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ItemIntegrating genomic and multiomic data for computational analysis of gene regulation in circulating immune cells(Georgia Institute of Technology, 2024-04-26) Brown, Margaret R.In the post-GWAS era, genetic associations with pathology have sparked interest in gene regulatory mechanisms since the majority of GWAS variants are located in noncoding regions. This idea fuels the hypothesis that trait associated variants are causal to gene expression variability. The primary question driving this thesis, is whether distinct gene regulatory mechanisms associated with genetics can be identified in circulating immune cells. First, eQTL fine mapping was performed using an all-but-one conditional analysis approach to prioritize putatively causal variants by disentangling the effects of linkage disequilibrium in peripheral blood. Identified eQTL for genes associated with inflammatory bowel disease were observed in immune cell populations, suggesting a functional relationship between genetics and gene expression variability. Next, heterogeneous gene regulatory mechanisms were observed in single nuclear multiomic data of circulating immune cells from individuals with Crohn’s disease and healthy donors. Paralleled heterogeneity was observed in both arms of the adaptative immune system, including an inflammatory signature within a subset of Crohn’s disease donors. Finally, an unprecedented approach to explain gene expression was implemented by training machine learning models on chromatin accessibility data, which demonstrated that ATAC peaks which are important for explaining gene expression are enriched with inflammatory disease GWAS variants. Altogether, this thesis highlights the genetic relevance of gene regulation in circulating immune cells for inflammatory disease and suggests that the interplay of genetics and pathology with respect to gene regulation is complex and heterogeneous among individuals.
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ItemGenetic influences of fatty acid metabolism and ancestral origins on disease(Georgia Institute of Technology, 2023-05-23) Astore, Courtney AlexandraAlthough there have been extensive efforts to study the influence of environmental factors and their interactions with various diseases, the contribution of metabolomic imbalances in the development and pathogenesis of disease is still not fully understood. Furthermore, despite numerous attempts to replicate the findings of genome-wide association studies (GWAS) in diverse populations, little progress has been made in replicating the associations of rare variants with complex diseases. The objective of this thesis is to leverage a combination of biobank-level phenotype and genetic data to investigate the effects of metabolites as well as explore the ancestral origins of rare variants in inflammatory bowel disease (IBD) and in other disease areas. Thus, the three chapters of this thesis investigate the roles of two main areas in human diseases: (1) fatty-acid metabolism, and (2) admixture and rare variants. The first study investigates the causal association between circulating metabolites and IBD. This study leverages the use of Mendelian Randomization (MR), a method that uses significant genetic variants from the GWAS of the exposure trait as instrumental variables to assess the causal relationship between a modifiable exposure and an outcome. In this case, we assessed over 200 metabolites and evaluated their relationship to IBD via MR. Omega-3 fatty acids were found to be one of the most significant protective associations with IBD, which was replicated in three independent GWAS. The second study is an extension of the first study, which further evaluates the disease architectures of 3 polyunsaturated fatty acids (PUFAs), omega-3 fatty acids, omega-6 fatty acids, and docosahexaenoic acid (DHA). The objective of this study is to demonstrate integrative PheWAS approaches using the metabolite levels and their polygenic scores to assess the association between the three PUFAs and over 1,300 disease endpoints. Using the metabolite-disease associations with concordant significant evidence from both approaches, we applied MR to assess causality. Protective associations with concordant evidence from all three PUFAs were identified. The last study assesses the role of admixture on the rare variant contribution to IBD. In this chapter we investigated the impact of 25 rare, European ascertained, Crohn’s disease (CD) variants identified by Sazonovs et.al. on IBD in African American whole genome sequencing data. Our findings showed a consistent four-to-five-fold reduction in allele frequency in African Americans when compared to European. Further, phasing on the WGS data confirmed that the CD risk alleles discovered in Europeans contributes to the risk in African Americans due to admixture. Additionally, we found that 45 rare variants discovered by a meta-analysis of UK Biobank and FinnGen spanning ten disease classes from Sun et.al. are also mostly present due to admixture in the African American cohort. These results highlight the importance of conducting whole exome and genome sequencing studies on large, diverse cohorts to gain a better understanding on the role of rare variants in disease and promote equitable research practices.
