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

描述(申请人提供)：视力障碍影响全球超过1.6亿人，其中约3700万人是盲人(1)。导致失明的主要原因包括白内障、青光眼、视网膜色素变性和老年性黄斑变性，为这些疾病开发有效、低成本的治疗方法是当务之急。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背后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Danio rerio是模拟人类疾病的理想系统，利用斑马鱼阐明先天性眼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，结合正向遗传筛选来识别患有眼睛缺陷的斑马鱼突变，以及分析突变表型的快速技术进步，使斑马鱼系统处于有助于我们了解人类先天性眼病的机制基础的前沿。这项拨款的研究将利用一种创新的全基因组测序和SNP作图方法，快速且负担得起地克隆从我们实验室最近完成的正向遗传筛查中识别出的隐性斑马鱼视觉系统突变体。在我们的筛选中鉴定出23个突变，表现为先天性白内障、眼前段发育不全、缺损、眼皮肤白化、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克隆这些突变中的大多数(如果不是全部)的受影响的基因座，然后进行有针对性的、假设驱动的实验，以确定导致眼睛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上。先天性白内障发生在每100,000名新生儿中约40人，是发达国家儿童失明的最常见原因。白内障也是近200种不同人类遗传病的共同临床特征，使其成为各种疾病的常见组成部分。虽然人们对年龄相关和环境引起的白内障知之甚多，但对先天性白内障的病因却知之甚少。因此，这里提出的研究将具有重大的科学价值，因为它们将识别正常晶状体发育所需的基因产物，并将提供动物先天性白内障模型，通过该模型可以进一步了解疾病的发病机制，并提供一个可以开发和测试新疗法的模型。

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.