TCEB3突变通过抑制巨核细胞成熟诱发IT的作用及其机制研究

Inherited thrombocytopenia (IT) is a kind of rare single-gene hereditary diseases. Since its highly genetic heterogeneity, the pathogenic genes are still not completely identified so far. We discovered an autosomal dominant IT parentage and the patients showed maturation disorder of megakaryocytes. By using exome sequencing and whole-genome genetic linkage analysis, we identified a new IT pathogenic gene TCEB3 and its pathogenic mutation p.S732N. The mutant can inhibit maturation of megakaryocyte through preventing the formation of polyploidy. However, the precise pathogenic mechanism remains to be elucidated. As the mutation was located in the elongation domain of TCEB3, which domain was critical for up-regulating the transcription of cyclin E, and cyclin E was indispensible for the maturation of megakaryocytes, we thus raised one hypothesis as follow. Through down-regulating the transcription of cyclin E, TCEB3-S732N impairs the maturation of megakaryocyte, and then causes reduced thrombopoiesis. We will investigate such a potential pathogenic mechanism of TCEB3-S732N in this novel IT by using point mutation mice and in vitro induced megakaryocytes from both human and mice. The results of the study will not only help to elucidate the pathogenesis of this new IT type, but can also provides a new direction for investigating the key regulations in the megakaryopoiesis and thrombopoiesis.

遗传性血小板减少症（IT）是一类罕见单基因遗传病。因具有高度遗传异质性，其致病基因至今仍未完全阐明。课题组发现一个常染色体显性遗传IT家系，患者表现出巨核细胞成熟障碍。通过全外显子组测序技术及全基因组连锁分析发现一个新的IT基因TCEB3，并鉴定出一个新致病变异p.S732N，该变异可通过阻止巨核细胞多倍体形成而抑制其成熟，但具体机制有待阐明。分析显示该变异位于TCEB3转录延长功能结构域，该区域对Cyclin E转录水平具有正向调控作用，而Cyclin E是巨核细胞成熟关键调控因子。因此我们推测，p.S732N可能通过下调Cylin E转录水平，抑制巨核细胞成熟，从而导致血小板生成减少。本研究拟通过定点突变小鼠及体外诱导人/鼠源巨核细胞对TCEB3突变体的致病机制开展进一步研究，研究结果不仅有助于阐明这一新型IT发病机制，也可为揭示巨核细胞发育与血小板生成的关键调控机制提供新的方向。

遗传性血小板减少症（Inherited thrombocytopenia, IT）是以血小板数量减少为主要特征，常伴有血小板功能障碍以及凝血功能异常的一组罕见性疾病。其由与巨核细胞（MKs）分化、血小板形成或清除有关的基因突变引起，现已证实超过30种单基因突变均可致病。课题组在前期发现的IT家系基础上，进一步明确了该IT致病基因并不是TCEB3，而是ANKRD26，突变位点为5’UTR区域c.-128G>T，该IT被命名为Ⅱ型血小板减少症（THC2）。该家系是目前我国报道的最大THC2家系，该突变类型在全球范围一共只有2个家系，在我国属于首次报道。已有研究显示THC2患者MKs在发育后期不能沉默ANKRD26表达，但异常表达ANKRD26导致血小板减少机制不明。课题组利用骨髓病理、体外巨核细胞诱导分化、原代巨核细胞单细胞测序、以及诱导多能干细胞等试验证实，异常表达ANKRD26通过中心体作用上调P21表达，导致MKs不能顺利启动S期，DNA合成障碍，从而抑制了MKs多倍体形成的分子机制。同时，体外细胞试验发现TCEB3的转录延长作用与泛素化作用均参与巨核细胞多倍体形成的调控。其中，TCEB3可通过促进ERK信号通路上游基因表达而促进巨核细胞多倍体形成。此外，THC2因只具有单一血小板表型，容易被误诊为免疫性血小板减少症，为此，课题组利用该家系的血浆蛋白组学数据筛选并鉴定出一组具有潜在诊断价值的血浆蛋白标志物。综上，本课题围绕我国较为罕见的THC2大家系，结合临床病人的离体研究与体外细胞模型研究，阐明了THC2致病机制，为THC2的治疗提供了新的靶点，并鉴定出潜在血浆蛋白标志物用于THC2诊断。