Deep Phenotyping of Human Knockouts and Population Studies of the APOC3 Pathway

人类基因敲除的深度表型分析和 APOC3 通路的群体研究

• 批准号: 9902507
• 负责人: Daniel James Rader
• 金额: $ 67.9万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9902507
• 关键词: Address; Ankle; Apolipoprotein A-I; Apolipoproteins B; Apolipoproteins C; Atherosclerosis; Biological; Biological Markers; Biology; Catabolism; Cells; Cholesterol; Consanguinity; Coronary heart disease; Data; Disease; Dose; Event; Fatty acid glycerol esters; Functional disorder; Genes; Genetic; Genotype; Glucose; Heparin; Hepatic; Hepatocyte; Heterozygote; High Density Lipoprotein Cholesterol; High Density Lipoproteins; High Prevalence; Homeostasis; Homozygote; Human; Individual; Inflammation; Inflammatory; Insulin; Insulin Resistance; Isotopes; Kinetics; Knock-out; Knowledge; Label; Lipase; Lipids; Lipolysis; Lipoprotein (a); Lipoproteins; Liver Function Tests; Loss of Heterozygosity; Low-Density Lipoproteins; Measures; Mediating; Mediation; Metabolic; Metabolic Clearance Rate; Metabolic Diseases; Metabolic Pathway; Metabolism; Modeling; Mutation; Non-Insulin-Dependent Diabetes Mellitus; OGTT; Oral; Participant; Pathway interactions; Patients; Phenotype; Physiology; Plasma; Population; Population Study; Production; Proteins; Risk; Risk Factors; Role; Safety; Subgroup; Testing; Therapeutic; Thick; Thromboplastin; Tracer; Triglycerides; Uncertainty; Very low density lipoprotein; X-Ray Computed Tomography; apolipoprotein C-III; atherosclerosis risk; cardiometabolism; cardiovascular risk factor; cohort; extracellular; genomic locus; glucose metabolism; heart disease risk; human model; indexing; induced pluripotent stem cell; insight; lipid metabolism; lipoprotein lipase; liver function; loss of function; loss of function mutation; particle; rare variant; recruit; response; stable isotope; therapeutic target; uptake

Several recent studies indicate that loss of function (LoF) mutations in the apolipoprotein C-III (APOC3) gene offer protection from coronary heart disease (CHD) risk, possibly by reducing circulating triglyceride-rich lipoproteins (TRLs). Inhibition of the apoC-III pathway can provide an attractive therapeutic mechanism to lower CHD risk. There are however many outstanding uncertainties that exist, including impact of apoC-III deficiency on (i) lipoprotein kinetics; (ii) high density lipoprotein remodeling and functionality; (iii) non-lipid related pathways; (iv) the mechanisms by which apoC-III inhibition may reduce CHD risk, (v) the relative contribution of different lipoprotein- associated apoC-III levels on CHD; (vi) liver function and insulin resistance from a safety perspective. In this application, we propose studies to address these gaps by leveraging natural human models of apoC-III deficiency. We have identified the world’s first humans with homozygous APOC3 genetic deficiency who are from an isolated Pakistani village with a high prevalence of consanguinity. Recruitment of all inhabitants in this village (~5000 people) has already been completed; genotyping in a subset has already identified 113 APOC3 knockouts with complete apoC-III deficiency providing opportunities for detailed phenotyping. Specifically, in AIM-1, we will conduct deep phenotyping studies in 113 human APOC3 knockouts (n = 113) and in an equal number of heterozygotes and non-carriers to address the following: (i) role of apoC-III deficiency in modulating protein and lipid composition of TRLs to impact the activity of extracellular lipases; (ii) effect of apoC-III deficiency on subclasses of LDL; (iii) role of apoC-III in modulating Lp(a) levels and composition; and (iv) contribution of apoC-III deficiency to modulation of HDL composition and function. We will also address the consequences of genetic APOC3 deficiency on (iv) systemic lipid and glucose metabolism and a range of proteins related to inflammation and other pathways (v) dose-response association with atherosclerosis. In AIM-2, we will conduct lipoprotein kinetic studies using isotope tracers on 18 trios of APOC3 null homozygotes, heterozygotes, and non-carriers to evaluate the impact of apoC-III deficiency on kinetics of apoB- containing lipoproteins and production and clearance of HDL apoA-I. We will also generate human iPS cells from 5 APOC3 knockouts and 5 matched/related non-carriers and differentiate them into hepatocytes to evaluate (a) VLDL apoB and TG production (b) TRL and LDL uptake and (c) SRB1 mediated HDL uptake. In AIM-3, we will measure total plasma apoC-III and lipoprotein-associated (apoB lipoproteins, HDL, and Lp[a]) apoC-III in 5,000 participants from the EPIC-Norfolk cohort (2,500 with incident CHD events). These studies will enable assessment of: (i) disease mediation; (ii) CHD risk progressively adjusted for lipoprotein-associated apoC-III levels and other factors; (iii) dose-response association; (iv) relevance of apoC-III in subgroups (e.g., by T2D); (v) risk prediction beyond traditional CHD risk factors; and (vi) genetic loci associated with apoC-III levels.

