Cloning zebrafish visual system mutants by whole-genome sequencing & SNP mapping

描述（由申请人提供）：视力障碍影响全球超过1.6亿人，其中大约3700万人是盲人。失明的主要原因包括白内障、青光眼、色素性视网膜炎和与年龄有关的黄斑变性，为这些疾病开发有效、低成本的治疗方法是最高优先事项。具有与人类相似的解剖学和生理学的模式生物对于理解这些疾病的分子和细胞机制以及开发和测试潜在的治疗方法至关重要。斑马鱼是模拟人类疾病的理想系统，利用斑马鱼阐明先天性眼部疾病的分子和细胞基础的研究在该领域产生了重大影响。事实上，前向遗传筛选相结合，以确定斑马鱼突变体与眼部缺陷，并在分析突变表型的技术进步的快速步伐定位斑马鱼系统的最前沿的那些有助于我们的知识的机制基础的人类先天性眼病。这项资助的研究将利用创新的全基因组测序和SNP作图方法，快速、经济地克隆从我们实验室最近完成的正向遗传筛选中鉴定的隐性斑马鱼视觉系统突变体。在我们的筛选中鉴定了23个突变体，其表现为先天性白内障、眼前节发育不全、缺损、眼皮肤白化病、视网膜变性和其他发育缺陷。我们的新作图技术将使我们能够在大多数（如果不是全部）这些突变体中克隆受影响的位点，然后进行有针对性的假设驱动的实验，以确定导致眼部缺陷的潜在分子和细胞机制。我们将把这些进一步的研究工作集中在先天性白内障突变体上。先天性白内障的发生率约为每10万人中有40人，是发达国家儿童失明的最常见原因。白内障也是近200种不同人类遗传疾病的常见临床特征，使其成为其他异质性疾病集合的常见组成部分。虽然对年龄相关性和环境诱发性白内障了解很多，但对先天性白内障的病因学了解较少。因此，本文提出的研究将具有显著的科学价值，因为它们将鉴定正常透镜发育所需的基因产物，并且它们将提供动物先天性白内障模型，通过该模型可以促进对疾病机制的理解，并且通过该模型可以开发和测试新的疗法。

DESCRIPTION (provided by applicant): Visual impairments affect over 160 million people worldwide, and of these, roughly 37 million are blind (1). Major causes of blindness include cataracts, glaucoma, retinitis pigmentosa and age-related macular degeneration and the development of effective, low-cost therapies for these disorders is of the highest priority. Model organisms with similar anatomy and physiology to humans are vital to understand the molecular and cellular mechanisms underlying these diseases and in which to develop and test potential therapies. The zebrafish, Danio rerio, is an ideal system for modeling human disease, and studies utilizing zebrafish to elucidate the molecular and cellular underpinnings of congenital ocular disorders have made a significant impact in the field. Indeed, the combination of forward genetic screens to identify zebrafish mutants with ocular defects, and the rapid pace of technological advancement in analyzing the mutant phenotypes has positioned the zebrafish system at the forefront of those that contribute to our knowledge of the mechanistic underpinnings of human congenital eye diseases. Research in this grant will utilize an innovative whole-genome sequencing and SNP mapping approach to rapidly and affordably clone recessive zebrafish visual system mutants identified from a recently completed forward genetic screen in our lab. 23 mutants were identified in our screen that presented with congenital cataracts, anterior segment dysgenesis, colobomas, oculocutaneous albinism, retinal degeneration and other developmental defects. Our novel mapping technique will enable us to clone the affected loci in most, if not all, of these mutants and then perform targeted, hypothesis-driven experiments to determine the underlying molecular and cellular mechanisms that lead to ocular defects. We will focus these further research efforts on the congenital cataract mutants. Congenital cataracts occur in ~40 per 100,000 human births and represent the most common cause of childhood blindness in the developed world. Cataracts are also a common clinical feature in nearly 200 different human genetic diseases making them a frequent component of an otherwise heterogeneous collection of disorders. While much is known about age-related and environmentally induced cataracts, less is known about the etiology of congenital cataracts. Thus, the studies proposed here will have significant scientific merit as they will identify gene products required for normal lens development and they will provide animal congenital cataract models through which an understanding of disease mechanism can be can be advanced and a model through which novel therapies can be developed and tested.