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

• 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
• 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

 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(申请人提供)：病理性血栓形成是发达国家发病率和死亡率的主要原因。虽然易感遗传风险因素已被确定，但未知的修饰基因导致患者和有或无血栓风险因素的患者和家庭的疾病严重程度和外显率不同。了解这些修饰物有助于将血栓形成和复发风险较高的患者分类，并可能作为调节出血性疾病严重程度的危险因素提供信息。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，对可能改变人类凝血障碍严重程度的止血调节基因进行大规模评估。在初步研究中，我们通过使用锌指核酸酶对斑马鱼抗凝血酶III(At3)基因进行定向突变，建立了一种诱导和自发血栓形成的模型，并表明这会导致消耗性凝血障碍和自发血栓形成。我们已经利用强大的基因组编辑核酸酶(TALENS和CRISPR/CAS)开发了一些额外的凝血因子突变体，并进行了化学诱变筛选，发现了含有潜在血栓形成修饰物基因的潜在抑制突变系。在具体目标1中，我们将使用CRISPR/CAS来评估在AT3缺乏的情况下个体凝血因子降低对自发性血栓形成的影响。在具体目标2中，我们将使用CRISPR/CA评估这些凝血因子分子之间复杂的多基因上位性相互作用，其水平和吞吐量在目前技术的哺乳动物系统中是不可能的。特定目标3将使用下一代测序技术分离在我们的突变实验中产生的抑制突变的潜在修饰基因。这些研究将确定已知凝血因子之间的关键相互作用以及这些途径的未知修饰物，这将是加强人类血栓和出血性疾病诊断和治疗的候选对象。