Validating novel candidate genes for normal-tension glaucoma

摘要 我的最终职业目标是成为一名独立的科学家，研究遗传机制， 希望能改善病人的治疗方法。具体来说，我专注于疾病 在家族性队列中发现基因，这仍然是研究正常人的最有效手段之一， 张力性青光眼（NTG）。青光眼描述了一组眼部病症，其特征在于： 视网膜神经节细胞（RGC）的进行性变性，包括视神经， 占成年人口的1-3%。传统的治疗方法集中在降低眼内压 (IOP)，是最大的可改变风险因素。然而，患有NTG的一部分人-一种诊断， 占所有青光眼病例的近三分之一，并表明视神经萎缩，尽管IOP在 正常范围-降低IOP治疗后继续显示疾病进展。一个关键的障碍， 开发新的治疗方法是了解NTG的分子发病机制。为此，我们集团 鉴定了一个常染色体显性NTG家系，该家系对降IOP治疗反应不良。 连锁分析、全外显子组分析和全基因组分析显示， 在个别家庭成员中的高效隔离，尽管绝大多数情况下似乎是 解释为在支架/伴侣蛋白中高度保守的残基处的非常罕见的错义取代 蛋白RPAP 3。该变体在计算机模拟中具有高致病性分数，预计会破坏N-末端 蛋白质的结构域。N-末端结构域对于刺激HSP 90的ATP酶活性至关重要， 另一种调节细胞外基质（ECM）产生的支架/伴侣蛋白 件.此外，来自家族血液样本的RNA-seq揭示了ECM相关基因是由基因组中的一个基因决定的。 在差异基因表达分析中是最失调的。这些观察结果支持 假设RPAP 3的N-末端的破坏损害了HSP 90的调节，导致缺陷 ECM生产/维护。这可能会导致正常眼压性青光眼， 支撑视神经的结构比如筛板我的建议旨在：（一）确定功能 RPAP 3编码变体对HSP 90相互作用/刺激和ECM基因表达的影响， (ii)确定诊断为青光眼的患者队列中有害RPAP 3变体的流行率， 和（iii）阐明在这个大家族中分离的其他青光眼风险变体。结果可能导致 改善NTG患者的筛查和治疗选择。通过实现这些目标，我还将 拓宽了我在疾病基因发现和遗传变异功能表征方面的技能， 职业生涯专注于人类疾病遗传学和分子诊断。

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.