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

描述(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。许多人类疾病，包括癌症，最终都是由特定基因的突变引起的。这些基因的发现是改进疾病检测和诊断以及设计基于分子的靶向治疗的关键一步。然而，疾病基因的发现受到与人类群体合作的困难和许多致病等位基因的低外显率的阻碍。为了改进疾病基因的发现，我们正在使用斑马鱼，这是一个很好的人类疾病模型，包括癌症、微生物发病机制和出生缺陷。此前，我们使用正向基因筛查来确定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“敏感株”通常需要“二次打击”--一个特定位点的突变--才能表现出疾病，因此可能是识别人类疾病中关键的疾病修正基因的宝贵资源。由于疾病的低外显性和长潜伏期，以及缺乏针对人类最常见癌症--上皮癌的斑马鱼模型，阻碍了利用易感菌株进行基因发现的进展。我们已经成功地利用单倍型图谱确定了导致斑马鱼睾丸肿瘤的一个低外显率、成体发病的疾病基因。在这里，我们建议建立一个强大的、用户友好的单倍型作图小组，作为整个斑马鱼群落的资源。利用这个小组和斑马鱼分离癌症敏感株的优势，我们将识别与上皮癌变有关的新基因突变。单倍型图谱方法和这些上皮性癌症菌株的可获得性将显著扩大斑马鱼在人类疾病研究中的力量。 与公共健康相关：在美国，每年有超过50万人死于癌症，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，我们正在使用脊椎动物的遗传模型系统--斑马鱼。在这项建议中，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.