Unexpected mechanism underlying mislocalization of thrombocytopenia-associated ETV6 point mutation

血小板减少症相关 ETV6 点突变错误定位的意外机制

• 批准号: 10605685
• 负责人: Michael R McConville
• 金额: $ 3.88万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-31 至 2026-01-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605685
• 关键词: Affect; Amino Acids; Animal Model; Binding; Bioinformatics; Biological Models; Blood Platelets; Cell Maintenance; Cell Nucleus; Cell model; Cells; Chemicals; Childhood Precursor B Lymphoblastic Leukemia; Chronic Myelomonocytic Leukemia; ClinVar; Cytoplasm; DNA Binding; DNA Binding Domain; Data; Development; Disease; Dysmyelopoietic Syndromes; ETV6 gene; Exhibits; Family; Functional Imaging; Functional disorder; Genetic; Genetic Markers; Genetic Variation; Germ-Line Mutation; Goals; Hematologic Neoplasms; Hematopoietic; Heterozygote; Hour; In Vitro; Induced Mutation; Inherited; Knockout Mice; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Megakaryocytes; Missense Mutation; Mutate; Mutation; Nuclear; Nuclear Export; Penetrance; Peripheral; Platelet Count measurement; Point Mutation; Production; Proteins; RUNX1 gene; Recurrence; Risk; Signal Transduction; Somatic Mutation; Tertiary Protein Structure; Thrombocytopenia; Thrombopoiesis; Transcription Repressor; Transgenic Mice; Variant; Work; biophysical techniques; conditional knockout; exportin 1 protein; gene repression; genetic approach; human disease; inhibitor; leptomycin B; leukemia; meter; mouse model; mutant; mutation carrier; novel; novel marker; protein function; protein reconstitution; public database; stem cells; tool; transcription factor

PROJECT SUMMARY/ABSTRACT ETV6 is a transcriptional repressor involved inhematopoietic stem cell maintenance and terminal differentiation of megakaryocytes. ETV6 conditional knockout mice demonstrate a marked decrease in peripheral platelet counts and a compensatory increase in immature megakaryocytes. In concordance with these findings, in recent years, a number of germline mutations in ETV6 that result in mislocalization of the protein from the nucleus to the cytoplasm have been associated with inherited thrombocytopenia. Carriers of these mutations are also at an increased risk of hematologic malignancies as ~30% have gone on to develop myelodysplastic syndrome or leukemia. Most of these germline mutations are found in the DNA-binding domain (DBD) of ETV6. Functional studies of these DBD mutations demonstrate a loss of DNA-binding capacity in vitro and a loss of transcriptional repression in cells. However, one mutation, the Pro214Leu missense mutation identified in 5 families thus far, occurs in the long intrinsically disordered central domain of ETV6. It too demonstrates a loss of transcriptional repression in vitro, but the mechanism explaining this loss has not yet been established. Preliminary data I have gathered demonstrates that this Pro214Leu missense mutation creates a de novo nuclear export signal (NES) leading to exportin 1 (XPO1) mediated nuclear export. This constitutes the first described instance of a point mutation creating a de novo NES. We intend to develop cellular and animal model systems to probe the effects of this unexpected disease mechanism on thrombopoiesis. We are developing a homologous ETV6 P214L transgenic mouse line will validate its suitability as an animal model of ETV6-related thrombocytopenia. This will allow us to use genetic and chemical tools to study the effects of ETV6 P214L nuclear relocalization on megakaryocyte and platelet development. Lastly, a preliminary bioinformatics search utilizing ClinVar, a publicly available database of genetic variation, and an NES prediction server has yielded additional candidate mutations that may also create de novo NESs. We intend to show that missense mutation dependent nuclear export is a general mechanism of disease, and characterization of candidate NESs may yield novel biomarkers of disease.

项目摘要/摘要 ETV6是一种转录阻遏物，涉及无局部干细胞维持和末端分化 巨核细胞。 ETV6有条件的敲除小鼠表明周围血小板明显下降 未成熟的巨核细胞的计数和补偿性增加。与这些发现一致 近年来，ETV6中的许多种系突变导致蛋白质错误定位 细胞质的细胞核与遗传性血小板减少症有关。这些突变的载体 血液学恶性肿瘤的风险也增加，因为约有30％的人继续发展骨髓增生 综合征或白血病。这些种系突变中的大多数都在ETV6的DNA结合结构域（DBD）中发现。 这些DBD突变的功能研究表明，体外DNA结合能力的丧失，并且丧失了 细胞中的转录抑制。然而，一个突变，Pro214LEU的错义突变在5中鉴定出 迄今为止，家族发生在ETV6的长期本质上无序的中心结构域中。这也表明了损失 在体外的转录抑制作用，但尚未确定解释这种损失的机制。 我收集的初步数据表明，这种Pro214LEU错义突变创建了一个从头开始 导致Exportin 1（XPO1）介导的核出口的核出口信号（NES）。这构成了第一个 描述了一个点突变的实例。我们打算发展细胞和动物 模型系统以探测这种意外的疾病机制对血小板的影响。我们是 开发同源的ETV6 P214L转基因小鼠系将验证其作为动物模型的适用性 ETV6相关的血小板减少症。这将使我们能够使用遗传和化学工具来研究 ETV6 p214l核细胞和血小板发育的核重定位。最后，初步 生物信息学搜索利用Clinvar，遗传变异的公开数据库和NES 预测服务器产生了其他候选突变，这些突变也可能创建从头开始。我们打算 表明错义突变取决于核出口是疾病的一般机制，而 候选人的表征可能会产生新颖的疾病生物标志物。