Cloning & Functional Studies of Deafness Mutations

克隆

• 批准号: 7782039
• 负责人: KENNETH R JOHNSON
• 金额: $ 54.88万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7782039
• 关键词: Affect; Age; Anatomy; Animal Model; Applications Grants; Auditory; Biological Assay; Candidate Disease Gene; Child; Chromosome Mapping; Chromosomes; Cloning; Co-Immunoprecipitations; Communities; DNA Sequence; DNA Sequence Analysis; Development; Diagnostic; Ear; Electron Microscopy; Endolymph; Functional disorder; Future; Gene Expression; Gene Mutation; Genes; Goals; Hair Cells; Hearing; Hearing Impaired Persons; Hearing problem; Heritability; Homeostasis; Human; Immunohistochemistry; In Situ Hybridization; Individual; Inherited; Knock-out; Laboratories; Labyrinth; Light; Location; Maintenance; Maps; Molecular; Morphogenesis; Mus; Mutant Strains Mice; Mutation; Names; Pathology; Pathology processes; Pathway interactions; Pattern; Physiology; Population; Positioning Attribute; Process; Proteins; Public Health; Research; Resolution; Role; Screening procedure; Sensory; Spatial Distribution; Study models; Testing; Time; Transcript; deafness; experience; hearing impairment; improved; mRNA Expression; mouse model; mutant; positional cloning; programs; public health relevance; research study

DESCRIPTION (provided by applicant): The mouse is an excellent model for studying human hearing disorders because of the similarities in inner ear anatomy, function, and pathology. Our research program seeks to identify molecules and pathways that are important in the normal development and physiology of the ear by discovering and studying gene mutations in mice that disrupt these processes. To achieve this goal, our research includes both discovery and hypothesis driven components. We identified five new hearing-related genes by the positional cloning of spontaneous, deafness-causing mutations named hurry-scurry (hscy), jitterbug (jbg), roundabout (rda), hyperspin (hspn), and deaf wanderer (dwnd). None of these five genes was previously known to be associated with hearing or deafness in humans or mice. The first aim of this renewal application is to determine the specific roles of these genes in inner ear development and auditory function. Inner ears of mutant mice at various ages will be examined by light and electron microscopy for structural anomalies, and temporal and spatial distributions of gene transcripts and proteins will be determined by in situ hybridization and immunohistochemistry. Potential interactions among the proteins encoded by these newly identified genes and other inner ear-expressing genes will be analyzed by subcellular co-localizations, GST pull-down assays and co-immunoprecipitation experiments. Other experiments will test specific hypotheses about the proposed functions of each gene, as they relate to such processes as inner ear development, endolymph homeostasis, hair cell morphogenesis, and mechanotransduction. The second aim of this application is to identify, by high resolution mapping and candidate gene analysis, the genes underlying six newly mapped deafness mutations: tilt-a-whirl (tow), variable circling (Varc), propeller (pir), figure eight (fgt), windmill (wdml), and helter-skelter (hsk). These new mutations were mapped to chromosome regions that are devoid of known deafness-causing mutations and therefore are likely to identify genes not previously known to be involved in the hearing process. Inner ear pathologies associated with the new mutations will be characterized to provide clues to gene identity and function. When the responsible genes are identified, gene expression patterns will be determined in mutant and control mice. The third aim of this proposal is to continue our screening program to discover new mouse deafness mutations and determine their inheritance and chromosomal locations. Since the last grant application, we have discovered 26 new heritable mutations with associated hearing impairment. We propose to genetically map each of these mutations to a resolution of less than 5 cM. The mutations that map to the same chromosome locations as known deafness mutations or hearing-related genes will be tested for potential allelism by functional complementation and DNA sequence analysis, and those that map to new locations will become subjects for future positional cloning efforts to identify their underlying genes. PUBLIC HEALTH RELEVANCE: Impairment of hearing is the most common sensory deficit in human populations and affects about one of every 1,000 children. We use the mouse as a model for studying human hearing disorders because of the anatomical, functional, and pathological similarities between mouse and human inner ears. Accomplishment of the goals stated in this grant application will (1) improve our understanding of the molecular mechanisms that underlie the hearing process and pathologies that cause deafness and (2) provide the scientific community with new mouse models that could contribute to the development of diagnostics, treatments and therapies for human hereditary hearing impairment.

描述（由申请人提供）：小鼠是研究人类听力障碍的优秀模型，因为它们在内耳解剖、功能和病理上具有相似性。我们的研究项目旨在通过发现和研究小鼠中破坏这些过程的基因突变来识别在耳朵正常发育和生理中重要的分子和途径。为了实现这一目标，我们的研究包括发现和假设驱动两部分。我们通过对自发性耳聋突变的定位克隆，鉴定出5个新的听力相关基因，分别是：匆匆（hscy）、吉特巴（jbg）、回旋（rda）、超旋（hspn）和失聪流浪者（dwnd）。在此之前，人们并不知道这五种基因与人类或小鼠的听力或耳聋有关。这项更新应用的第一个目的是确定这些基因在内耳发育和听觉功能中的具体作用。不同年龄的突变小鼠内耳将通过光镜和电镜检查结构异常，并通过原位杂交和免疫组织化学确定基因转录本和蛋白质的时空分布。这些新发现的基因编码的蛋白与其他内耳表达基因之间的潜在相互作用将通过亚细胞共定位、GST下拉实验和共免疫沉淀实验进行分析。其他实验将测试关于每个基因功能的具体假设，因为它们与内耳发育、内淋巴稳态、毛细胞形态发生和机械转导等过程有关。本应用程序的第二个目的是通过高分辨率定位和候选基因分析，确定六种新定位的耳聋突变的基因：倾斜旋转（tow），可变旋转（Varc），螺旋桨（pir），数字八（fgt），风车（wdml）和混乱（hsk）。这些新的突变被定位到染色体区域，这些区域缺乏已知的导致耳聋的突变，因此很可能识别出以前不知道的与听力过程有关的基因。与新突变相关的内耳病理将被表征，以提供基因身份和功能的线索。当确定了相关基因后，将确定突变小鼠和对照小鼠的基因表达模式。本提案的第三个目标是继续我们的筛选计划，以发现新的小鼠耳聋突变，并确定其遗传和染色体位置。自上次拨款申请以来，我们已经发现了26个与听力障碍相关的新的遗传突变。我们建议将这些突变的遗传图谱绘制到小于5厘米的分辨率。与已知的耳聋突变或听力相关基因位于相同染色体位置的突变将通过功能互补和DNA序列分析来检测潜在的等位基因，而那些位于新位置的突变将成为未来定位克隆工作的对象，以确定其潜在基因。