Exploring the use of forward genetics to search for novel genetic associations to glaucoma

Abstract

Purpose : Glaucoma can result from any disruption of the complex pathways controlling aqueous humor dynamics or the susceptibility of retinal ganglion cells to intraocular pressure. Although it seems that a large proportion of early onset glaucoma cases have a genetic basis, only 12 genes have been described so far for primary forms of the disease, compared to hundreds for retinal dystrophies. We propose to use a state-of-the-science forward genetics protocol in an unbiased manner to identify non-redundant genes involved in the modulation of glaucoma.

Methods : We used OCT images to screen mice in a forward genetics pipeline searching for genes associated to changes in inner retinal thickness to look for candidate genes that may be associated to glaucoma. N-Ethyl-N-Nitrosourea (ENU)-mutated founder mice underwent whole exome sequencing and G3 mice included in the study were pre-genotyped at all mutant loci. We have obtained OCT scans at a standard location of 1.5 disc diameter superior to the optic disc. All investigators obtaining OCT measurements underwent careful training and intra- and inter-observer variability was determined. Thickness of the inner retinal layers (IRT, GCC) was measured and the data was analyzed using Linkage Analyzer and Candidate Explorer to find potential gene-phenotype associations.

Results : Testing of measurement variability revealed that ganglion cell complex (GCC) measurements resulted in a 64-66% reduction in inter- and intra-observer variability when compared to nerve fiber layer measurements. Thus, two measurements of the inner retina were obtained: GCC and IRT (inner retinal thickness; top of the internal limiting membrane [ILM] to the top of the outer nuclear layer [ONL]) for preliminary data on 5 pedigrees of ENU-mutated mice (total of 227 mice). These have led to the identification of 4 statistically significant gene-phenotype associations to GCC and 4 associations to IRT. An additional 5 pedigrees will be included in this pilot study for a projected 0.6% saturation of the genome.

Conclusions : Forward genetics screening using inner retinal thickness measurements is identifying potential candidate genes that may be associated to modulation of glaucoma. Selected genes may be targeted using CRISPR mutagenesis in order to corroborate the screening findings and further look at mechanisms.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.