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.

项目概要/摘要 ETV6 是一种转录抑制因子，参与造血干细胞维持和终末分化 巨核细胞。 ETV6条件敲除小鼠外周血小板明显减少 计数和未成熟巨核细胞的代偿性增加。根据这些发现，在 近年来，ETV6 中的许多种系突变导致该蛋白从 细胞核到细胞质与遗传性血小板减少症有关。这些突变的携带者 患血液系统恶性肿瘤的风险也增加，因为约 30% 的人会发展为骨髓增生异常 综合症或白血病。大多数种系突变都存在于 ETV6 的 DNA 结合域 (DBD) 中。 这些 DBD 突变的功能研究表明，体外 DNA 结合能力丧失，并且 细胞中的转录抑制。然而，在 5 种病毒中发现了一种突变，即 Pro214Leu 错义突变。 迄今为止，家族发生在 ETV6 长期本质上无序的中心域中。这也说明了一种损失 体外转录抑制，但解释这种损失的机制尚未确定。 我收集的初步数据表明，这种 Pro214Leu 错义突变会产生从头突变 核输出信号 (NES) 导致输出蛋白 1 (XPO1) 介导的核输出。这构成了第一个 描述了从头创建 NES 的点突变实例。我们打算开发细胞和动物 模型系统来探讨这种意想不到的疾病机制对血小板生成的影响。我们是 开发同源 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 核重新定位对巨核细胞和血小板发育的影响。最后，初步 利用 ClinVar（遗传变异的公开数据库）和 NES 进行生物信息学搜索 预测服务器已经产生了额外的候选突变，这些突变也可能从头创建 NES。我们打算 表明错义突变依赖的核输出是疾病的一般机制，并且 候选 NES 的表征可能会产生新的疾病生物标志物。