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

项目摘要/摘要 这项建议的目标是利用临床、基因组和基因组学方法来描述遗传性耳聋的新候选基因， 和生物工具。该方法结合了对信息家系的临床评估、基因组测序来识别 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，以及在 携带人类候选基因的CRISPR-Cas9小鼠的活体重建和表型的全面评估 基因分型。识别听力损失的新基因将扩大诊断基因测试，获得对细胞的洞察 和分子听力机制，并允许未来开发针对这些机制的治疗。 我的提案描述了这种方法在一个有两个孩子患有先天性心脏病的近亲家庭中的应用。 双侧、深度、非综合征性听力损失。这些儿童是错义突变ALDOC的纯合子 P.R149C。ALDOC编码醛缩酶C，三种醛缩酶同工酶之一(A、B和C)对糖酵解和 果糖代谢。ALDOC在大脑和内耳的感觉上皮细胞中高度表达，并在 在神经发育方面。其类似基因ALDOB的突变导致遗传性果糖不耐受，在那里积累 底物1-磷酸果糖会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 在内耳中发现与ALDOC相关的听力损失，并概述检验这一假设的实验方法。 目的1.对突变株ALDOC p.R149C的生化性质进行初步研究 C，突变蛋白的催化活性几乎完全丧失。为了进一步量化这一点 突变的影响，我将完成热稳定性测试，Michael-Menten酶动力学计算，以及 通过创建和分析醛缩酶A/C杂交组来评估其对醛缩酶异构体的影响。 目的2.为了评价Aldocp.R149C的表型效应，我设计了一只携带人的CRISPR-Cas9小鼠 突变。来自纯合子小鼠的初步结果显示有轻微的听力损失。接下来，我将彻底评估 通过听性脑干反应(ABR)测试纯合子在补充果糖和不补充果糖的情况下的听力 并用免疫组织化学和扫描电子显微镜对耳蜗外植体进行分析。 目的3.为了确定ALDOC p.R149C听力损失所涉及的细胞类型(S)，我将利用单细胞rna测序 目的：鉴定野生型和Aldocp.R149C纯合子小鼠内耳细胞群体的差异。 我的项目将提供听神经科学、生物化学和基因组学方面的培训。它将负担得起足够的资金 科学素养和交流、实验设计和实施方面的增长机会，以及 跨学科协作。它与耳鼻喉科的临床暴露相辅相成，并嵌入到UW MSTP拥有50年培训内科科学家的经验。这一综合培训计划将提供 作为一名内科科学家开始成功的职业生涯所必需的经验和指导。

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.