A Genomics Approach to Gamma-Globin Regulation

伽马珠蛋白调控的基因组学方法

• 批准号: 9164151
• 负责人: Vivien Andrea Sheehan
• 金额: $ 14.54万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-16 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9164151
• 关键词: Accounting; Adenosine Monophosphate; Adult; Affect; American; Binding; Binding Sites; Biochemistry; Bioinformatics; Biometry; Boxing; Budgets; ChIP-seq; Childhood; Chronic; Clinical; Clinical Trials; Country; DNA Binding; Data; Data Analyses; Development; Development Plans; Disease; Doctor of Philosophy; Dose; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; Ethics; Fellowship; Fetal Hemoglobin; Funding; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Determinism; Genetic Variation; Genetic screening method; Genetic study; Genomic approach; Genomics; Globin; Goals; Gold; Grant; Hematology; Hemoglobin concentration result; Hemoglobinopathies; Heritability; Human; Human Genetics; In Vitro; Individual; Knowledge; Laboratories; Lead; Measures; Medical Students; Medicine; Mentors; Mentorship; Methods; Modality; Molecular; Molecular Biology; Molecular Biology Techniques; Morbidity - disease rate; Neoadjuvant Therapy; Pathway Analysis; Pathway interactions; Patients; Pediatric Hematology; Pediatric Hospitals; Pharmaceutical Preparations; Pilot Projects; Play; Population; Protein Kinase; Qualifying; RNA; Regulation; Regulator Genes; Research; Research Assistant; Research Project Grants; Role; Science; Sickle Cell; Sickle Cell Anemia; Single Nucleotide Polymorphism; Staging; System; Techniques; Technology; Texas; Time; Training; United States Food and Drug Administration; Variant; Work; Writing; base; beta Thalassemia; career; career development; cohort; college; design; exome sequencing; experience; follow-up; gamma Globin; genetic manipulation; genetic variant; genome wide association study; hydroxyurea; in vivo; innovation; insight; knock-down; medical schools; meetings; mortality; novel; novel therapeutic intervention; novel therapeutics; oncology; pediatric patients; population based; professor; programs; rare variant; research study; small hairpin RNA; tenure track; therapy design; therapy development; transcriptome; transcriptome sequencing

The Candidate: I am highly motivated and exceptionally qualified to pursue a career in academic medicine in the field of Hematology. I earned a PhD in Biochemistry prior to attending medical school, completing a 6 year program in three and a half years. As a third year medical student, I decided to pursue a combined research and clinical career in sickle cell disease (SCD), including adult and pediatric subspecialty training, and carried out this plan over twelve years of training. My fellowship research introduced me to the powerful potential of genomics, and I have applied this technology to identify rare variants associated with fetal hemoglobin levels (HbF, α22), in order to understand –globin regulation and develop novel, intelligently designed therapies for individuals with hemoglobinopathies. I have capitalized on the genetics and genomics strengths of Baylor College of Medicine (BCM), the large SCD population of Texas Children's Hospital, and the exceptional mentorship in hematology available at Baylor and across the country to develop my research plan. I have promising results indicating a role for FOXO3 as a positive regulator of –globin, supported by gold standard in vitro functional studies. I intend to apply my expertise in molecular biology, genomics and SCD to unravel the mechanism by which FOXO3 and other gene sin its pathway regulate –globin. This research will add to our understanding of globin switching and erythropoiesis, and is likely to lead us to novel HbF induction therapies. Research Career Development Plan: I will utilize my mentorship team and the wide array of educational and research opportunities in Houston to become an expert in globin switching and erythropoiesis. I will gain expertise in the cutting edge molecular techniques needed to accomplish my goal of investigating the role of FOXO3 in –globin regulation. The graduate programs at Baylor in biomedical science provide the courses I need to train in molecular biology methods, biostatistics, bioinformatics and genomics, as well as ethics and scientific writing. I will meet frequently with my co-primary mentor, Dr. Goodell, to review data from ChIP-Seq (Aim 1c) and RNA-Seq (Aim 2b) experiments, and will discuss the interpretation of the results and the insights they provide into erythropoiesis and –globin regulation with my co-primary mentor, Mitch Weiss. Dr. Boerwinkle will continue to provide guidance for my genomic analysis of whole exome sequencing (WES) data (Aim 1). I will continue to enjoy exceptional support from BCM, and my division of Pediatric Hematology/Oncology, with 85% protected time guaranteed as a tenure-track assistant professor, with laboratory space, a research assistant, and department funds for research materials that may exceed the K08 budget throughout the duration of the grant. My mentorship team will help me accomplish my goals of making a significant contribution to our understanding of –globin regulation and erythropoiesis, and submit a competitive R01 application by the end of year 3. Research Project: Several hemoglobinopathies, most notably SCD and beta thalassemia, could be effectively treated by increasing –globin expression. A more complete understanding of –globin regulation could facilitate targeted design of a HbF inducer. In the preliminary data of this proposal, I describe the innovative gene-bases analysis of rare variants identified by WES of 171 patients with SCD. This analysis identified FOXO3 as a positive regulator of –globin. I then verified the association with functional studies in the best in vitro system for studying –globin regulation, human primary erythroid culture. I now propose to use the WES data from a much larger cohort of patients (n=1000) with sickle cell disease to confirm the relationship between FOXO3 and –globin levels, and identify additional FOXO3 pathway genes that play a role in –globin regulation. I will measure the effect of various degrees of FOXO3 knockdown on –globin expression, to replicate the in vivo heterozygous FOXO3 variant state, and determine the dose effect of FOXO3 on –globin levels. The function of FOXO3 pathway genes in –globin will be investigated through shRNA knockdown in human primary erythroid culture. I will also begin to analyze the functionality of the seven unique FOXO3 variants identified in our pilot study. The mechanisms by which FOXO3 and FOXO3 pathway genes regulate –globin, will be elucidated through several modalities. I will determine the effect of FOXO3 on the expression levels of other erythroid genes throughout erythroid maturation by performing RNA-Seq on RNA from erythroid precursors with and without FOXO3 knockdown at all five stages of erythropoiesis. Erythroid specific FOXO3 binding sites will be identified by global ChIP-Seq performed in human primary erythroid cells. Expression data, DNA binding site data and pathway analysis of genes associated with HbF levels identified through analysis of WES data will be combined to produce a complete picture of the factors involved in FOXO3 regulation of –globin levels. My work and future career will focus on –globin regulation, and applying this information to developing new therapies for individuals with hemoglobinopathies.

