Project Grant R21EY036238

USING FORWARD GENETICS TO EXPLORE THE GENETICS AND MECHANISMS OF EARLY ONSET GLAUCOMA - PROJECT SUMMARY THE PATHWAYS REGULATING THE DEVELOPMENT OF EARLY-ONSET GLAUCOMA ARE NOT WELL UNDERSTOOD. DESPITE EVIDENCE SUGGESTING THAT A LARGE PROPORTION OF EARLY-ONSET GLAUCOMA CASES MAY HAVE A GENETIC BASIS, KNOWN GENES ONLY ACCOUNT FOR ABOUT 20% OF CASES. IN FACT, ONLY 12 GENES HAVE BEEN DESCRIBED SO FAR, FOR PRIMARY FORMS OF THIS DISEASE, COMPARED TO HUNDREDS FOR RETINAL DYSTROPHIES. IN THIS COMPLEX BIOLOGICAL SYSTEM, A FORWARD GENETICS APPROACH IS AN IDEAL STRATEGY TO ASK, WITHOUT PRECONCEPTIONS, WHICH GENES/MOLECULES ARE IMPORTANT IN REGULATING EARLY-ONSET GLAUCOMA. OUR SHORT-TERM GOAL IS TO IDENTIFY AND CHARACTERIZE GENE/PROTEIN DEFECTS AND MOLECULAR PATHWAYS THAT LEAD TO EARLY-ONSET GLAUCOMA. THE LONG-TERM GOAL IS TO LEVERAGE OUR RESEARCH DISCOVERIES TO UNDERSTAND THIS BLINDING DISEASE, IMPROVE SCREENING STRATEGIES, AND IDENTIFY NOVEL THERAPEUTIC OPPORTUNITIES. WE PROPOSE THAT A HIGH-THROUGHPUT AND UNBIASED STRATEGY PROVIDES AN IDEAL APPROACH TO DISCOVERY OF GENE/PHENOTYPE ASSOCIATIONS FOR EARLY-ONSET GLAUCOMA. IN COLLABORATION WITH NOBEL LAUREATE BRUCE BEUTLER, WE HAVE BEEN EMPLOYING A ROBUST STATE-OF-THE-SCIENCE AND UNBIASED FORWARD GENETICS PIPELINE IN WHICH RANDOM MUTATIONS ARE GENERATED AND MICE CAN BE SCREENED FOR SIGNS OF GLAUCOMA. WE HAVE ALREADY COLLECTED RETINAL IMAGES FROM 6000 MUTAGENIZED MICE AND PROPOSING TO USE THIS EXTENSIVE DATABASE TO SCREEN FOR GENES THAT LEAD TO INNER RETINAL THINNING. OUR APPROACH HAS SIGNIFICANT ADVANTAGES COMPARED TO OTHER EXISTING PROTOCOLS. MOST IMPORTANTLY, OURS IS THE FIRST AND ONLY PROTOCOL IN WHICH ALL MICE HAVE BEEN PRE-GENOTYPED AT ALL MUTANT LOCI. IN ADDITION, THE LARGE SCALE OF OUR SYSTEM AND THE LARGE PEDIGREE SIZE WILL ALSO ADD TO THE DISCOVERY POWER. TOGETHER, THESE ADVANTAGES WILL ALLOW US TO IDENTIFY AND PURSUE NOVEL GENE/PHENOTYPE ASSOCIATIONS RELATED TO GLAUCOMA. IN OUR RETINAL STUDIES WE HAVE IDENTIFIED OVER 45 GENE-PHENOTYPE ASSOCIATIONS AFTER COVERING JUST 5%-8% OF THE MOUSE GENOME. OF THESE, 20 GENES HAVE NOT BEEN REPORTED TO BE ASSOCIATED WITH THE RETINA. THESE RESULTS ATTEST TO THE STRENGTH OF OUR PIPELINE. HAVING AN EXCELLENT CONTINUOUS VARIABLE PARAMETER TO MONITOR FOR EARLY-ONSET GLAUCOMA (GANGLION CELL COMPLEX OCT MEASUREMENTS) ALSO SUPPORTS THE FEASIBILITY OF OUR PROPOSAL. WE WILL HARNESS THE POWER OF CRISPR/CAS9 GENE EDITING, SINGLE CELL RNA SEQUENCING, AND CO- IMMUNOPRECIPITATION EXPERIMENTS WITH HIGHLY SENSITIVE MASS SPECTROMETRY AND PROTEOMICS ANALYSIS, AND OTHER TECHNIQUES TO EXPLORE THE MECHANISMS OF THESE ASSOCIATIONS. THIS PROPOSED RESEARCH WILL ADVANCE OUR KNOWLEDGE OF THE GENETIC BASIS IN EARLY-ONSET GLAUCOMA. WE ALSO ANTICIPATE THAT OUR RESULTS WILL LEAD TO THE IDENTIFICATION OF NOVEL DIAGNOSTIC AND THERAPEUTIC AVENUES.