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

遗传性听力损失的新候选基因的发现和表征

基本信息

批准号：
10228506
负责人：
Ryan James Carlson
金额：
$ 3.99万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-16 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10228506
关键词：
Adult Affect Age Aldehyde-Lyases Aldolase A Aldolase C Alleles Auditory Auditory Brainstem Responses Basic Science Bilateral Biochemical Biochemistry Biological Biological Assay Biological Models Biology Brain CRISPR/Cas technology Candidate Disease Gene Cell Death Cell Survival Cells Cellular Morphology Cellular biology Child Clinical Clinical Data Cochlea Cochlear Implants Communication Complement Data Science Development Diagnostic Diet Enzyme Kinetics Enzymes Epithelial Equilibrium Escherichia coli Evaluation Experimental Designs Exposure to Family Fructose Future Gene Mutation Genes Genetic Genetic Enhancement Genetic Transcription Genomics Genotype Glycolysis Goals Growth Hair Cells Hearing Hearing Aids Hereditary fructose intolerance syndrome Homozygote Human Hybrids Immunohistochemistry In Vitro Individual International Isoenzymes Kinetics Laboratories Labyrinth Lead Learning Mammals Measures Mentorship Metabolic Metabolism Middle East Missense Mutation Molecular Morphology Mus Mutate Mutation Neurosciences Organ of Corti Otolaryngology Phenotype Physicians Play Population Privatization Property Proteins Public Health Quality of life Recording of previous events Recreation Role Scanning Electron Microscopy Scientist Sensory Siblings Supplementation Techniques Temperature Testing Time Training Translational Research Universities Visit Washington Wild Type Mouse base career cell type consanguineous family design dietary exome experience experimental study fructose-1-phosphate gene discovery gene function genetic disorder diagnosis genetic testing genome sequencing genomic tools hearing impairment hepatocyte injury hereditary hearing loss in vitro Assay in vivo in vivo Model insight interdisciplinary collaboration interest melting mouse model mutant neurodevelopment new therapeutic target novel novel therapeutics paralogous gene postnatal protein function research clinical testing restoration scientific literacy single-cell RNA sequencing social success targeted treatment therapy development thermostability tool transcriptomics whole genome

项目摘要

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.

项目概要/摘要该提案的目标是利用临床、基因组、该方法结合了信息丰富的家庭的临床评估、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，以及对携带人类候选者的 CRISPR-Cas9 小鼠中观察到的表型进行体内重建和全面评估听力损失新基因的鉴定将扩大诊断基因测试，深入了解细胞。和听力的分子机制，并允许未来开发针对这些机制的疗法。我的提案描述了这种方法在有两个患有先天性、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 编码醛缩酶 C，它是对糖酵解至关重要的三种醛缩酶同工酶（A、B 和 C）之一。 ALDOC 在大脑和内耳感觉上皮中高度表达，并发挥作用。其旁系同源物 ALDOB 的突变会导致遗传性果糖不耐受，其中积累底物果糖-1-磷酸会导致肝细胞损伤和细胞死亡。在这里，我们提出了类似的机制。内耳中与 ALDOC 相关的听力损失，并概述了检验这一假设的实验方法。目的 1. 为了评估突变型 ALDOC p.R149C 的生化特性，我首先纯化了野生型和突变型醛缩酶来自大肠杆菌的 C 并证明突变蛋白的催化活性几乎完全丧失，以进一步量化这一点。突变的影响，我将完成热稳定性测试、Michael-Menten 酶动力学计算，以及通过创建和测定醛缩酶 A/C 混合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，我设计了一只携带人类基因的 CRISPR-Cas9 小鼠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。通过听觉脑干反应 (ABR) 测试，在补充或不补充果糖的情况下纯合子的听力并通过免疫组织化学和扫描电子显微镜分析耳蜗外植体。目标 3. 为了确定 ALDOC p.R149C 听力损失所涉及的细胞类型，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。科学素养和交流、实验设计和执行的增长机会，以及它以耳鼻喉科的临床接触为补充，并嵌入华盛顿大学。 MSTP 拥有 50 年培训医师科学家的经验。该综合培训计划将提供以下服务：作为一名医师科学家开始成功的职业生涯所必需的经验和指导。