Myosin 15:Genetics, Pathology and Therapeutic Potential

描述(由申请人提供)：遗传性内耳疾病很常见，对生活质量有重大影响。遗传性内耳疾病目前还没有可用的临床治疗方法。小鼠是了解遗传性内耳疾病和开发治疗措施的理想哺乳动物模型。小鼠模型促进了人类遗传性疾病基础基因的发现，使研究这些基因在内耳发育和功能中的作用成为可能，并有望成为开发遗传性内耳疾病治疗方法的模型。这项拨款申请建立在我们的发现基础上，即非传统肌球蛋白基因Myo15的突变导致了两个自发突变小鼠的严重先天性耳聋和前庭功能障碍：Shaker 2和Shaker 2J，以及DFNB3人类。我们使用这些小鼠模型来展示表达Myo15的转基因对耳聋的长期结构和功能表型的纠正。我们描述了Myo15、Myo6、Myo7A、旋转体和Whirlin缺陷突变、双杂合子和双突变的病理发展。虽然在双重杂合子中没有增加与年龄相关的听力损失的风险，但这些研究揭示了每个肌球蛋白基因的独特功能，并提示MYO15除了将Whirlin运输到立体纤毛尖端之外，还可能具有其他功能。我们建立了用于基因治疗的腺病毒载体，并建立了正常小鼠和Myo15突变小鼠在断奶和成年期耳蜗中差异表达基因的数据库。这些研究为这笔赠款的目标奠定了坚实的基础。MYO15有多种异构体，它们是通过选择性剪接产生的，包括MYO15的运动区N末端是否存在一个大的富含Pro的区域。我们假设这个富含脯氨酸的区域对于听力所必需的蛋白质-蛋白质相互作用是重要的。我们已经用敲入技术生成了一个小鼠模型，它重现了人类富含脯氨酸区域的突变。这些突变体具有严重的先天性耳聋，发束病理与Shaker 2和Shaker 2J小鼠不同，前庭功能明显正常。我们提出了一种结构-功能分析，通过突变等位基因、细胞培养和耳蜗外植体分析，揭示MYO15亚型在耳蜗发育和功能中的重要性。我们将进行经典的遗传分析，以评估突变等位基因之间的相互作用，并识别相互作用的蛋白质。我们的调查团队在跨学科合作方面取得了成就，汇集了耳鼻喉科、显微镜、生理学和发育遗传学方面的专家。这个团队将使我们能够充分利用动物模型来了解内耳疾病的机制，并有可能识别对正常听力至关重要的新基因。 公共卫生相关性：耳聋是一种常见的出生缺陷，在2000年影响了大约1名新生儿。这些儿童中约有一半是因为遗传原因而受到影响，目前还没有已知的治疗或预防措施。肌球蛋白15是一种分子马达蛋白，对正常听力很重要。该基因的突变会导致人类和小鼠耳聋。在这项拨款提案中，我们寻求识别肌球蛋白15与之相互作用的其他蛋白质，并使用小鼠模型来识别由人类突变引起的病理变化。

DESCRIPTION (provided by applicant): Hereditary inner ear disease is prevalent and has significant implications for quality of life. There is currently no available clinical cure for hereditary inner ear disease. The mouse serves as an ideal mammalian model for understanding genetic inner ear disease and for developing therapeutic measures. Mouse models have facilitated the discovery of genes that underlie hereditary disease in humans, have made it possible to study the role of these genes in inner ear development and function, and hold great promise as models for developing treatments for hereditary inner ear disease. This grant application builds on our discovery that mutations in the unconventional myosin gene, Myo15, are responsible for profound congenital deafness and vestibular dysfunction in two spontaneous mouse mutants: shaker 2 and shaker 2J, and in humans with DFNB3. We used these mouse models to demonstrate the long-term structural and functional phenotypic correction of deafness with a transgene expressing Myo15. We characterized the development of pathology in Myo15, Myo6, Myo7a, pirouette, and whirlin deficient mutants, double heterozygotes and double mutants. Although there is no enhanced risk of age related hearing loss in double heterozygotes, these studies revealed unique functions of each myosin gene, and suggested the possibility that MYO15 has other functions besides transportation of whirlin to the stereocilia tips. We established adenoviral vectors for gene therapy and a database of genes exhibiting differential expression in the cochlea between weaning and adulthood in normal and Myo15 mutant mice. These studies laid a sound foundation for the goals of this grant. There are multiple isoforms of MYO15 that are generated by alternative splicing, including the presence or absence of a large proline-rich region N-terminal to the motor domain of MYO15. We hypothesize that this proline-rich region is important for protein-protein interactions necessary for hearing. We have generated a mouse model that recapitulates a human mutation in the proline-rich domain using knock-in technology. These mutants have profound congenital deafness, hair bundle pathology that is distinct from shaker 2 and shaker 2J mice, and apparently normal vestibular function. We propose a structure-function analysis that will reveal the importance of MYO15 isoforms in the development and function of the cochlea using mutant alleles, cell culture and cochlear explant assays. We will conduct a classical genetic analysis to evaluate interactions between mutant alleles and identify interacting proteins. Our investigative team has a track record for accomplishments resulting from cross-disciplinary collaboration, bringing together experts in otolaryngology, microscopy, physiology, and developmental genetics. This team will enable us to exploit the animal models fully to understand the mechanisms of inner ear disease and has the potential to identify novel genes essential for normal hearing. PUBLIC HEALTH RELEVANCE: Deafness is a common birth defect, affecting about 1 birth in 2000. About half of these children are affected because of genetic reasons, and there is no known cure or prevention. Myosin 15 is a molecular motor protein that is important for normal hearing. Mutations in this gene cause deafness in humans and mice. In this grant proposal we seek to identify other proteins that Myosin 15 interacts with and to use mouse models to identify the pathological changes caused by human mutations.