Identifying and Characterizing Novel Genes for Inherited Bone Marrow Failure

我的博士研究是遗传性骨髓衰竭基因的鉴定和表征， 骨髓增生异常综合征（BMF/MDS）。自从我上次提交F30以来，我已经确定了两个 这些条件的候选基因的基因组分析严重影响的家庭。其目的是 新的建议是测试每个基因作为其宿主家族中遗传性BMF/MDS的可能原因，然后 以评估每个基因在造血中的可能作用。 在目标1中，我将测试GALE（UDP-半乳糖-4-差向异构酶）中的错义突变是否可能是 导致严重血小板减少症，发热性中性粒细胞减少症和贫血，在一个大的血缘关系 血族在Aim 1a中，我将通过对GALE基因的DNA进行完全测序来筛选GALE的其他突变， 来自BMF/MDS储存库的无关患者。在目标1b中，我将比较 野生型和突变型GALE。我也会比较野生型和突变型的晶体结构 与NADH和核苷酸糖复合的GALE。在目标1c中，我将在原代人类中抑制GALE CD34+造血干细胞和测量细胞增殖、分化和存活。我也会 探索内质网应激和其他可能是GALE损失损害的机制的影响 造血发育 在目标2中，我将测试转录因子TFDP2中的错义突变（E2F二聚化）是否 伴侣-2）可能导致严重的血小板减少症，贫血和颅缝早闭， 血缘家庭中的兄弟姐妹。在目标2a中，我将在DNA中筛选TFDP2的其他突变， 来自BMF/MDS储存库的患者和独立的颅缝早闭患者。在目标2b中， 我将比较突变型和野生型TFDP2与E2F伴侣的结合亲和力，并将 比较野生型和突变型E2F/TFDP2复合物对靶基因转录活性的影响。 在目标2c中，我将测试TFDP2对CD34+细胞的抑制作用，并测试细胞周期中的缺陷 通过比较核内复制水平和BrdU掺入，研究了核内复制和DNA复制的关系。 阐明GALE和TFDP2在造血中的作用将为该领域增加重要的知识。 研究GALE的作用可以帮助解释糖基化的变化如何影响造血 增殖和分化。E2F途径在细胞周期调控中是众所周知的，但在细胞周期调控中却不那么重要。 发展研究TFDP2基因突变将有助于我们更好地理解E2F/TFDP2的作用 转录因子复合物在造血和人类发育中的作用。

My PhD research is the identification and characterization of genes for inherited bone marrow failure and myelodysplastic syndromes (BMF/MDS). Since my previous F30 submission, I have identified two candidate genes for these conditions by genomic analysis of severely affected families. The aims of this new proposal are to test each gene as the possible cause of inherited BMF/MDS in its host family, then to evaluate the possible role of each gene in hematopoiesis. In Aim 1, I will test whether a missense mutation in GALE (UDP-galactose-4-epimerase) may be responsible for severe thrombocytopenia, febrile neutropenia, and anemia in a large consanguineous kindred. In Aim 1a, I will screen for other mutations in GALE by fully sequencing the gene in DNA from unrelated patients from the BMF/MDS repositories. In Aim 1b, I will compare enzymatic activities of wildtype and mutant GALE by HPLC. I will also compare the crystal structures of wildtype and mutant GALE in complex with NADH and nucleotide sugars. In Aim 1c, I will suppress GALE in primary human CD34+ hematopoietic stem cells and measure cell proliferation, differentiation, and survival. I will also explore ER stress and other effects that may be mechanisms by which loss of GALE impairs hematopoietic development. In Aim 2, I will test whether a missense mutation in transcription factor TFDP2 (E2F Dimerization Partner-2) may be responsible for severe thrombocytopenia, anemia, and craniosynostosis in affected siblings in a consanguineous family. In Aim 2a, I will screen for other mutations in TFDP2 in DNA from patients from the BMF/MDS repositories and, independently, in patients with craniosynostosis. In Aim 2b, I will compare mutant and wildtype TFDP2 with respect to binding affinity to E2F partners and will compare effects of wildtype and mutant E2F/TFDP2 complexes on transcription activity of target genes. In Aim 2c, I will test the effects of suppression of TFDP2 on CD34+ cells, and test for defects in cell cycle transition and in DNA replication by comparing endoreduplication levels and BrdU incorporation. Elucidating roles for GALE and TFDP2 in hematopoiesis will add important knowledge to the field. Studying the role of GALE can help explain how changes in glycosylation affect hematopoietic proliferation and differentiation. E2F pathways are well-known in cell cycle regulation, but less so in development. Studying mutation in TFDP2 will improve our understanding of the role of E2F/TFDP transcription factor complexes in hematopoiesis and human development.