Date of Award

Winter 12-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type



This thesis sought to provide a better understanding of clonality in various malignant and non-malignant settings using a variety of genomic analytical tools. Clonality is pre-defined as the presence of a mixed population of cells in which each sub-population has distinct somatic mutation profile. It is a common feature in cancers where subpopulations of cells arise as a result of independent, yet continual acquisition of somatic mutations. The clonal architecture of cancers can be used as a diagnostic and prognostic biomarker as well as to monitor disease progression or resolution. Besides cancer, clonal variability and expansion is also implicated in various non-malignant settings where somatically acquired mutations play a role in disease ontogeny and contribute to clinical morbidities. A prime example is clonal hematopoiesis of indeterminate potential (CHIP), which is associated with age-related, atherosclerotic cardiovascular disease due to aberrant inflammatory responses from TET2 mutated hematopoietic clones. Specifically, these clones increase the disease risk by modulating the interleukin-1β secretion pathway presumably resulting in direct endothelial damage that acts as a nidus for an atherosclerotic plaque.Given the clinical importance of clonal variability, clonal profiling is a powerful method to study diseases resulting from acquired somatic mutation. However, our understanding of clonal variability in disease is generally limited by, 1) the relatively high error rate of high-throughput, next-generation sequencing (NGS) methods (approximately 2%), which obfuscates the detection of somatic mutations at low variant allele frequencies, 2) a lack of longitudinal data that would allow one to track the evolutionary dynamics of these somatic mutations, and 3) a lack of comprehensive multi-regional sampling, especially in the case of solid tumors that would enable one to define clonal heterogeneity spatially.In this thesis, we first optimized an error-corrected sequencing (ECS) strategy that has approximately 100-fold higher limit of detection than standard NGS. We then applied ECS to longitudinally survey physiologic clonal hematopoiesis in healthy individuals aged 0 – 24. According to the thresholds defined by CHIP, which is limited to mutations at ≥2% variant allele frequency (VAF), this age group would not be expected to harbor any clonal hematopoietic mutations, but many researchers, including our group considered this information to be the result of technical inability to detect mutations with low variant allele frequency rather than a true absence of mutations. As a result, our group previously used ECS to determine that clonal hematopoiesis (CH) <0.02 VAF was ubiquitous in individuals at middle age, causing us in this thesis to examine and characterize CH in newborns, children, adolescent and young adults. With ECS, we examined the evolutionary dynamics of clonal mutations during normal hematopoiesis from birth to young adulthood and established that 30% of healthy infants were born with clonal hematopoietic somatic mutations in genes associated with leukemia.Second, after we found that many healthy young individuals harbored potentially pathogenic somatic mutations in blood, we moved on to examine clonal transfer and clonal dynamics in the context of unrelated allogeneic hematopoietic stem cell transplantation (HSCT) where the majority of healthy HLA-matched, unrelated donors is between ages of 20 to 40. Our lab has previously shown that pre-existing hematopoietic progenitors with pathogenic mutations could be selected by chemotherapy and result in therapy-related AML, and as mentioned above, we have demonstrated that a significant percentage of healthy individuals of all ages harbored hematopoietic progenitors with pathogenic mutations. We therefore hypothesized that healthy donors would harbor mutated clones in blood that engraft the recipients, and the process of HSCT presents a potent selection pressure for donor clones. The most compelling finding was that 100% of the donor clones engrafted and the 84% of these clones harbored mutations that were pathogenic Our results also suggested a possible link between these engrafted pathogenic mutations and the development of chronic graft-versus-host disease in the recipients.We next characterized clonal hematopoiesis in the background of Down Syndrome (DS) where individuals have approximately 150-fold increased risk of developing leukemia. This was done by comparing CH in DS children who were otherwise healthy with those that had developed myeloid leukemia of Down Syndreom (ML-DS). Besides trisomy 21, ML-DS is characterized by mutations in the X-linked transcription factor, GATA1, but GATA1 mutations have recently been demonstrated in about 30% of umbilical cord blood samples from DS children suggesting that additional lesions were required for leukemic transformation. We demonstrated that the clonal profiles in ML-DS differ from those in DS children without leukemia. Our results also suggest an alternative route in which GATA1 mutated clones contribute to leukemogenesis via oligoclonal, cell extrinsic interactions.Lastly, I investigated the tumor ontogeny of metastatic glioblastoma in a young adult (aged 27) with neurofibromatosis (NF1) using multi-region sequencing on widely disseminated tumor cells across different brain lesions. Glioblastoma in NF1 is rare, and has conventionally been thought to arise as a result of bi-allelic loss of the NF1 gene. However, by examining the spatial genetic heterogeneity and the tumor phylogeny, our results suggested that the somatic loss of the second NF1 allele occurred much later during disease progression, and pathogenic mutations in other genes such as KMT2B were involved in initial oncogenic transformation instead.Collectively, these findings have augmented understanding of clonal hematopoiesis from birth through young adulthood, clonal variability in metastatic glioblastoma, and provide a foundational basis for further explorations in establishing causal links between clonal profiles and disease ontogenies.


English (en)

Chair and Committee

Todd Druley

Committee Members

John Welch, Grant Challen, Meagan Jacoby, Jamie Blundell,


Permanent URL: https://doi.org/10.7936/scfg-zs49