候选人：我非常有动力并且非常有资格从事学术事业 在进入医学院之前，我获得了生物化学博士学位， 作为一名三年级医学生，我决定在三年半内完成六年的课程。 镰状细胞病 (SCD) 的综合研究和临床职业，包括成人和儿科亚专业 培训，并在十二年的培训中执行了这个计划。我的奖学金研究向我介绍了 基因组学的巨大潜力，我已经应用这项技术来识别与胎儿相关的罕见变异 血红蛋白水平（HbF、α22），以便了解 - 珠蛋白调节并开发新颖、智能的产品 我利用遗传学和基因组学为血红蛋白病患者设计了治疗方法。 贝勒医学院 (BCM) 的优势、德克萨斯儿童医院大量的 SCD 患者以及 贝勒和全国范围内的血液学方面的杰出指导可以帮助我制定研究计划。 我得到了有希望的结果，表明 FOXO3 在金的支持下作为珠蛋白的正调节剂发挥着作用 我打算将我在分子生物学、基因组学和 SCD 方面的专业知识应用于标准体外功能研究。 这项研究将揭示 FOXO3 及其途径中的其他基因调节 γ-球蛋白的机制。 增加了我们对珠蛋白转换和红细胞生成的理解，并可能引导我们进行新的 HbF 诱导 疗法。 研究职业发展计划：我将利用我的导师团队和广泛的教育资源 我将获得在休斯顿成为珠蛋白转换和红细胞生成方面的专家的研究机会。 实现我研究作用的目标所需的尖端分子技术专业知识 FOXO3 在 β-珠蛋白调控中的作用 贝勒生物医学科学研究生项目提供了课程 I。 需要接受分子生物学方法、生物统计学、生物信息学和基因组学以及伦理和道德方面的培训 我会经常与我的共同主要导师 Goodell 博士会面，审查 ChIP-Seq 的数据。 （目标 1c）和 RNA-Seq（目标 2b）实验，并将讨论结果的解释和见解 它们与我的共同主要导师 Mitch Weiss 博士一起参与红细胞生成和 β-珠蛋白调节。 Boerwinkle 将继续为我的全外显子组测序 (WES) 数据的基因组分析提供指导 （目标 1）我将继续享受 BCM 和我的儿科部门的特殊支持。 血液学/肿瘤学，作为终身教授助理教授，有 85% 的受保护时间， 实验室空间、一名研究助理以及可能超过 K08 的研究材料的部门经费 在整个资助期间的预算我的导师团队将帮助我实现我的目标。 对我们理解 -珠蛋白调节和红细胞生成做出了重大贡献，并提交了一份 在第 3 年年底之前提交具有​​竞争力的 R01 申请。 研究项目：几种血红蛋白病，尤其是 SCD 和 β 地中海贫血，可能与 通过增加 γ-珠蛋白表达来有效治疗 更全面地了解 γ-珠蛋白调节。 可以促进 HbF 诱导剂的针对性设计。在该提案的初步数据中，我描述了 对 171 名 SCD 患者的 WES 鉴定的罕见变异进行创新的基因基础分析。 确定 FOXO3 是 γ-珠蛋白的正调节因子，然后我验证了其与功能研究的关联。 研究 -珠蛋白调节的最佳体外系统，人类原代红系培养物，我现在建议使用。 来自更大的镰状细胞病患者队列 (n=1000) 的 WES 数据证实了这种关系 FOXO3 和 γ-珠蛋白水平之间的关系，并确定在 γ-珠蛋白中发挥作用的其他 FOXO3 途径基因 我将测量不同程度的 FOXO3 敲低对 -珠蛋白表达的影响。 复制体内杂合 FOXO3 变异状态，并确定 FOXO3 对 -珠蛋白 的剂量效应 水平中 FOXO3 通路基因的功能将通过 shRNA 敲低进行研究。 我还将开始分析七种独特的 FOXO3 的功能。 我们的试点研究中发现了变异。 FOXO3和FOXO3途径基因调节-珠蛋白的机制将被阐明 我将通过多种方式确定 FOXO3 对其他红细胞表达水平的影响。 通过对来自红细胞前体的 RNA 进行 RNA-Seq 来研究红细胞成熟的整个基因 如果红细胞生成的所有五个阶段没有 FOXO3 敲低，则红细胞特异性 FOXO3 结合位点将被抑制。 通过在人原代红系细胞中进行的全局 ChIP-Seq 鉴定表达数据、DNA 结合位点。 通过 WES 数据分析确定的与 HbF 水平相关的基因的数据和途径分析将 结合起来可以全面了解 FOXO3 调节 γ-珠蛋白水平的因素。 工作和未来的职业生涯将重点关注-球蛋白调节，并将这些信息应用于开发新的 针对血红蛋白病患者的治疗。