Generation and Rapid Mapping of Low-Penetrance Disease Alleles in Zebrafish

描述（由申请人提供）：这项工作的长期目标是通过发现致病突变来改善人类健康。包括癌症在内的许多人类疾病最终都是由特定基因突变引起的。发现这些基因是改善疾病检测和诊断以及设计基于分子的靶向治疗的关键步骤。然而，疾病基因的发现受到人类群体工作的困难和许多致病等位基因的低多态性的阻碍。为了改善疾病基因的发现，我们正在使用斑马鱼，这是一种研究人类疾病的极好模型，包括癌症，微生物致病和出生缺陷。以前，我们使用正向遗传筛选来识别导致斑马鱼癌症易感性增加的基因突变。我们和其他人还表明，特定人类疾病等位基因的转基因表达使斑马鱼对相关人类疾病易感。这些“易感菌株”通常需要“第二次打击”-特定位点的突变-才能表现出疾病，因此可能是鉴定人类疾病中关键疾病修饰基因的宝贵资源。利用易感菌株进行基因发现的进展受到疾病的低发病率和长潜伏期以及缺乏人类最常见的癌症--上皮癌的斑马鱼模型的阻碍。我们已经成功地使用单倍型定位，以确定一个低突变率，成年发病的疾病基因引起的斑马鱼睾丸肿瘤。在这里，我们建议建立一个强大的，用户友好的单倍型映射面板作为整个斑马鱼社区的资源。利用这个面板和斑马鱼分离酶癌症易感性菌株，我们将确定新的基因突变负责上皮癌变。单倍型作图方法和这些上皮癌菌株的可用性将显着扩大斑马鱼用于人类疾病研究的能力。 公共卫生相关性：美国每年有50多万人死于癌症，需要更好的治疗。为了发现癌症中突变的基因并开发更有效的治疗方法，我们正在使用遗传脊椎动物模型系统，斑马鱼。在这项提案中，我们描述了显着提高斑马鱼癌症基因发现效率的方法，以及准确模拟人类癌症的斑马鱼的产生。

DESCRIPTION (provided by applicant): The long-term goal of this work is to improve human health by enabling the discovery of disease-causing mutations. Many human diseases, including cancer, are ultimately caused by mutations in specific genes. Discovery of these genes is a critical step in improving detection and diagnosis of disease, as well as for the design of molecularly-based, targeted therapies. However, disease gene discovery is hampered by the difficulties of working with human populations and by the low penetrance of many disease-causing alleles. To improve disease gene discovery, we are using the zebrafish, an excellent model of human diseases including cancer, microbial pathogenesis and birth defects. Previously, we used forward genetic screens to identify gene mutations that cause increased cancer susceptibility in zebrafish. We and others have also shown that transgenic expression of specific human disease alleles makes zebrafish susceptible to the relevant human disease. These "susceptibility strains" generally require a "second hit"-a mutation at a specific locus-to manifest disease, and thus could be an invaluable resource for identifying critical disease- modifying genes in human disease. Progress in using the susceptibility strains for gene discovery is hampered by the low penetrance and long latency of disease, and by the lack of zebrafish models for epithelial cancers, the most common cancers in humans. We have successfully used haplotype mapping to identify a low-penetrance, adult onset disease gene causing testicular tumors in zebrafish. Here we propose to establish a robust, user-friendly haplotype mapping panel as a resource for the entire zebrafish community. Taking advantage of this panel and the zebrafish separase cancer-susceptibility strain, we will identify novel gene mutations responsible for epithelial carcinogenesis. The availability of haplotype mapping methods and these epithelial cancer strains will significantly expand the power of the zebrafish for human disease research. PUBLIC HEALTH RELEVANCE: More than half a million people die of cancer each year in the US, and better treatments are needed. To discover the genes mutated in cancers and allow the development of more effective therapies, we are using the genetic vertebrate model system, the zebrafish. In this proposal we describe methods to significantly increase the efficiency of cancer gene discovery in zebrafish and the generation of zebrafish that accurately model human cancers.