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

通过全基因组测序克隆斑马鱼视觉系统突变体

• 批准号: 8358939
• 负责人: Jeffrey Gross
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8358939
• 关键词: Affect; Age related macular degeneration; Anatomy; Animal Model; Animals; Anterior; Biochemical; Biological Assay; Biological Models; Birth; Blindness; Caenorhabditis elegans; Cataract; Cell physiology; Cells; Childhood; Chromosome Mapping; Clinical; Cloning; Collection; Coloboma; Congenital Disorders; Data; Defect; Development; Developmental Biology; Disease; Disease model; Ethylnitrosourea; Etiology; Eye Abnormalities; Genetic; Genetic Screening; Genome; Glaucoma; Grant; Hereditary Disease; Human; Human Genetics; Image; Laboratories; Lead; Maps; Mediating; Methods; Modeling; Molecular; Mutate; Mutation; Ocular Albinism; Oculocutaneous Albinism; Organ; Patients; Phenotype; Physiology; Positioning Attribute; Process; Proteins; Reporting; Research; Research Personnel; Resolution; Retinal Degeneration; Retinitis Pigmentosa; Single Nucleotide Polymorphism Map; System; Techniques; Testing; Time; Tissues; Validation; Visual impairment; Visual system structure; Zebrafish; age related; base; blind; cell type; congenital cataract; cost; gene discovery; genome sequencing; human disease; in vivo; in vivo Model; innovation; interest; lens; malformation; mutant; novel; positional cloning; protein function; research study; simple sequence length polymorphism

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. PUBLIC HEALTH RELEVANCE: Model organisms with similar ocular anatomy and physiology to humans are vital to understand the molecular and cellular mechanisms underlying congenital disorders of the visual system and in which to develop and test potential therapies. The zebrafish is an ideal system for such studies, and research in this grant will utilize an innovativ whole-genome sequencing approach to identify affected loci in zebrafish models of human ocular diseases. We will clone the mutations in ~20 different mutant lines and then perform targeted experiments on congenital cataract mutants in order to determine the mechanistic underpinnings of their lens defects.

描述（由申请人提供）：视觉障碍影响全球超过1.6亿人，其中约3700万是盲人（1）。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相关的黄斑变性以及为这些疾病的有效低成本疗法的发展是最优先的。与人类相似的解剖结构和生理学的模型生物对于了解这些疾病的基础和开发和检验潜在疗法的分子和细胞机制至关重要。斑马鱼Danio Rerio是建模人类疾病的理想系统，并利用斑马鱼阐明先天性眼疾病的分子和细胞基础的研究对该领域产生了重大影响。的确，向前遗传筛选鉴定出具有眼缺损的斑马鱼突变体的组合，以及分析突变体表型在分析突变体表型方面的快速发展，将斑马鱼系统定位在那些有助于我们对人类先天性眼疾病的机械基础知识的人的最前沿。该赠款中的研究将利用创新的全基因组测序和SNP映射方法来快速且负担得起的克隆隐性斑马鱼视觉系统突变体，这些斑马视觉系统突变体从我们实验室中最近完成的前向遗传筛选中鉴定出来。在我们的屏幕上鉴定出23个突变体，并出现了先天性白内障，前节失调，colobobomas，眼皮白血病，视网膜变性和其他发育缺陷。我们的新型映射技术将使我们能够在这些突变体的大多数（如果不是全部）中克隆受影响的基因座，然后执行靶向假设驱动的实验，以确定导致眼部缺陷的基本分子和细胞机制。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。先天性白内障发生在每100,000人的大约40次，是发达国家儿童失明的最常见原因。白内障也是近200种不同的人类遗传疾病的常见临床特征，使其成为原本异质性疾病的常见组成部分。尽管对年龄相关和环境引起的白内障的了解众所周知，但对先天性白内障的病因却少知道。因此，此处提出的研究将具有重要的科学功绩，因为它们将确定正常晶状体发育所需的基因产品，并且可以提供动物先天性白内障模型，通过该模型可以提出对疾病机制的理解，并可以开发和测试新的疗法。 公共卫生相关性：具有类似的眼部解剖结构和生理学的模型生物对于了解视觉系统的先天性疾病的分子和细胞机制至关重要。斑马鱼是此类研究的理想系统，该赠款的研究将利用一种创新的全基因组测序方法来识别斑马鱼模型的人类眼部疾病模型中受影响的基因座。我们将在约20种不同的突变线中克隆突变，然后对先天性白内障突变体进行靶向实验，以确定其晶状体缺陷的机械基础。