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ItemMechanisms of Coherence and Incoherence Between GWAS and Single-Cell eQTL Effects in Crohn's Disease(Georgia Institute of Technology, 2023-05-02) Collins, Jared BlakeThe integration of expression quantitative trait loci with GWAS data has proven invaluable in the exploration of mechanisms through which genetic variants influence complex traits. However, it has also highlighted instances of incoherence in which the eQTL effects of GWAS risk variants seemingly contradict observed case and control expression. Patterns of incoherence may indicate variants associated with disease via protection, but due to the highly heterogenous nature of varying cell-types, may also indicate cell-type specific associations with disease that are convoluted by bulk RNA sequencing. Here, we conduct exploratory analysis integrating sceQTL and GWAS data associated with Crohn’s Disease to assess patterns of coherence and incoherence, using both bulk RNA-seq and predicted single-cell gene expression for case-control expression. We show that integration of GWAS summary statistics with single-cell eQTL data is a promising approach for uncovering cell type specific patterns of coherence and incoherence, and may suggest functional mechanisms underlying these associations.
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ItemInvestigation of maturation and survival of human long-lived plasma cells using integrated single-cell analysis(Georgia Institute of Technology, 2022-08-29) Duan, MeixueSingle-cell transcriptomic sequencing (scRNA-seq), first introduces in 2009, provides an unprecedented opportunity to profile gene expression at the level of individual cells. This approach has rapidly become the favored technique to address fundamental cell biological phenomena such as the pathways of differentiation and maturation of cell types, as well as to explore and elucidate the pathology of disease. Long-lived plasma cells (LLPC), which are quiescent and terminally differentiated B lineage cells, primarily function as antibody (Ab) manufacturing factories that secrete of Abs, which are the primary contributor to durable humoral-mediated immunity. The Lee Lab at Emory University has been working on elucidating of heterogeneity of plasma cells. Using cell surface protein levels of CD19, CD38 and CD138 to define subtypes, they have demonstrated transcriptional and functional differences between short-lived plasma cell (SLPC) and LLPC using bulk RNA-seq techniques. They have also devised a human in vitro antibody-secreting cell (ASC) culture media that supports experimental manipulations. The first aim of the thesis utilized scRNA-seq combined with single cell V(D)J sequencing (scVDJ-seq) to characterize the heterogeneity, maturation, and survival of human bone marrow plasma cells (BMPC). These analyses first demonstrated that there are two maturation stages and four maturation trajectories associated with 15 robust human BMPC subgroups. High resolution bioinformatics analysis of the data provided by scRNA-seq identified critical pathways associated with maturation, and notably, manual curation of all annotated apoptosis/apoptotic related pathways led to discovery of a novel ASC pro-survival factor, MDK. Therefore, the second aim of my thesis was to explore the genes and pathways that MDK regulates so as to infer the intracellular downstream signaling it activates by gene regulatory network analysis. Integration with experimental manipulation validated the identities of active receptors for MDK, and these results are expected to support engineering of ASC in future research. Previous studies from the Sanz lab at Emory University observed that a subgroup of circulating pathogenic autoreactive ASCs expand during systemic lupus erythematosus (SLE) patient flares. A distinct subset of class-switched B cells lacking the memory B cell markers CD27, IgD and CXCR5, termed DN2 (CXCR5-CD21-CD11c+) was also reported to be expanded in the peripheral blood of SLE patients. Considering the success of applying scRNA-seq and scVDJ-seq to elucidate fundamental biology with respect to human BMPC, the third aim of my thesis utilized my analytical pipeline to explore the potential pathogenesis of SLE by comparing B cell heterogeneity in human BM between healthy controls and SLE patients. Unfortunately, the results reported here were compromised due to a sample mixing issue that was only detected late in the analysis. However, the enhanced methodology I constructed for the study appears to have improved good performance by discovering that 1) the general genes and functions that shift during B cell development between HD and SLE; 2) most SLE B cells prior to the immature B stage are less differentiated from normal B cells, except for a subgroup of Pro B and immature B cells; 3) SNP-heritability of SLE disease is enriched at a central tolerance checkpoint in the immature B stage; and 4) multiple key transcription factors exhibit turnover in gene expression that differs between SLE and controls at the central tolerance checkpoint.
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ItemSalivary Micro RNA as a Biomarker for Concussion Symptoms in Pediatric Patients(Georgia Institute of Technology, 2021-05) Bryant, Graham G.This research project employs an untargeted approach using RNA sequencing to identify miRNA in saliva following a traumatic brain injury, specifically a concussion, to better understand how acute brain injuries can be diagnosed and treated. There is currently no biological test in place for diagnosing concussions, which are one of the most frequently observed sport related injuries. This study is invested in determining novel molecular biomarkers in saliva that are associated with concussion so that they can then be correlated with symptoms of concussion. The study predicted that salivary miRNA levels are altered in children with concussion, and there will be a significant difference in levels of miRNA at baseline (pre-injury) and post-injury in athletes. By determining novel biomarkers, noninvasive saliva testing may help to better identify concussions and prevent premature return to play as well as a better understanding of injury mechanisms.
