between the three lines. While homozygous Crb1^rd8/rd8 mice show a high number of these characteristic lesions, the two other lines revealed significantly fewer lesions. In line 1, typical lesions were difficult to identify and located far inferiorly, while line 2 showed a variable and intermediate number of lesions. OCT, semithin histology and immunohistochemistry revealed that retinal pathology in severely affected animals was associated with photoreceptor nuclei dropout and microglia activation, which was gradually reduced in both weaker affected lines even at older ages. SNP data suggests that the reduced phenotype correlates with an increase in C57Bl6/J background.

Conclusions: This data highlights the crucial role of the genetic background in determining the phenotype of homozygous rd8 mice. Similar genetic background effects should be more widely considered, not only in rd8 mice, but also in other retinal degeneration models and human disease.

Keywords: 696 retinal degenerations: hereditary • 536 gene modifiers • 695 retinal degenerations: cell biology