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.

项目摘要/摘要该建议的目的是表征一个新型候选基因，用于使用临床，基因组，遗传性听力损失，和生物工具。该方法结合了信息丰富家庭的临床评估，基因组测序以识别候选基因，在体外分析候选突变等位基因对基因和蛋白质功能的后果以及体内娱乐和对具有人类候选者的CRISPR-CAS9小鼠观察到的表型的全面评估基因型。鉴定新的听力损失基因将扩大诊断基因检测，使人深入了解细胞和听力的分子机制，并允许未来针对这些机制的疗法发展。我的提议描述了这种方法在与两个先天孩子的亲密家庭中的应用，双边，深刻的，非合成的听力损失。孩子们对错义突变Aldoc纯合子 P.R149C。 ALDOC编码藻酶C，这是三个藻酶同工酶之一（A，B和C），对糖酵解至关重要果糖代谢。 ALDOC在大脑和内耳的感觉上皮中高度表达，并起作用旁系同源物Aldob的突变导致了雌树的肠道肠道。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在内耳内与ALDOC相关的听力损失和轮廓实验方法检验该假设。目的1。为了评估突变体aldoc p.R149c的生化特性，我首先纯化了野生型和突变的藻酶 C来自大肠杆菌，并证明突变蛋白的催化活性几乎完全丧失。进一步量化这一点突变的效果，我将完成热稳定性测试，Michael-Menten酶动力学计算，还通过创建和断言藻酶A/C混合动力组来评估其对醛酶异构体的影响。目的2。为了评估Aldoc P.R149C的表型效应，我设计了一种携带人类的CRISPR-CAS9小鼠突变。纯合小鼠的初步结果显示出轻度的听力损失。接下来，我将彻底评估通过听觉脑干反应（ABR）测试，听到纯合子的纯合子，并没有补充果糖并通过免疫组织化学和扫描电子显微镜分析人工耳蜗外植体。目标3。确定ALDOC P.R149C听力损失中涉及的细胞类型，我将使用单细胞RNA测序确定野生细胞群体和Aldoc P.R149C纯合小鼠之间的内耳细胞群体的差异。我的项目将在听觉神经科学，生物化学和基因组学方面提供培训。它将负担得起足够的科学素养和沟通，实验设计和执行以及跨学科合作。它是通过临床暴露于耳鼻喉科来完成的，并嵌入了UW中 MSTP，拥有50年的体验培训身体科学家。该集成的培训计划将为开始从事物理科学家的成功职业所必需的经验和心态。