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ItemTranscriptomic profiling of human cells destined for therapeutic applications(Georgia Institute of Technology, 2021-03-23) Medrano Trochez, CamilaCell therapy is a growing field as many diseases are still untreatable. Potential applications of cell therapies include treating cancers, autoimmune disease, urinary problems, and infectious disease, rebuilding damaged cartilage in joints, repairing spinal cord injuries, improving a weakened immune system, and helping patients with neurological disorders. Numerous clinical trials have recently focused on the use of mesenchymal stromal cells (MSCs) as a cell therapy for various diseases with unmet medical challenges, including graft-vs-host disease, osteoarthritis, autism, and auto-immune diseases. MSCs are multipotent cells that have both regenerative and immunomodulatory capacity, and which are being developed for therapeutic intervention across a variety of inflammatory and immune conditions. MSCs can be isolated from various tissues, such as bone marrow, umbilical cord, and bone marrow aspirate concentrate (BMAC), which introduces tissue dependent variability between MSC-based cell products that also differ according to donor. Furthermore, manufacturing processes vary between sites (both clinical and commercial), leading to process-dependent variability. These sources of variability across the MSC field compound the ability to compare clinical trial results and have contributed to a lack of conclusive historical data to support the potential for clinical efficacy. Overall, there is an obvious need for deep phenotypic characterization of MSCs to compare heterogeneity as a function of tissue-of-origin as well as donor, and to identify potential phenotypic signatures that can be used for biomarkers or quality attributes for MSC-based cell products. My research focused on the study of MSCs as well as BMACs, which contain small amounts of MSC, but are still used in cell therapy owing to their MSC-like properties. This study aims to quantify the impact that diverse cell preparation methods may have on patient outcomes, the transcriptional variability due to the tissue of origin of the cells used, and the mechanism by which these cells modulate the immune system. I also evaluate the gene expression profiles of BMAC cell types from osteoarthritic (OA) patients and non-OA donors. 1. Characterization of the gene expression profiles of bone marrow and umbilical cord tissue-derived MSC. I assess the variability due to tissue of origin, batch effects, and donor effects. Samples derived from the same tissue separate in two major groups corresponding to high quality cells and low-quality cells. The high-quality cells further sub-divide in two subgroups. Cells from each donor belong to one or the other of these subgroups. One of the subgroups contains active cells expressing immunosuppressive genes, while the other subgroup cells overexpress cell cycle genes, suggesting these cells may be more actively undergoing mitosis. The samples from the two different tissues also express qualitatively different gene expression profiles. 2. MSC immunosuppressive capacities. I describe how MSC immunosuppressive capacities are enhanced at the transcriptional level by proinflammatory cytokines. I characterize the gene expression profiles of bone marrow derived MSC exposed to TNFα and IFNγ, showing that the MSC activated with IFNγ express higher levels of immunosuppressive genes. Additionally, single cell RNAseq analysis performed on T cells exposed to IFNγ -enhanced MSC reveals enrichment in gene activity related to T cell suppression and cell stress related pathways. 3. Characterization of BMAC samples form osteoarthritic patients and non- osteoarthritic donors. Osteoarthritis is a common degenerative disease, with no lasting cure. Even though it is considered a non-inflammatory disease, the immune system plays an important role in OA progression. The constant activation of pro-inflammatory cytokines due to tissue damage provokes deterioration of the cartilage. Cell therapy has been proposed as a treatment for this disease. MSC from different sources are being used to treat OA, such as commercial MSC, bone marrow aspirates concentrate (BMAC) or autologous MSC. This chapter describes my characterization of the transcriptomic profiles of BMAC from OA patients and healthy donors. The results suggest overexpression of immune related pathways in BMAC samples from OA patients compared to samples from non-OA donors. Since pro-inflammatory cytokines play a role in progression of the disease, this study suggests a possible benefit to the use of allogeneic BMAC from non-OA donors over samples from OA patients.
