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Identification of Genetic Factors in Atherosclerosis Using an Apoe Mouse Model

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Identification of Genetic Factors in Atherosclerosis Using an Apoe Mouse Model Identification of Genetic Factors in Atherosclerosis Using an Apoe Mouse Model Andrew Todd Grainger Los Gatos, California Bachelors of Science in Molecular Biology, University of California San Diego, San Diego, California, 2013 Masters of Science in Biology, University of California San Diego, San Diego, California, 2014 A Dissertation Presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Department of Biochemistry and Molecular Genetics University of Virginia December 2019 Dr. Weibin Shi Dr. Charles Farber Dr. Aakrosh Ratan Dr. Norbert Leitinger I Abstract Atherosclerosis is the primary cause of coronary artery disease (CAD), ischemic stroke and peripheral arterial disease. Despite major achievements made in the past few decades, CAD and atherosclerosis-related events remain the number one cause of death in the United States and other developed countries. Therefore, there is a critical medical need to develop novel and effective therapies. An effective way to find new targets for intervention is through conducting genetic studies in animal models. When deficient in Apoe, mouse strains BALB/cJ and SM/J exhibit distinct differences in atherosclerosis and its associated risk factors. We hypothesized that linkage analysis of progeny derived from these inbred strains would lead to the discovery of new genes and new pathways in atherosclerosis and its associated cardiometabolic phenotypes. F2 mice were generated from an intercross between the two Apoe-/- strains and fed 12 weeks of Western diet. Many QTL loci were mapped for plasma lipids and glucose, carotid lesion size, and aortic lesion size. This included a significant QTL for aortic atherosclerosis, Ath49, which was mapped to the major histocompatibility region. Moreover four novel QTLs for carotid atherosclerosis, two significant QTLs named Cath7 on chromosome 5 and Cath8 on chromosome 9 and two suggestive QTLs, Cath5 and Cath6 on chromosomes 15 and 18 respectively, were mapped. Through a combination of haplotype analysis and a novel strategy employing gene expression, aortic lesion size correlation, and eQTL data, we prioritized Mep1α as a promising candidate gene for Ath49. We generated double knockouts and found that Mep1α is a novel gene negatively affecting plaque formation. Finally, we developed a method utilizing machine learning-based segmentation to accurately quantify subcutaneous and visceral fat volumes in mice using MRI and humans using CT. We found that BMI, a commonly used measure for diagnosing obesity, is only moderately associated with subcutaneous fat and has no association with visceral fat volume in humans. II Dedication To my parents, Jim and Claire: For giving me unconditional love and providing me with the care and support I needed to follow my dreams. I could never have gotten where I am today without you, and I love you. To my mentor, Weibin Shi: For believing in me and providing me with the platform and trust to thrive. You have given me the ability to grow into a scientist I can be proud of. III Table of Contents Title I Abstract II Dedication III List of Figures VIII List of Abbreviations XIII Chapter 1: Introduction 1 1.1 Atherosclerosis Pathogenesis........................................................................................................................2 1.1.1 Atherosclerosis Is The Leading Health Burden Worldwide.........................................2 1.1.2 Current Treatment Strategies For Atherosclerosis..........................................................4 1.1.3 Atherosclerotic Plaque Formation...........................................................................................6 1.2 Genetics of Atherosclerosis............................................................................................................................9 1.2.1 Before GWAS.....................................................................................................................................9 1.2.2 Human Genome-Wide Association Studies (GWAS) for Coronary Artery Disease............................................................................................................................................................9 1.2.3 Linkage Analysis in Mice for Aortic Atherosclerosis.....................................................11 1.3 Going from QTL to gene: Past and Current Strategies......................................................................19 1.3.1 Comparative Genomics Between Mouse and Human...................................................19 1.3.2 Haplotype Analysis......................................................................................................................31 1.3.3 Gene Expression Changes Through eQTLs........................................................................31 1.4 Aims........................................................................................................................................................................33 Chapter 2: Genetic linkage of hyperglycemia and dyslipidemia in an intercross between BALB-cJ and SM-J Apoe-deficient mouse strains 38 2.1 Abstract................................................................................................................................................................39 2.2 Introduction.......................................................................................................................................................39 2.3 Results..................................................................................................................................................................41 2.3.1 Trait value distributions...........................................................................................................41 2.3.2 Fasting glucose levels.................................................................................................................44 2.3.3 Fasting lipid levels........................................................................................................................47 2.3.4 Coincident QTLs for fasting glucose and lipids................................................................51 2.3.5 Correlations between plasma glucose and lipid levels................................................53 IV 2.3.6 Prioritization of positional candidate genes for Chr9 coincident QTLs................54 2.4 Discussion............................................................................................................................................................55 2.5 Methods ...............................................................................................................................................................59 Chapter 3: Polygenic Control of Carotid Atherosclerosis in a BALB-cJ × SM-J Intercross and a Combined Cross Involving Multiple Mouse Strains 63 3.1 Abstract................................................................................................................................................................64 3.2 Introduction.......................................................................................................................................................65 3.3 Results..................................................................................................................................................................66 3.3.1 Trait value frequency distribution.......................................................................................66 3.3.2 QTL analysis of carotid lesion sizes.....................................................................................67 3.3.3 Combined cross analysis for overlapping QTL...............................................................71 3.3.4 Candidate genes for Cath1.......................................................................................................74 3.3.5 Relationships of carotid atherosclerosis with plasma lipids and glucose..........76 3.4 Discussion..........................................................................................................................................................78 3.5 Methods...............................................................................................................................................................81 Chapter 4: Genetic analysis of atherosclerosis identifies a major susceptibility locus in the major histocompatibility complex of mice 84 4.1 Abstract................................................................................................................................................................85 4.2 Introduction.......................................................................................................................................................85 4.3 Results...................................................................................................................................................................87 4.3.1 QTL analysis of atherosclerotic lesions...............................................................................87 4.3.2 Candidate genes for Ath49.......................................................................................................93 4.3.3 Correlations of atherosclerotic lesion sizes with plasma lipid and glucose levels...........................................................................................................................................................105 4.4
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