Identification and characterization of functional genetic variants associated with human longevity

摘要 人类遗传学和基因组学的最新进展为识别和 药物靶点验证。对模式生物的研究表明， 与年龄相关的病理的频率和严重程度受保守的遗传途径和因素的影响 当通过基因、免疫或饮食调节达到目标时，可以延长寿命， 健康寿命。这一压倒性的证据为延缓衰老过程和减弱衰老的新药带来了希望。 通过调节这些保守的衰老途径来治疗人类的年龄相关疾病。在项目2（原项目1）中， 我们一直在进行旨在实现这一目标的实验：识别和功能表征 与人类健康衰老和极端衰老相关的衰老保守途径中的遗传变异 长寿用于治疗调节，以改善人类健康寿命和寿命。我们通过细胞展示了 SIRT 6、USP 35和UBE 3C中长寿相关的罕见编码变体改变蛋白质功能的模型和/或 在人体组织和细胞衰老过程中拮抗与年龄相关的有害变化的表达。 我们发现SMAD 3和3个与长寿相关的非编码变异体具有相同的抗衰老作用。 参与PKC/NF-κ B B信号通路的基因（NFKBIA、CLU和PRKCH）。在更新申请中， 项目2建议通过采取系统的多学科方法来确认和扩展我们的观察结果。 项目2的目标是识别和直接测试因果变异的影响，其遗传扰动 与长寿的联系。我们的方法是使用高通量筛选方法，如 用于编码变体的整合蛋白质互作组扰动筛选（InPOINT）和大规模平行 报告基因分析（MPRA）的非编码变体，并使用CRISPR- 工程化人多能干细胞（hPSC），多细胞分化模式，多组学 方法，以及由分子、生物化学和细胞测定组成的功能分析。我们的功能 对SIRT 6、USP 35和UBE 3C中长寿相关变异的分析表明，它们在保护中具有潜在作用。 对抗与年龄相关的认知能力下降和阿尔茨海默病（AD）的风险，以及对细胞表型的影响。 衰老，这越来越多地涉及神经退行性疾病。因此，我们将重点放在老龄化- SIRT 6、UBE 3C和USP 35编码变体在认知健康背景下的拮抗作用， 保护AD。项目2的最终影响在于它有可能揭示保守的途径， 人类衰老的基本机制，并作为健康衰老的治疗靶点， 项目1、3、4和核心B。

Abstract Recent advances in human genetics and genomics provide novel opportunities for the identification and validation of drug targets. Studies in model organisms have demonstrated that the rate of aging and the frequency and severity of age-related pathologies are influenced by conserved genetic pathways and factors which, when targeted genetically, pharmacologically, or through dietary modulation, can extend lifespan and healthspan. This overwhelming evidence raises hopes for new drugs that slow the aging process and attenuate age-related disease in humans by modulating these conserved pathways of aging. In Project 2 (old Project 1), we have been conducting the experiments aimed at exactly this goal: To identify and functionally characterize genetic variants in the conserved pathways of aging that are associated with human healthy aging and extreme longevity for therapeutic modulation to improve human healthspan and lifespan. We demonstrated through cell models that longevity-associated rare coding variants in SIRT6, USP35, and UBE3C alter protein function and/or expression which antagonize age-related deleterious changes in human tissues and during cellular senescence. We found the same aging-antagonizing effects of longevity-associated non-coding variants in SMAD3 and the 3 genes (NFKBIA, CLU and PRKCH) involved in the PKC/NF-B signaling pathways. In the renewal application, Project 2 proposes to confirm and extend our observations by taking a systematic multidisciplinary approach. The objective of Project 2 is to identify and directly test the impact of causal variants, whose genetic perturbations underlie the association with longevity. Our approach is to use high-throughput screening methods, such as INtegrated PrOtein INteractome perTurbation screening (InPOINT) for coding variants and Massively Parallel Reporter Assay (MPRA) for non-coding variants, and to investigate the underlying mechanisms using CRISPR- engineered human pluripotent stem cells (hPSC), multiple cellular differentiation paradigms, multiomics approaches, and functional analysis composed of molecular, biochemical, and cellular assays. Our functional analyses of longevity-associated variants in SIRT6, USP35, and UBE3C point to their potential role in protection against age-related cognitive decline and risk of Alzheimer’s disease (AD) and on the phenotypes of cellular senescence, which are increasingly implicated in neurodegenerative disease. Thus, we will focus on the aging- antagonizing effects of the SIRT6, UBE3C, and USP35 coding variants in the context of cognitive health and protection from AD. The ultimate impact of Project 2 lies in its potential to reveal conserved pathways as fundamental mechanisms of aging in humans and as therapeutic targets for healthy aging, in close collaboration with Projects 1, 3, 4 and Core B.