MECHANISMS IN HAEMOPOIETIC DIFFERENTIATION: INSIGHTS FROM NOVEL LOCI IN GENETIC THROMBOCYTOPENIA

我的总体研究目标是提高对血液或血管等部位的特化细胞如何从干细胞发育的理解。这些知识对于心脏病和癌症等常见疾病的新干细胞治疗至关重要。我的研究将集中在干细胞如何发展成血小板。然而，我的研究结果将适用于其他细胞类型，特别是其他血细胞和形成新血管的细胞。血小板是血液中的细胞样成分，在骨髓中通过分化过程产生，其中干细胞逐渐发育成更专门的细胞类型。这一通路中的最后一个细胞是巨核细胞，然后巨核细胞分裂形成血小板。血小板的形成由许多蛋白质控制，每一种蛋白质都由不同的基因组成。目前还不完全清楚哪些蛋白质和基因参与血小板形成，或者这些是如何调节的。我的研究背后的原理是，参与血小板形成的基因自然发生故障的人血液中的血小板数量会减少（血小板减少症）。因此，我将仔细选择可能由遗传原因引起的血小板减少症的研究对象，寻找新的遗传缺陷，然后通过实验测试这些缺陷如何影响血小板形成。这一策略使我的共同导师Ouwehand教授和其他人发现，RBM 8A和NBEAL 2基因的缺陷导致了罕见的灰色血小板和血小板减少症，其中血小板形成减少。这项研究还阐明了这些基因如何控制正常的血小板形成。我将通过从两个来源中识别血小板计数低的研究受试者来扩展对导致血小板减少症的缺陷基因的搜索：1）在英格兰西南部血友病中心登记的重度血小板减少症导致出血的患者;和2）来自布里斯托、牛津和剑桥产前诊所的妇女，在常规产前血液检查中检测到轻度血小板减少症。在这一组中，血小板减少症的非遗传原因是常见的。然而，我将使用额外的血液检查和产科血液学家的专家意见，以帮助确定遗传性血小板减少症的可能性很高的妇女。在NIHR-BioResource罕见疾病中心的帮助下，我将邀请参与者提供DNA样本，供Wellcome Trust桑格研究所分析。我将使用“下一代测序”技术来研究所有制造蛋白质的人类基因（ExoSeq）中的DNA序列和一些调节这些基因的DNA序列。我将把我的结果与欧洲范围的BRIGDE研究（由Ouwehand教授协调）联系起来，该研究将提供1000名出血性疾病患者的Exoseq结果，包括一些遗传性血小板减少症患者。这将有助于证实在研究对象中发现的基因缺陷是血小板减少症的原因。我将花大部分时间研究一个新的缺陷基因在血小板形成中的确切作用。这将涉及由我的主管和合作者开发的几种最先进的实验室技术，包括对选定研究参与者的血细胞进行详细的重新分析。我将在经过充分测试的模型系统和从血液样本中纯化并在实验室中分化为巨核细胞和其他细胞的干细胞中分析“关闭”基因的影响。我将确定由该基因产生的蛋白质如何与其他蛋白质相互作用。我的研究将揭示对血小板生成很重要的新基因。它还将确定一个新基因在干细胞分化形成血小板和其他特化细胞中的确切作用。这些发现将与干细胞生物学有更广泛的相关性，并将有助于开发常见疾病的细胞再生疗法。

My overall research objective is to improve understanding of how specialised cells in sites such as the blood or blood vessels develop from stem cells. This knowledge is essential for new stem cell treatments for common illnesses such as heart disease and cancer. My research will focus on how stem cells develop into blood platelets. However, my research findings will be applicable to other cell types, particularly other blood cells and cells that form new blood vessels. Platelets are cell-like components of blood that are made in the bone marrow by the process of differentiation, in which stem cells develop into progressively more specialised cell types. The final cell in this pathway is the megakaryocyte which then fragments to form platelets. Platelet formation is controlled by many proteins, each of which is made from a different gene. It is not fully understood which proteins and genes are involved in platelet formation, or how these are regulated.The principle behind my research is that people with naturally occurring faults in genes involved in platelet formation will have reduced numbers of platelets in the blood (thrombocytopenia). Therefore, I will carefully select study subjects with thrombocytopenia of likely genetic cause, search for new genetic faults and then test experimentally how these affect platelet formation. This strategy enabled my co-supervisor Prof Ouwehand and others to discover that faults in the RBM8A and NBEAL2 genes cause the rare Gray Platelet and Thrombocytopenia Absent Radii syndromes in which platelet formation is reduced. This research also clarified how these genes control normal platelet formation. I will extend the search for faulty genes that cause thrombocytopaenia by identifying study subjects with low platelet counts from two sources: 1) patients with severe thrombocytopenia causing bleeding registered at Haemophilia centres in SW England; and, 2) women from antenatal clinics in Bristol, Oxford and Cambridge with mild thrombocytopenia, detected on routine antenatal blood tests. In this group, non-genetic causes of thrombocytopenia are common. However, I will use additional blood tests and expert input from obstetric haematologists to help identify women with a high likelihood of genetic thrombocytopenia. With help from the NIHR-BioResource centre for rare diseases, I will invite participants to give DNA samples for analysis at The Wellcome Trust Sanger Institute. I will use 'next generation sequencing' technology to look at the DNA sequence in all human genes that make proteins (ExoSeq) and some DNA sequences that regulate these genes. I will link my results with the Europe-wide BRIGDE study (co-ordinated by Prof Ouwehand) which will provide Exoseq results from 1000 patients with bleeding disorders, including some with genetic thrombocytopenia. This will help confirm that gene faults identified in the study subjects are the cause of thrombocytopenia.I will spend most of my research study investigating the precise role of one new faulty gene in platelet formation. This will involve several state-of-the-art laboratory techniques developed by my supervisors and collaborators, including detailed re-analysis of blood cells from selected study participants. I will analyse the effect of 'switching off' the gene in well-tested model systems and in stem cells purified from blood samples and differentiated in the laboratory to form megakaryocytes and other cells. I will identify how the protein made from the gene interacts with other proteins. My research will reveal new genes that are important for platelet production. It will also identify the precise role of one new gene in how stem cells differentiate to form platelets and other specialised cells. These findings will have broader relevance to stem cell biology and will assist development of cellular regenerative therapies for common disorders.