A Genomics Approach to Gamma-Globin Regulation

我非常有动力，也非常有资格从事学术事业

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.