Cloning & Functional Studies of Deafness Mutations

描述(申请人提供)：小鼠是研究人类听力障碍的极佳模型，因为它在内耳解剖、功能和病理上有相似之处。我们的研究计划旨在通过发现和研究小鼠中扰乱这些过程的基因突变，来确定在耳朵的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个部分。我们通过对自发的、导致耳聋的突变进行定位克隆，发现了5个新的听力相关基因，它们分别是：匆忙突变(Hscy)、吉特巴突变(Jbg)、环行突变(Rda)、超旋突变(Hspn)和聋人漫游者(Dwnd)。这五个基因之前都不知道与人类或老鼠的听力或耳聋有关。这次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的内耳将通过光学和电子显微镜检查结构异常，并将通过原位杂交和免疫组织化学来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质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下拉试验和免疫共沉淀实验来分析。其他实验将测试关于每个基因所提出的功能的具体假设，因为它们与内耳发育、内淋巴动态平衡、毛细胞形态发生和机械转导等过程有关。这项应用的第二个目的是通过高分辨率作图和候选基因分析，确定六个新定位的耳聋突变的基因：倾斜-a-Whirl(TOW)、可变旋转(VARC)、螺旋桨(PIR)、8字形(FGT)、风车(WdML)和杂乱无章(HSK)。这些新的突变被映射到染色体区域，这些区域没有已知的导致耳聋的突变，因此很可能识别出以前未知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，以提供基因识别和功能的线索。一旦确定了相关基因，就可以确定突变小鼠和对照小鼠的基因表达模式。这项建议的第三个目标是继续我们的筛查计划，以发现新的小鼠耳聋突变，并确定它们的遗传和染色体位置。自从上一次拨款申请以来，我们已经发现了26个与听力障碍相关的可遗传突变。我们建议将这些突变中的每一个基因定位到低于5厘米的分辨率。映射到与已知耳聋突变或听力相关基因相同的染色体位置的突变将通过功能互补和DNA序列分析来测试潜在的等位基因，而映射到新位置的突变将成为未来位置克隆工作的对象，以确定其潜在基因。 公共卫生相关性：听力障碍是人类最常见的感觉缺陷，大约每1000名儿童中就有一人受到影响。我们使用小鼠作为研究人类听力障碍的模型，因为小鼠和人类的内耳在解剖、功能和病理上都有相似之处。完成这项拨款申请中规定的目标将(1)提高我们对导致耳聋的听力过程和病理基础的分子机制的了解，以及(2)为科学界提供新的小鼠模型，这可能有助于人类遗传性听力障碍的诊断、治疗和治疗的发展。

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.