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ItemTranscriptomic profiling of human cells destined for therapeutic applications(Georgia Institute of Technology, 2021-03-21) Medrano Trochez, CamilaCell therapy is a growing field as many diseases are still untreatable. Potential applications of cell therapies include treating cancers, autoimmune disease, urinary problems, and infectious disease, rebuilding damaged cartilage in joints, repairing spinal cord injuries, improving a weakened immune system, and helping patients with neurological disorders. Numerous clinical trials have recently focused on the use of mesenchymal stromal cells (MSCs) as a cell therapy for various diseases with unmet medical challenges, including graft-vs-host disease, osteoarthritis, autism, and auto-immune diseases. MSCs are multipotent cells that have both regenerative and immunomodulatory capacity, and which are being developed for therapeutic intervention across a variety of inflammatory and immune conditions. MSCs can be isolated from various tissues, such as bone marrow, umbilical cord, and bone marrow aspirate concentrate (BMAC), which introduces tissue dependent variability between MSC-based cell products that also differ according to donor. Furthermore, manufacturing processes vary between sites (both clinical and commercial), leading to process-dependent variability. These sources of variability across the MSC field compound the ability to compare clinical trial results and have contributed to a lack of conclusive historical data to support the potential for clinical efficacy. Overall, there is an obvious need for deep phenotypic characterization of MSCs to compare heterogeneity as a function of tissue-of-origin as well as donor, and to identify potential phenotypic signatures that can be used for biomarkers or quality attributes for MSC-based cell products. My research focused on the study of MSCs as well as BMACs, which contain small amounts of MSC, but are still used in cell therapy owing to their MSC-like properties. This study aims to quantify the impact that diverse cell preparation methods may have on patient outcomes, the transcriptional variability due to the tissue of origin of the cells used, and the mechanism by which these cells modulate the immune system. I also evaluate the gene expression profiles of BMAC cell types from osteoarthritic (OA) patients and non-OA donors. 1. Characterization of the gene expression profiles of bone marrow and umbilical cord tissue-derived MSC. I assess the variability due to tissue of origin, batch effects, and donor effects. Samples derived from the same tissue separate in two major groups corresponding to high quality cells and low-quality cells. The high-quality cells further sub-divide in two subgroups. Cells from each donor belong to one or the other of these subgroups. One of the subgroups contains active cells expressing immunosuppressive genes, while the other subgroup cells overexpress cell cycle genes, suggesting these cells may be more actively undergoing mitosis. The samples from the two different tissues also express qualitatively different gene expression profiles. 2. MSC immunosuppressive capacities. I describe how MSC immunosuppressive capacities are enhanced at the transcriptional level by proinflammatory cytokines. I characterize the gene expression profiles of bone marrow derived MSC exposed to TNF⍺ and IFN𝛾, showing that the MSC activated with IFN𝛾 express higher levels of immunosuppressive genes. Additionally, single cell RNAseq analysis performed on T cells exposed to IFN𝛾-enhanced MSC reveals enrichment in gene activity related to T cell suppression and cell stress related pathways. 3. Characterization of BMAC samples form osteoarthritic patients and non- osteoarthritic donors. Osteoarthritis is a common degenerative disease, with no lasting cure. Even though it is considered a non-inflammatory disease, the immune system plays an important role in OA progression. The constant activation of pro-inflammatory cytokines due to tissue damage provokes deterioration of the cartilage. Cell therapy has been proposed as a treatment for this disease. MSC from different sources are being used to treat OA, such as commercial MSC, bone marrow aspirates concentrate (BMAC) or autologous MSC. This chapter describes my characterization of the transcriptomic profiles of BMAC from OA patients and healthy donors. The results suggest overexpression of immune related pathways in BMAC samples from OA patients compared to samples from non-OA donors. Since pro-inflammatory cytokines play a role in progression of the disease, this study suggests a possible benefit to the use of allogeneic BMAC from non-OA donors over samples from OA patients.
