Unexpected mechanism underlying mislocalization of thrombocytopenia-associated ETV6 point mutation

项目总结/摘要 ETV 6是一种转录抑制因子，参与造血干细胞的维持和终末分化 巨核细胞ETV 6条件性基因敲除小鼠显示外周血小板显著减少 计数和未成熟巨核细胞的代偿性增加。根据这些调查结果，在 近年来，ETV 6中的许多种系突变导致蛋白质的错误定位， 细胞核向细胞质的转移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了，因为约30%的人发展为骨髓增生异常 综合征或白血病。这些种系突变中的大多数发现于ETV 6的DNA结合结构域（DBD）中。 这些DBD突变的功能研究表明，在体外DNA结合能力的丧失和在体外DNA结合能力的丧失。 细胞中的转录抑制。然而，一个突变，Pro214 Leu错义突变在5例中被发现， 迄今为止，在ETV 6的长的内在无序的中心结构域中发生。这也表明了一种损失 的转录抑制在体外，但解释这种损失的机制尚未建立。 我收集的初步数据表明，这种Pro214 Leu错义突变会导致一个新的 核输出信号（内斯）导致输出蛋白1（XPO 1）介导的核输出。这构成了第一个 描述了产生从头内斯的点突变的实例。我们打算开发细胞和动物 模型系统，以探索这种意想不到的疾病机制对血小板生成的影响。我们 开发同源ETV 6 P214 L转基因小鼠系将验证其作为以下动物模型的适用性： ETV 6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 ETV 6 P214 L在巨核细胞和血小板发育中的核再定位。最后，初步 利用ClinVar（一种公开可用的遗传变异数据库）和内斯进行生物信息学检索 预测服务器已经产生了额外的候选突变，这些突变也可能产生新的NES。我们打算 显示错义突变依赖核输出是疾病的一般机制， 候选NES的表征可以产生新的疾病生物标志物。

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.