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（由适用提供）：病理血栓形成是发达国家发病率和死亡率的主要原因。虽然已经确定了诱发的遗传危险因素，但未知的修饰基因促进了患有和没有血栓性危险因素的患者和家庭中观察到的可变疾病严重程度和渗透率。了解这种修饰符可以帮助对患者进行较高的血栓形成和复发风险分类，该项目将利用强大的遗传工具，包括基因组编辑核武器，下一代测序和斑马鱼模型，以对止血调节基因进行大规模评估，以使人类凝结分离的潜力改变。在初步研究中，我们通过使用锌指核定的斑马鱼抗凝血酶III（AT3）基因的靶向诱变产生了诱导和赞助血栓形成模型，并表明这会导致易消耗的凝血病和赞助血栓形成。我们使用可靠的基因组编辑核（Talens和CRISPR/CAS）开发了许多其他凝血因子突变体，并进行了一个化学诱变筛选，该筛选已确定了具有前瞻性血栓形成基因的潜在抑制突变线。在特定目标1中，我们将使用CRISPR/CAS来评估在AT3缺乏症的背景下，单个凝血因素减少赞助血栓形成的影响。在特定的目标2中，我们将使用CRISPR/CAS在具有当前技术的哺乳动物系统中使用CRISPR/CAS来评估这些凝血因子分子的复杂多基因上皮相互作用。特定的目标3将隔离使用下一代测序技术在我们的诱变实验中产生的抑制突变体的基因。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符，这将是增强人类血栓形成和出血性疾病的诊断和治疗的候选者。