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

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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.