Unexpected mechanism underlying mislocalization of thrombocytopenia-associated ETV6 point mutation

项目摘要/摘要 ETV6是一种参与造血干细胞维持和终末分化的转录抑制因子 巨核细胞的数量。ETV6条件性基因敲除小鼠外周血小板显著减少 未成熟的巨核细胞计数和补偿性增加。与这些发现一致的是，在 近年来，ETV6中的一些种系突变导致蛋白质错误定位 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 患恶性血液病的风险也会增加，因为大约30%的人会发展成骨髓增生异常 综合症或白血病。这些胚系突变大多在ETV6的DNA结合域(DBD)中发现。 这些DBD突变的功能研究表明，在体外失去了DNA结合能力，并失去了 细胞中的转录抑制。然而，一种突变，Pro214Leu错义突变在5 到目前为止，这些家族发生在ETV6的长期内在无序的中心区域。它也表明了一种损失 在体外转录抑制，但解释这种损失的机制尚未建立。 我收集的初步数据表明，Pro214Leu错义突变产生了从头开始的 核输出信号(NES)导致Exportin 1(XPO1)介导的核输出。这构成了第一个 描述了一个点突变产生新的NES的实例。我们打算发展细胞和动物 探索这种意想不到的疾病机制对血小板生成的影响的模型系统。我们是 建立同源ETV6 P214L转基因小鼠品系将验证其作为猪传染性支气管炎动物模型的可行性 ETV6相关性血小板减少症。这将使我们能够使用遗传和化学工具来研究 ETV6P214L在巨核细胞上的核重新定位与血小板发育。最后，一个初步的 利用可公开获得的遗传变异数据库ClinVar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，这些突变也可能产生从头开始的Ness。我们打算 表明错义突变依赖的核输出是疾病的一般机制，以及 对候选Ness的表征可能会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.