Genomic Locus Modulating Corneal Thickness in the Mouse IdentifiesPOU6F2as a Potential Risk of Developing Glaucoma

Abstract

Purpose: Central corneal thickness (CCT) is one of the most heritable ocular traits and it is also a phenotypic risk factor for primary open angle glaucoma (POAG). The present study uses the BXD Recombinant Inbred (RI) strains to identify novel quantitative trait loci (QTLs) modulating CCT in the mouse with the potential of identifying a molecular link between CCT and risk of developing POAG.Methods: The BXD RI strain set was used to define mammalian genomic loci modulating CCT, with a total of 818 corneas measured from 61 BXD RI strains (between 60-100 days of age). The mice were anesthetized and the eyes were positioned in front of the lens of the Phoenix Micron IV Image-Guided OCT system or the Bioptigen OCT system. CCT data for each strain was averaged and used to identify quantitative trait loci (QTLs) modulating this phenotype using the bioinformatics tools on GeneNetwork (www.genenetwork.org). The candidate genes and genomic loci identified in the mouse were then directly compared with the summary data from a human primary open-angle glaucoma (POGA) genome wide association study (NEIGHBORHOOD) to determine if any genomic elements modulating mouse CCT are also risk factors for POAG.Results: This analysis revealed one significant QTL on Chr 13 and a suggestive QTL on Chr 7. The significant locus on Chr 13 (13 to 19 Mb) was examined further to define candidate genes modulating this eye phenotype. For the Chr 13 QTL in the mouse, only one gene in the region (Pou6f2) contained nonsynonymous SNPs. Of these five nonsynonymous SNPs in Pou6f2, two resulted in changes in the amino acid proline which could result in altered secondary structure affecting protein function. The 7 Mb region under the mouse Chr 13 peak distributes over 2 chromosomes in the human: Chr 1 and Chr 7. These genomic loci were examined in the NEIGHBORHOOD database to determine if they are potential risk factors for human glaucoma identified using meta-data from human GWAS. The top 50 hits all resided within one gene (POU6F2), with the highest significance level of p = 10−6 for SNP rs76319873. POU6F2 is found in retinal ganglion cells and in corneal limbal stem cells. To test the effect of POU6F2 on CCT we examined the corneas of a Pou6f2-null mice and the corneas were thinner than those of wild-type littermates. In addition, these POU6F2 RGCs die early in the DBA/2J model of glaucoma than most RGCs.Conclusions: Using a mouse genetic reference panel, we identified a transcription factor, Pou6f2, that modulates CCT in the mouse. POU6F2 is also found in a subset of retinal ganglion cells and these RGCs are sensitive to injury.Authors SummaryGlaucoma is a complex group of diseases with several known causal mutations and many known risk factors. One well-known risk factor for developing primary open angle glaucoma is the thickness of the central cornea. The present study leverages a unique blend of systems biology methods using BXD recombinant inbred mice and genome-wide association studies from humans to define a putative molecular link between a phenotypic risk factor (central corneal thickness) and glaucoma. We identified a transcription factor, POU6F2, that is found in the developing retinal ganglion cells and cornea. POU6F2 is also present in a subpopulation of retinal ganglion cells and in stem cells of the cornea. Functional studies reveal that POU6F2 is associated the central corneal thickness and with susceptibility of retinal ganglion cells to injury.

POU6F2 Positive Retinal Ganglion Cells a Novel Group of ON-OFF Directionally Selective Subtypes in the Mouse Retina

“…The thinner CCT is also associated with an increased severity of visual field loss and a more rapid progression of the disease [11][12][13] . We use a systems genomics approach (the BXD recombinant mouse strain and the powerful bioinformatics available on GeneNetwork.org) to define genomic elements modulating CCT [14][15][16][17][18][19] . We examine the BXD strains and map genomic loci modulating CCT in the mouse 15 .…”

“…We use a systems genomics approach (the BXD recombinant mouse strain and the powerful bioinformatics available on GeneNetwork.org) to define genomic elements modulating CCT [14][15][16][17][18][19] . We examine the BXD strains and map genomic loci modulating CCT in the mouse 15 . CCT was measured in 818 eyes in 61 BXD strains 15 and the data mapped to a single locus in the mouse.…”

“…We examine the BXD strains and map genomic loci modulating CCT in the mouse 15 . CCT was measured in 818 eyes in 61 BXD strains 15 and the data mapped to a single locus in the mouse.…”

“…Our collaborator, Dr. Janey Wiggs examined these syntenic regions in the human NEIGHBORHOOD database 20 to determine if there are any potential risk factors for glaucoma within these loci. The top 50 hits are all one gene, POU6F2 15 . At the time POU6F2 was a potential risk factor for human glaucoma 21,22 ; however a subsequent study from the UK Biobank 23 demonstrates that POU6F2 is a glaucoma risk factor in humans even after correcting for multiple tests.…”

“…In the embryonic eye, strong POU6F2 staining was observed in neuroblasts destined to become RGCs. There is also staining of the developing cornea and corneal stem cells 15 . The present study identifies multiple POU6F2-positive RGC subtypes, and a portion of these cells form a novel group of ON-OFF directionally selective RGCs.…”