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ItemFine-mapping of human genetic regulatory variants(Georgia Institute of Technology, 2020-05-13) Tian, RuoyuThe majority of GWAS (Genome-Wide Association Study) identified common genetic variants map to regulatory regions of gene, and are likely to influence disease risk by affecting gene expression. One of the most important challenges is to experimentally fine-map causal regulatory variants that typically lie in credible intervals of 100 or more variants. Another large proportion of genetic variants, rare variants, are expected to have large effects causing disease in individual, but are not detectable in GWAS. Herein, I provide both experimental and computational approaches for fine-mapping common and rare genetic variants accounting for medium and large effect on population or individual. First, I describe a single cell clone-based strategy for targeted single-nucleotide polymorphism (SNP) evaluation wherein microindels are introduced by CRISPR/Cas9. Multiple constraints, including the variability in mutability, clonal genotype and expense, render this approach infeasible for fine-mapping 10%-20% moderate effect size expression SNPs (eSNPs), which is also validated in a simulation study. Subsequently, I switch to a moderate-throughput parallel screening tool that characterizes multiplexed CRISPR/Cas9 perturbed transcriptomes by single-cell RNA-seq, called “expression CROP-seq”. Two causal SNPs, rs2251039 and rs35675666, are identified that significantly alter the expression of CISD1 and PARK7, respectively. The sites overlap with chromatin accessibility peaks and are risk loci of inflammatory bowel disease. Expression CROP-seq reduces the variability identified in previous method and is powerful to screen genetic regulatory variants within credible intervals. Finally, to extend its application to rare variants, I develop a novel gene categorization system according to gene intolerance to promoter polymorphism and depletion of rare regulatory variants with GTEx v8 data. 49 GTEx tissues are clustered into functional groups with gene features. It supports the use of tissue-gene genomic annotation for prioritization of GWAS tagged risk loci. In summary, this work comprehensively describes and evaluates two CRISPR/Cas9-based eSNP screening systems. The use of rare regulatory variants in gene classification with tissue information demonstrates its potential in rare disease diagnoses. Both researches inevitably contribute to the genetic interpretation of human complex disease and personalized medicine in post-GWAS era.
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ItemIntegrative immunotranscriptomic analysis of long-lived plasma cells(Georgia Institute of Technology, 2019-05-21) Garimalla, Swetha N.Long-lived plasma cells are a key component of serological memory encoded by the adaptive immune response. To date, prior studies of these cell types have largely assessed plasma cells as one homogenous population. The Lee Lab at Emory University provided strong evidence for the presence of subpopulations of plasma cells in the bone marrow and peripheral blood. In this work, I analyze six of these populations: pops 2, 3, and 5 in the peripheral blood and pops A, B, and D in the bone marrow sampled from live human donors. Whole-transcriptome analysis of these six flow-sorted plasma cell populations is compared within and across tissue within this study. Further, I present 4 possible models of development of these plasma cells based on the results. Finally, I performed a singlecell RNA sequencing analysis of the long-lived plasma cells (popD) and present evidence for the possibility of subpopulations of this cell type. This work describes many mechanisms of molecular development of long-lived plasma cells, some of which have been experimentally validated by Dr. Doan Nguyen as a result of these analyses, however, further experimental validation is required to validate all findings in this work.
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ItemBlood eQTL detection in structured populations and its application to interpretation of genetic association studies(Georgia Institute of Technology, 2018-11-09) Zeng, BiaoExpression QTL (eQTL) detection has emerged as an important tool for unravelling the relationship between genetic risk factors and disease or clinical phenotypes. Most studies focus on analyses predicated on the assumption that only a single causal variant explains the association signal in each interval. This greatly simplifies the statistical modeling, but is liable to biases in scenarios where multiple linked causal-variants are responsible. Here in this thesis, my primary goal was to address the prevalence of secondary cis-eQTL signals regulating peripheral blood gene expression locally, utilizing two large human cohort studies, each greater than 2,500 samples with accompanying whole genome genotypes. The CAGE dataset is a compendium of Illumina microarray studies, and the Framingham Heart Study (FHS) is a two-generation Affymetrix dataset. I firstly describe performing simulation to reveal the potential interference of causal variants in LD regions. I then also describe a Bayesian co-localization analysis of the extent of sharing of cis-eQTL detected in both studies as well as with the BIOS RNA-seq dataset. Stepwise conditional modeling demonstrates that multiple eQTL signals are present for ~40% of over 3,500 eGenes in both microarray datasets, and that the number of loci with additional signals reduces by approximately two-thirds with each conditioning step. Although fewer than 20% of the peak signals across platforms fine-map to the same credible interval, the co-localization analysis finds that as many as 50%~60% of the primary eQTL are actually shared. Subsequently, co-localization of eQTL signals with GWAS hits detected 1,349 genes whose expression in peripheral blood is associated with 591 human phenotype traits or diseases, including enrichment for genes with regulatory functions such as protein kinase activity and DNA binding. Just one quarter of these co-localization signals are replicated, further highlighting the technological and methodological barriers to reconciliation of GWAS and eQTL signals. My results are provided as a web-based resource for visualization of multi-site regulation of gene expression and their association with human complex traits and disease states. In addition to the cis-eQTL study, as a member of the eQTLgen consortium, I also conduct trans-eQTL detection in multiple cohorts, including FHS, which contains related individuals, and performed cis-trans eQTL mediation analysis, which I will report as a side project. This thesis provides novel insights into the complexity of gene regulation and the low consistency of fine mapping across studies, and introduces new software, PolyQTL, for co-localization of genetic signals in structured populations.