Chemical and Genetic Modifiers of the Protein C Pathway

描述（由申请人提供）：静脉血栓形成每年影响约1/1，000人。大多数病例涉及1个或多个复杂的遗传因素以及一系列环境触发因素。家族性血栓形成的大多数单基因病例与蛋白C（PC）通路的缺陷有关。发现调控PC途径突变频率的基因将具有巨大的临床意义。PC敲除斑马鱼将由冷冻精子产生，所述冷冻精子对应于在来自ENU诱变斑马鱼的DNA文库中鉴定的2个先前鉴定的PC无效等位基因。这些鱼将用于敏化全基因组ENU诱变筛选，以鉴定致命血栓形成的抑制因子。将通过定位克隆鉴定可遗传突变。此外，PC敲除斑马鱼将用于第二次高通量筛选，以鉴定新型抗凝剂。将再次利用斑马鱼模式生物的优势来测定35，000种不同的小分子化合物，用于体内拯救致死表型。成功拯救的化合物可以在人血浆凝固测定和体内小鼠模型中快速测试，以确定它们的靶点并评估它们的治疗潜力。

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.