Dissecting the Genetics of Human Hemostatic Disorders Using Zebrafish

使用斑马鱼剖析人类止血疾病的遗传学

基本信息

批准号：
9243287
负责人：
JORDAN A SHAVIT
金额：
$ 38.75万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9243287
关键词：
Adult Affect Anticoagulants Anticoagulation Antifibrinolytic Agents Antithrombin III Antithrombin III Deficiency Biological Models Blood Coagulation Disorders Blood Coagulation Factor Blood coagulation Chemicals Clinical Clustered Regularly Interspaced Short Palindromic Repeats Coagulation Process Complex Data Development Diagnosis Disease Embryo Equilibrium Ethylnitrosourea Evaluation Event Family Fertilization Fishes Genes Genetic Genetic Screening Heart Diseases Hemorrhage Hemostatic Agents Hemostatic function Hospitalization Human Human Genetics Individual Induced Mutation Intervention Knowledge Larva Mammals Measures Methods Minority Modeling Modification Morbidity - disease rate Mutagenesis Nitrosourea Compounds Optics Organism Pathologic Pathway interactions Patients Penetrance Phenotype Plasma Recurrence Regulator Genes Research Risk Risk Factors Risk stratification Severities Severity of illness Stroke Suppressor Mutations System Technology Testing Thrombophilia Thrombosis Venous Thrombosis Zebrafish base biomarker panel cost enhancing factor experience experimental study genetic risk factor genome editing high risk individual patient mortality mutant next generation sequencing novel nuclease offspring patient stratification positional cloning prospective public health relevance therapeutic target tool zebrafish development zinc finger nuclease

项目摘要

DESCRIPTION (provided by applicant): Pathologic thrombosis is a leading cause of morbidity and mortality in the developed world. While predisposing genetic risk factors have been identified, unknown modifier genes contribute to the variable disease severity and penetrance observed among patients and families with and without thrombophilic risk factors. Understanding of such modifiers could help classify patients at higher risk for thrombosis and recurrence, and might also be informative as risk factors modulating the severity of bleeding disorders. This project will take advantage of powerful genetic tools, including genome editing nucleases, next generation sequencing, and the zebrafish model to conduct a large scale evaluation of hemostasis regulatory genes with the potential to modify the severity of human coagulation disorders. In preliminary studies, we have produced a model of induced and spontaneous thrombosis by targeted mutagenesis of the zebrafish antithrombin III (at3) gene using zinc finger nucleases, and shown that this results in consumptive coagulopathy and spontaneous thrombosis. We have developed a number of additional coagulation factor mutants using robust genome editing nucleases (TALENS and CRISPR/Cas), and conducted a chemical mutagenesis screen that has identified potential suppressor mutant lines harboring prospective thrombosis modifier genes. In Specific Aim 1, we will use CRISPR/Cas to evaluate the effect of reduction of individual coagulation factors on spontaneous thrombosis in the context of at3 deficiency. In Specific Aim 2, we will evaluate complex multigene epistatic interactions of these coagulation factor molecules using CRISPR/Cas at a level and throughput that is not possible in mammalian systems with current technology. Specific Aim 3 will isolate the modifier genes underlying the suppressor mutants generated in our mutagenesis experiment using next generation sequencing technologies. These studies will identify critical interactions amongst known coagulation factors as well as unknown modifiers of these pathways, which will be candidates for enhanced diagnosis and therapy of human thrombotic and hemorrhagic diseases.

描述（由申请人提供）：病理性血栓形成是发达国家发病和死亡的主要原因，虽然已经确定了诱发遗传风险因素，但未知的修饰基因导致在有或没有的患者和家庭中观察到的不同的疾病严重程度和外显率。了解这些修饰因素可以帮助对血栓形成和复发风险较高的患者进行分类，并且还可以作为调节出血性疾病严重程度的风险因素提供信息。该项目将利用强大的遗传工具，包括基因组。编辑核酸酶、下一代测序和斑马鱼模型对止血调节基因进行大规模评估，有可能改变人类凝血障碍的严重程度。在初步研究中，我们通过定向诱变建立了诱导和自发血栓形成的模型。使用锌指核酸酶对斑马鱼抗凝血酶 III (at3) 基因进行研究，并表明这会导致消耗性凝血病和自发性血栓形成。使用强大的基因组编辑核酸酶（TALENS 和 CRISPR/Cas）构建凝血因子突变体，并进行化学诱变筛选，识别出含有预期血栓形成修饰基因的潜在抑制突变体系。在特定目标 1 中，我们将使用 CRISPR/Cas 来评估其效果。在 at3 缺乏的情况下，单个凝血因子减少对自发性血栓形成的影响在特定目标 2 中，我们将使用这些凝血因子分子来评估复杂的多基因上位相互作用。 CRISPR/Cas 的水平和通量在现有技术的哺乳动物系统中是不可能的。Specific Aim 3 将使用下一代测序技术分离我们的诱变实验中产生的抑制突变体背后的修饰基因。凝血因子以及这些途径的未知调节剂，将成为增强人类血栓和出血性疾病的诊断和治疗的候选者。