Identifying and Characterizing Novel Genes for Inherited Bone Marrow Failure

遗传性骨髓衰竭新基因的鉴定和表征

• 批准号: 9389987
• 负责人: Aaron Seo
• 金额: $ 4.9万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-02 至 2018-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9389987
• 关键词: Affect; Affinity; Anemia; Aplastic Anemia; Binding; Blood Cells; Bone Marrow; Bone Marrow Cells; Bromodeoxyuridine; CD34 gene; Candidate Disease Gene; Cell Cycle; Cell Cycle Regulation; Cell Proliferation; Cell model; Cell physiology; Cells; Cellular biology; Child; Clinical; Clinical Management; Clonal Evolution; Complex; Congenital Abnormality; Craniosynostosis; Crystallization; DNA; DNA biosynthesis; Defect; Development; Diagnosis; Diamond; Dimerization; Disease; Doctor of Philosophy; Dyskeratosis Congenita; Dysmyelopoietic Syndromes; Early Diagnosis; Etiology; Failure; Family; Family history of; Fanconi' s Anemia; Fever; Gene Targeting; Genes; Genetic; Genetic Transcription; Genomics; Goals; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hereditary Disease; High Pressure Liquid Chromatography; Human; Human Development; Impairment; Inherited; Knowledge; Marrow; Measures; Medical; Missense Mutation; Molecular; Molecular Biology; Molecular and Cellular Biology; Morphology; Mutation; Myelogenous; NADH; Neutropenia; Pancytopenia; Pathway interactions; Patients; Production; Research; Role; Siblings; Stress; Structure; Syndrome; TFDP2 gene; Technology; Telomere Maintenance; Testing; Thrombocytopenia; Training; Uridine Diphosphate Galactose; accurate diagnosis; base; bone marrow failure syndrome; consanguineous family; design; dimer; epimerase; glycosylation; improved; insight; loss of function; mutant; novel; patient oriented research; protein function; repository; sugar nucleotide; targeted treatment; tool; transcription factor

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.

我的博士研究是遗传性骨髓衰竭基因的识别和表征 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 通过对受影响严重的家庭进行基因组分析，得出这些疾病的候选基因。本次活动的目的 新的提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 评估每个基因在造血中可能的作用。 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 导致大量近亲结婚的严重血小板减少症、发热性中性粒细胞减少症和贫血 亲属。在目标 1a 中，我将通过对 DNA 中的基因进行全面测序来筛选 GALE 中的其他突变。 来自 BMF/MDS 存储库的无关患者。在目标 1b 中，我将比较以下酶的活性 通过 HPLC 检测野生型和突变型 GALE。我还将比较野生型和突变型的晶体结构 GALE 与 NADH 和核苷酸糖形成复合物。在目标 1c 中，我将抑制原代人类中的 GALE CD34+ 造血干细胞并测量细胞增殖、分化和存活。我也会 探索 ER 应激和其他影响，这些影响可能是 GALE 丧失损害的机制 造血发育。 在目标 2 中，我将测试转录因子 TFDP2 中是否存在错义突变（E2F 二聚化） Partner-2）可能是受影响患者严重血小板减少、贫血和颅缝早闭的原因 近亲家庭中的兄弟姐妹。在目标 2a 中，我将筛选 DNA 中 TFDP2 的其他突变 来自 BMF/MDS 存储库的患者以及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，我将测试抑制TFDP2对CD34+细胞的影响，并测试细胞周期缺陷 通过比较核内复制水平和 BrdU 掺入来观察转变和 DNA 复制。 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 的作用有助于解释糖基化的变化如何影响造血功能 增殖和分化。 E2F 通路在细胞周期调控中众所周知，但在细胞周期调控中却鲜为人知。 发展。研究 TFDP2 突变将提高我们对 E2F/TFDP 作用的理解 造血和人类发育中的转录因子复合物。