最近的几项研究表明，载脂蛋白 C-III (APOC3) 基因中的功能丧失 (LoF) 突变会导致 可能通过减少循环中富含甘油三酯的脂蛋白来预防冠心病 (CHD) 风险 （TRL）。抑制 apoC-III 通路可以提供一种有吸引力的治疗机制来降低 CHD 风险。 然而，存在许多突出的不确定性，包括 apoC-III 缺陷对 (i) 的影响 脂蛋白动力学； (ii) 高密度脂蛋白重塑和功能； (iii)非脂质相关途径； (四) apoC-III 抑制可降低 CHD 风险的机制，(v) 不同脂蛋白的相对贡献 与 CHD 相关的 apoC-III 水平； (vi) 从安全角度看肝功能和胰岛素抵抗。 在此应用中，我们提出研究通过利用自然人类模型来解决这些差距 apoC-III 缺乏。我们发现了世界上第一例患有纯合 APOC3 遗传缺陷的人类 他们来自巴基斯坦一个偏僻的村庄，血缘关系盛行。招募本区所有居民 村庄（~5000人）已经建成；子集中的基因分型已鉴定出 113 个 APOC3 完全 apoC-III 缺陷的基因敲除为详细表型分析提供了机会。 具体来说，在 AIM-1 中，我们将对 113 个人类 APOC3 敲除（n = 113）进行深度表型研究 并在相同数量的杂合子和非携带者中解决以下问题：(i) apoC-III 缺陷在 调节 TRL 的蛋白质和脂质组成以影响细胞外脂肪酶的活性； (ii) apoC-III的作用 低密度脂蛋白亚类缺乏； (iii) apoC-III 在调节 Lp(a) 水平和组成中的作用； (四) apoC-III 缺乏对 HDL 组成和功能调节的贡献。我们还将解决 遗传性 APOC3 缺陷对 (iv) 全身脂质和葡萄糖代谢以及一系列蛋白质的影响 与炎症和其他途径有关（v）与动脉粥样硬化的剂量反应相关。 在 AIM-2 中，我们将使用同位素示踪剂对 18 个 APOC3 null 三重奏进行脂蛋白动力学研究 纯合子、杂合子和非携带者，以评估 apoC-III 缺陷对 apoB- 动力学的影响 含有脂蛋白以及 HDL apoA-I 的产生和清除。我们还将从 5 个细胞中产生人类 iPS 细胞 APOC3 敲除和 5 个匹配/相关非携带者并将其分化为肝细胞以进行评估 (a) VLDL apoB 和 TG 产生 (b) TRL 和 LDL 摄取以及 (c) SRB1 介导的 HDL 摄取。 在 AIM-3 中，我们将测量总血浆 apoC-III 和脂蛋白相关蛋白（apoB 脂蛋白、HDL 和 Lp[a]) apoC-III 是来自 EPIC-Norfolk 队列的 5,000 名参与者（其中 2,500 名发生了 CHD 事件）。这些研究 将能够评估：(i) 疾病调节； (ii) 根据脂蛋白相关因素逐步调整冠心病风险 apoC-III 水平和其他因素； (iii) 剂量-反应关联； (iv) apoC-III 在亚组中的相关性（例如，通过 T2D）； (v) 超越传统冠心病风险因素的风险预测； (vi) 与 apoC-III 水平相关的遗传位点。