Chemical and Genetic Modifiers of the Protein C Pathway

描述（由申请人提供）：静脉血栓每年影响约1/ 1000人。大多数病例涉及一种或多种复杂的遗传因素以及一系列环境触发因素。大多数家族性单基因血栓病例与蛋白C （PC）通路缺陷有关。发现调控PC通路突变外显率的基因具有重要的临床意义。PC敲除斑马鱼将从冷冻精子中产生，这些精子对应于先前在ENU诱变斑马鱼DNA文库中鉴定出的2个PC空等位基因。这些鱼将用于敏化全基因组ENU突变筛选，以确定致死性血栓形成的抑制因子。可遗传的突变将通过位置克隆来鉴定。此外，PC敲除斑马鱼将用于第二个高通量筛选，以鉴定新型抗凝血剂。斑马鱼模式生物的优势将再次被利用来分析35000种不同的小分子化合物，以在体内拯救致命的表型。成功挽救性化合物可以在人血浆凝固试验和体内小鼠模型中快速测试，以确定其靶点并评估其治疗潜力。

DESCRIPTION (provided by applicant): Venous thrombosis affects approximately 1/1,000 individuals per year. Most cases involve 1 or more complex genetic factors together with a range of environmental triggers. The majority of monogenic cases of familial thrombosis are associated with defects in the protein C (PC) pathway. It would be of tremendous clinical significance to find genes that modulate the penetrance of mutations in the PC pathway. PC knockout zebrafish will be generated from frozen sperm corresponding to 2 previously identified null alleles of PC, identified in a library of DNA from ENU mutagenized zebrafish. These fish will be used in a sensitized genome-wide ENU mutagenesis screen to identify suppressors of the lethal thrombosis. Heritable mutations will be identified by positional cloning. In addition, the PC knockout zebrafish will be used in a second high-throughput screen to identify novel anticoagulants. The strengths of the zebrafish model organism will again be exploited to assay a diverse set of 35,000 small molecule compounds for in vivo rescue of the lethal phenotype. Successfully rescuing compounds can be quickly tested in human plasma coagulation assays and in vivo mouse models to determine their target and evaluate their therapeutic potential.