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

最近的一些研究表明，载脂蛋白C-III(APOC3)基因的功能丧失(LoF)突变提供了 预防冠心病(CHD)风险，可能是通过减少循环中富含甘油三酯的脂蛋白 (TRLS)。抑制载脂蛋白C-III通路可以提供一种诱人的治疗机制来降低冠心病的风险。 然而，存在许多突出的不确定性，包括载脂蛋白C-III缺乏对(I)的影响。 脂蛋白动力学；(Ii)高密度脂蛋白重塑和功能；(Iii)非脂相关途径；(Iv) 抑制载脂蛋白C-III可降低冠心病风险的机制，(V)不同脂蛋白- 相关载脂蛋白C-III水平对冠心病的影响；(Vi)从安全的角度看肝功能和胰岛素抵抗。 在这项应用中，我们建议通过利用自然人类模型来解决这些差距的研究 载脂蛋白C-III缺乏。我们已经确认了世界上第一例APOC3纯合子基因缺陷的人类 来自一个血缘关系很普遍的与世隔绝的巴基斯坦村庄。招募这里的所有居民 村庄(约5000人)已经完成；一个子集的基因分型已经确定了113个APOC3 完全缺乏apoC-III的基因敲除为详细的表型鉴定提供了机会。 具体来说，在AIM-1中，我们将对113个人类APOC3基因敲除(n=113)进行深入的表型研究。 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缺乏在 调节TRL的蛋白质和脂肪组成以影响胞外脂肪酶的活性；(Ii)apoC-III的作用 低密度脂蛋白亚类缺乏；(3)载脂蛋白C-III在调节脂蛋白(A)水平和组成中的作用；以及(4) 载脂蛋白C-III缺乏对高密度脂蛋白组成和功能调节的贡献。我们还将解决 遗传性载脂蛋白C3缺乏对(Iv)全身脂肪和葡萄糖代谢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关系。 在AIM-2中，我们将使用同位素示踪剂对18个APOC3缺失的三联体进行脂蛋白动力学研究 纯合子、杂合子和非携带者评估apoC-III缺乏对apoB-3动力学的影响 含脂蛋白与高密度脂蛋白apoA-I的产生和清除我们还将从5个细胞中培养出人类iPS细胞 APOC3基因敲除和5个匹配/相关的非携带者，并将他们分化为肝细胞以评估(A) 极低密度脂蛋白载脂蛋白B和甘油三酯的产生(B)TRL和低密度脂蛋白摄取以及(C)SRB1介导的高密度脂蛋白摄取。 在AIM-3中，我们将测量总血浆apoC-III和脂蛋白相关(ApoB)脂蛋白，高密度脂蛋白，和 Lp[a])来自EPIC-诺福克队列的5,000名参与者中的载脂蛋白C-III(2,500名发生冠心病事件)。这些研究 将能够评估：(1)疾病调解；(2)逐步调整与脂蛋白相关的冠心病风险 载脂蛋白C-III水平和其他因素；(3)剂量-反应关系；(4)载脂蛋白C-III在亚组中的相关性(例如，通过 T2d)；(V)传统冠心病危险因素之外的风险预测；以及(Vi)与apoC-III水平相关的遗传位点。

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.