Discovery and characterization of a novel candidate gene for inherited hearing loss

项目总结/摘要 本提案的目标是利用临床、基因组、 和生物工具。该方法结合了临床评估信息的家庭，基因组测序，以确定 候选基因，候选突变等位基因对基因和蛋白质功能的影响的体外分析，以及 体内重建和在携带人类候选物的CRISPR-Cas9小鼠中观察到的表型的全面评估 基因型听力损失新基因的鉴定将扩大诊断基因检测， 和听力的分子机制，并允许未来开发针对这些机制的治疗方法。 我的建议描述了这种方法在一个有两个先天性， 双侧、深度、非综合征性听力损失这些孩子是错义突变ALDOC的纯合子 p.R149C. ALDOC编码醛缩酶C，这是对糖酵解至关重要的三种醛缩酶同工酶（A、B和C）之一， 果糖代谢ALDOC在脑和内耳的感觉上皮中高度表达，并发挥作用。 在神经发育中。它的副产物ALDOB的突变导致遗传性果糖不耐受， 底物果糖-1-磷酸导致肝细胞损伤和细胞死亡。在这里，我们提出了一个类似的机制， 在内耳内的ALDOC相关的听力损失，并概述实验方法来测试这一假设。 目标1。为了评价突变ALDOC p.R149C的生化特性，我首先纯化了野生型和突变型醛缩酶 C从E。大肠杆菌中，并表现出几乎完全丧失催化活性的突变蛋白。为了进一步量化 突变的影响，我将完成热稳定性测试，Michael-Menten酶动力学计算， 通过产生和测定醛缩酶A/C杂合体组来评估其对醛缩酶异聚体的影响。 目标2.为了评估Aldoc p.R149C的表型效应，我设计了一种CRISPR-Cas9小鼠， 突变纯合子小鼠的初步结果显示轻度听力损失。接下来，我将全面评估 通过听性脑干反应（ABR）测试，在有和没有果糖补充的情况下，纯合子的听力 用免疫组化和扫描电镜分析耳蜗组织块。 目标3：为了确定ALDOC p.R149C听力损失中涉及的细胞类型，我将利用单细胞RNA测序 鉴定野生型和Aldoc p.R149C纯合小鼠之间内耳细胞群的差异。 我的项目将提供听觉神经科学、生物化学和基因组学方面的培训。它将提供充足的 科学素养和沟通，实验设计和执行的增长机会，以及 跨学科合作。它是补充临床接触耳鼻喉科和嵌入在华盛顿大学 MSTP拥有50年的医生-科学家培训经验。该综合培训计划将提供 经验和指导必要的开始一个成功的职业生涯作为一个医生科学家。

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to characterize a novel candidate gene for inherited hearing loss using clinical, genomic, and biological tools. The approach combines clinical evaluation of informative families, genome sequencing to identify candidate genes, in vitro analysis of the consequences of candidate mutant alleles on gene and protein function, and in vivo recreation and full evaluation of the phenotype observed in CRISPR-Cas9 mice bearing the human candidate genotype. The identification of new genes for hearing loss will expand diagnostic genetic testing, yield insight into cellular and molecular mechanisms of hearing, and allow for future development of therapies targeting these mechanisms. My proposal describes the application of this approach to a consanguineous family with two children with congenital, bilateral, profound, non-syndromic hearing loss. The children are homozygous for the missense mutation ALDOC p.R149C. ALDOC encodes aldolase C, one of three aldolase isozymes (A, B, and C) that are critical for glycolysis and fructose metabolism. ALDOC is highly expressed in brain and in the sensory epithelium of the inner ear and plays a role in neurodevelopment. Mutations in its paralog, ALDOB, lead to hereditary fructose intolerance where accumulation of the substrate fructose-1-phosphate causes hepatocyte injury and cell death. Here, we propose a similar mechanism within the inner ear for ALDOC-related hearing loss and outline experimental approaches to test this hypothesis. Aim 1. To evaluate the biochemical properties of mutant ALDOC p.R149C, I first purified wild-type and mutated aldolase C from E. coli and demonstrated near complete loss of catalytic activity of the mutant protein. To further quantify this mutation’s effects, I will complete thermostability testing, Michael-Menten enzyme kinetics calculations, and also evaluate its effect on aldolase heteromers by creating and assaying an aldolase A/C hybrid set. Aim 2. To evaluate the phenotypic effects of Aldoc p.R149C, I designed a CRISPR-Cas9 mouse carrying the human mutation. Preliminary results from homozygous mice show a mild hearing loss. Next, I will thoroughly evaluate the hearing of the homozygotes, with and without fructose supplementation, by auditory brainstem response (ABR) testing and analyze cochlear explants by immunohistochemistry and scanning electron microscopy. Aim 3. To determine the cell type(s) involved in ALDOC p.R149C hearing loss, I will utilize single-cell RNA sequencing to identify differences in inner ear cell populations between wild type and Aldoc p.R149C homozygous mice. My project will provide training across auditory neuroscience, biochemistry, and genomics. It will afford ample opportunities for growth in scientific literacy and communication, experimental design and execution, and interdisciplinary collaboration. It is complemented by clinical exposure to otolaryngology and embedded in the UW MSTP, which has 50 years’ experience training physician-scientists. This integrated training plan will provide the experience and mentorship necessary to begin a successful career as a physician scientist.