Project Summary Almost all tissues acquire spontaneous somatic mutations that can result in subsequent clonal expansion. Such clonal expansions are readily observable in peripheral blood samples as a patient’s age increases. This phenomenon, referred to as clonal hematopoiesis (CH), increases an individual’s risk for hematologic malignancies and cardiovascular disease, and has a prevalence over 10% in patients over the age of 65. Amongst the more frequently mutated genes in CH, as well as in subsequent malignancies such as acute myeloid leukemia (AML), are genes associated with DNA methylation, such as DNMT3A. However, the process through which clonal hematopoiesis occurs and gives rise to these diseases is unknown. This project seeks to uncover the biological impacts of these somatic mutations on hematopoiesis, which allow hematologic cells to transform into malignancies and impact cardiovascular health. Specifically, this project aims to characterize the effects of DNMT3A mutations on gene transcription, DNA methylation, and chromatin accessibility, to provide insight into the progression from clonal hematopoiesis to disease. The main approach in this proposal centers around single-cell sequencing along with genotyping of individual cells. In Specific Aim 1, I will use our lab’s recently developed Genotyping of Transcriptomes method to compare the transcriptional landscape of wild-type and mutant hematopoietic progenitor cells. This will provide insight into differentiation fate skews as well as changes in gene expression associated with the somatic mutations. In Specific Aim 2, I will use similar methods developed in our lab to combine single-cell methylome and chromatin accessibility profiling with somatic genotyping in order to identify putative mechanisms that could explain the changes in transcription and cell-fate determination. The outcomes of this study will inform the pathogenesis of normal hematopoiesis to CH to malignancy, providing useful insights into the role of screening for CH as well as possible therapeutic options to prevent the progression of disease. This project will be ideal for an aspiring physician-scientist in training, given its blend of novel sequencing technologies applied to clinical samples analyzed using cutting-edge computational biological methods, along with its possible implications for clinical recommendations. With the mentorship of my sponsor, co-sponsor, thesis committee, leadership of the Tri-I MD- PhD program, and the support of this fellowship, I am confident I will be well prepared to pursue and achieve my goal of being a physician scientist and independent investigator.