Validating novel candidate genes for normal-tension glaucoma

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ABSTRACT My ultimate career goal is to become an independent scientist researching the genetic mechanisms of Mendelian disorders in hopes of improving patient therapeutics. Specifically, I am focused on disease gene discovery in familial cohorts, which remains one of the most effective means by which to study normal- tension glaucoma (NTG). Glaucoma describes a group of ocular conditions characterized by the progressive degeneration of the retinal ganglion cells (RGCs) that comprise the optic nerve and is present in 1-3% of the adult population. Traditional therapies have focused on lowering intraocular pressure (IOP), the largest modifiable risk factor. However, a subset of individuals with NTG – a diagnosis which accounts for nearly one-third of all glaucoma cases and denotes optic atrophy despite an IOP that is within normal range – continue to show disease progression after IOP-lowering treatment. A critical barrier to developing new treatments is understanding the molecular pathogenesis of NTG. To this end, our group identified a pedigree with autosomal dominant NTG that is poorly responsive to IOP-lowering therapy. Linkage, whole-exome, and whole-genome analyses revealed that there is likely more than one risk allele of high effect segregating in individual family members, though the vast majority of cases seem to be explained by a very rare missense substitution at a highly conserved residue in scaffolding/chaperone protein RPAP3. This variant, with high in silico pathogenicity scores, is predicted to disrupt the N-terminal domain of the protein. The N-terminal domain is critical for stimulating the ATPase activity of HSP90, another scaffolding/chaperone protein that modulates the production of extracellular matrix (ECM) components. Further, RNA-seq from familial blood samples revealed that ECM-associated genes were among the most dysregulated in a differential gene expression analysis. These observations support the hypothesis that disruptions in the N-terminus of RPAP3 impair HSP90 regulation, leading to defects in ECM production/maintenance. This could result in normal-tension glaucoma by compromising key optic nerve-supporting structures like the lamina cribrosa. My proposal seeks to: (i) determine the functional consequences of RPAP3 coding variants on HSP90 interaction/stimulation and ECM gene expression, (ii) determine the prevalence of deleterious RPAP3 variants in a cohort of patients diagnosed with glaucoma, and (iii) elucidate additional glaucoma risk variants segregating in this large family. The results may lead to improved screening and treatment options for NTG patients. By carrying out these aims, I will also broaden my skillset in disease gene discovery and functional characterization of genetic variants, poising me for a career focused on human disease genetics and molecular diagnostics.