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：我积极主动，非常有资格追求学术事业 血液学领域的医学。我在上医学院之前获得了生物化学博士学位， 在三年半的时间里完成了一个为期六年的计划。作为一个三年级的医学生，我决定追求一个 镰状细胞病（SCD）的综合研究和临床生涯，包括成人和儿科分科 培训，并在12年的培训中实施了这一计划。我的奖学金研究让我认识到 基因组学的强大潜力，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 β 2），以了解β-珠蛋白调节和开发新的，智能 为血红蛋白病患者设计的治疗方法。我利用了遗传学和基因组学 贝勒医学院（Baylor College of Medicine）的优势，德克萨斯州儿童医院的大型SCD人口，以及 在贝勒和全国范围内提供血液学方面的特殊指导，以制定我的研究计划。 我有一个有希望的结果表明，FOXO 3作为一个积极的调节-珠蛋白的作用，支持金 标准体外功能研究。我打算运用我在分子生物学、基因组学和SCD方面的专业知识， 阐明FOXO 3及其途径中的其他基因调控β-珠蛋白的机制。这项研究将 增加了我们对珠蛋白转换和红细胞生成的理解，并可能引导我们发现新的HbF诱导 治疗 研究职业发展计划：我将利用我的导师团队和广泛的教育 和研究机会，成为珠蛋白转换和红细胞生成方面的专家。我将获得 专业知识的尖端分子技术需要完成我的目标，调查的作用， FOXO 3参与β-球蛋白调节。贝勒大学生物医学科学研究生课程提供以下课程： 需要在分子生物学方法、生物统计学、生物信息学和基因组学以及伦理学和 科学写作。我将经常与我的共同主要导师Goodell博士会面，以审查ChIP-Seq的数据 (Aim 1c）和RNA-Seq（目标2b）实验，并将讨论结果的解释和见解 他们提供了红细胞生成和β-珠蛋白调节与我的共同主要导师，米奇韦斯。博士 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 (Aim 1）。我将继续享受来自儿科的特殊支持， 血液学/肿瘤学，有85%的保护时间保证作为终身助理教授， 实验室空间，研究助理和部门资金的研究材料，可能超过K 08 预算在整个拨款期间。我的导师团队将帮助我实现我的目标， 对我们理解β-珠蛋白调节和红细胞生成有重要贡献，并提交了一份 R 01竞争性应用程序在第3年年底前完成。 研究项目：几种血红蛋白病，最明显的是SCD和β地中海贫血，可能是 通过增加β-珠蛋白表达有效治疗。对β-珠蛋白调节的更全面理解 可以促进HbF诱导剂的靶向设计。在本提案的初步数据中，我描述了 对171例SCD患者WES鉴定的罕见变异的创新基因基础分析。该分析 确定FOXO 3为β-珠蛋白的正调节因子。然后，我验证了与功能研究的关联， 研究β-珠蛋白调节的最佳体外系统，人原代红细胞培养。我现在建议使用 来自更大规模镰状细胞病患者队列（n=1000）的WES数据，以确认 在FOXO 3和β-珠蛋白水平之间，并确定在β-珠蛋白中起作用的其他FOXO 3途径基因， 调控我将测量不同程度的FOXO 3敲低对β-珠蛋白表达的影响， 复制体内杂合FOXO 3变体状态，并确定FOXO 3对β-珠蛋白的剂量效应 程度. FOXO 3通路基因在β-珠蛋白中的功能将通过shRNA敲低来研究， 人原代红细胞培养物。我还将开始分析七个独特的FOXO 3 在我们的试点研究中发现的变体。 FOXO 3和FOXO 3途径基因调节β-珠蛋白的机制将被阐明 通过几种方式。我将确定FOXO 3对其他红细胞表达水平的影响。 通过对来自红系前体的RNA进行RNA-Seq， 在红细胞生成的所有五个阶段都没有FOXO 3敲低。红细胞特异性FOXO 3结合位点将是 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 通过分析WES数据确定的与HbF水平相关的基因的数据和途径分析， 结合以产生参与FOXO 3调节β-珠蛋白水平的因子的完整图像。我 工作和未来的职业生涯将集中在β-珠蛋白的调节，并应用这些信息开发新的 血红蛋白病患者的治疗方法。