Cloning & Functional Studies of Deafness Mutations

描述（由申请人提供）：由于内耳解剖学、功能和病理学的相似性，小鼠是研究人类听力障碍的极好模型。我们的研究计划旨在通过发现和研究小鼠中破坏这些过程的基因突变来确定在耳朵的正常发育和生理学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的组件。我们通过定位克隆自发的、引起耳聋的突变，鉴定了五个新的听力相关基因，分别是hurry-scurry（hscy）、jitterbug（jbg）、roundabout（rda）、hyperspin（hspn）和deaf wanderer（dwnd）。这五个基因中没有一个与人类或小鼠的听力或耳聋有关。这项更新申请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。将通过光学和电子显微镜检查不同年龄的突变小鼠内耳的结构异常，并通过原位杂交和免疫组织化学确定基因转录物和蛋白质的时空分布。这些新发现的基因和其他内耳表达基因编码的蛋白质之间的潜在相互作用将通过亚细胞共定位，GST下拉分析和免疫共沉淀实验进行分析。其他实验将测试关于每个基因的拟议功能的特定假设，因为它们涉及内耳发育，内淋巴稳态，毛细胞形态发生和机械转导等过程。本申请的第二个目的是通过高分辨率定位和候选基因分析来鉴定六个新定位的耳聋突变的潜在基因：倾斜旋转（tow）、可变盘旋（Varc）、螺旋桨（pir）、8字形（fgt）、风车（wdml）和helter-pronter（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听力下降的突变，因此很可能识别出以前不知道参与听力过程的基因。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。当鉴定出负责基因时，将在突变小鼠和对照小鼠中确定基因表达模式。本提案的第三个目的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定位。自上一次拨款申请以来，我们已经发现了26个新的与听力障碍相关的遗传突变。我们建议将这些突变中的每一个基因映射到小于5 cM的分辨率。与已知耳聋突变或听力相关基因映射到相同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性，而那些映射到新位置的突变将成为未来定位克隆工作的主题，以确定其潜在的基因。

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.