In 1948, the Framingham Heart Study (FHS) was commissioned to elucidate commonalities that may contribute to cardiovascular disease (CVD).¹ Through this work, many of the core risk factors for CVD were established by the late 1970s. Despite these major advances, it has long been recognized that the conventional modifiable risk factors incompletely account for the incidence of CVD.² In view of this, advanced age is the greatest CVD risk factor, but it is an elusive therapeutic target and has traditionally been viewed as an unmodifiable risk factor. Since the pioneering findings by the FHS, a great deal of work has been performed to better understand the molecular pathogenesis of CVD, spurred, in part, by the completion of the human genome project. Despite these important advances, little insight has been gained on novel and prevalent causes of age-related CVD until recent work uncovered age-related clonal hematopoiesis (CH) as a causal risk factor for CVD.

On a daily basis, billions of cells of the body turn over to generate new cells necessary to maintain proper homeostatic function. As these cells replicate, they can incur mutations due to the imperfect fidelity of DNA polymerase and repair processes. Frequently, these mutations are synonymous, intronic, or dispensable and are, as a consequence, well tolerated. However, as individuals age, this mutational burden accumulates, particularly in tissues with high turnover rates such as hematopoietic cells. In the process of CH, hematopoietic stem cells incur these mutations in particular loci-denoted driver genes, which when mutated impart a competitive growth advantage that allows mutant cells to outcompete wild-type cells and clonally expand.³ Unlike other forms of somatic mosaicism such as cancer, CH is a premalignant state that becomes a near-ubiquitous phenomenon with advanced age. Because this condition is not associated with overt hematologic changes, it generally has been referred to as CH of indeterminate potential or CHIP.

Before the seminal reports on CH, age was a well-known risk factor of all-cause mortality and CVD.⁴ However, the underpinnings of this additive risk remained unknown. In 2014, 2 pioneering studies on CH were published by Genovese et al⁵ and Jaiswal et al.⁶ In both reports, peripheral blood samples from over 10 000 patients underwent whole-exome sequencing to identify and quantify CH by conventional next-generation sequencing methodology. Both studies found that CH prevalence increased with age and that this condition was associated with all-cause mortality. Expectedly, the premalignant state of CH was associated with increased risk of hematologic cancer. However, Jaiswal et al⁶ also noted an increased risk of incident coronary heart disease and ischemic stroke. In response, Fuster et al⁷ investigated whether deficiency of the CH driver gene Tet2 (tet methylcytosine dioxygenase 2) modified atherosclerosis progression. Indeed, in a murine model of atherosclerosis, the competitive bone marrow transplantation of Tet2-deficient cells led to exacerbated atherosclerosis progression through a myeloid-driven and Nlrp3 (NLR family pyrin domain containing 3) inflammasome/IL-1β (interleukin 1 beta)–mediated mechanism. Following the establishment of this causal association, Jaiswal et al corroborated Fuster’s findings, verified the association between CH and coronary heart disease, and found that CH was associated with increased coronary artery calcification and increased risk of myocardial infarction.⁸ Furthermore, after driver gene stratification, mutations in DNMT3A, TET2, ASXL1, and JAK2 were individually associated with increased risk of coronary heart disease. Since these groundbreaking studies, additional work has demonstrated CH is associated with increased atherosclerotic CVD event rate and increased all-cause mortality in a cohort of 13 129 individuals with established atherosclerotic CVD.⁹ Given the pronounced and well-established association of CH with coronary macrovascular disease, Akhiyat et al¹⁰ recently reported that CH was associated with worse coronary flow reserve and increased major adverse cardiovascular event (MACE) rate in patients with coronary microvascular disease.

In light of the initial association of CH with ischemic stroke and atherosclerotic disease, Bhattacharya et al¹¹ analyzed 86 178 patients from 8 prospective studies for CH. In this report, CH was associated with increased risk of total stroke. Moreover, DNMT3A- and TET2-medidated CH was associated with increased incidence of hemorrhagic stroke while TET2-mediated CH was also associated with increased incidence of ischemic stroke. Extending upon these findings, Arends et al¹² analyzed peripheral blood samples from 581 patients with first-ever ischemic stroke and a 3-year follow-up via error-corrected, targeted DNA sequencing, which permitted greater sensitivity in CH clone detection. In this cohort, CH was associated with large-artery atherosclerosis and increased white matter lesion load. Furthermore, patients with CH had a greater risk of MACEs. Of the different diver gene mutations, TET2- and PPM1D-mediated CH exhibited the greatest risk of a vascular event or death. Despite the wide accessibility and well-characterized models of ischemic and hemorrhagic stroke in mice, there has been no published causal evidence connecting CH to stroke, and the mechanistic role of CH in modifying outcomes after stroke remains outstanding.

The pathological role of CH has been well documented in the context of heart failure (HF). In a cohort of 56 597 individuals from 5 prospective studies, CH was associated with a 25% increased risk of HF incidence.¹³ Clonal mutations in ASXL1, TET2, and JAK2 were each separately associated with increased HF incidence. These findings were further corroborated by Shi et al,¹⁴ who found that CH was associated with higher HF incidence and specifically, higher HF with preserved ejection fraction (HFpEF) incidence. CH has also been robustly associated clinically and experimentally with worse prognosis in heart failure with reduced ejection fraction (HFrEF). In patients with HFrEF irrespective of the etiology, CH, specifically DNMT3A- and TET2-driven CH, was associated with increased risk of HF-related death or HF-related hospitalization.¹⁵ Furthermore, Assmus et al¹⁶ performed targeted error-corrected DNA sequencing on bone marrow–derived mononuclear cells or peripheral blood mononuclear cells from 419 patients with chronic ischemic HF that was sufficient to discern mutations in driver genes at a variant allele fraction ≥0.5% (ie, 1% of cells harboring a mutation). At optimized variant allele fraction cutoffs of 0.73% and 1.15%, patients with TET2- or DNMT3A-mediated CH were at a 77% increased risk of death. Expanding upon these findings, Kiefer et al¹⁷ demonstrated that mutations in other CH driver genes, which included CBL, CEBPA, EZH2, GNB1, PHF6, SMC1A, and SRSF2, were associated with increased mortality in patients with chronic ischemic HF. Finally, in patients who developed cardiogenic shock following an acute myocardial infarction, TET2- and ASXL1-mediated CH was associated with decreased 30-day survival.¹⁸ Translating these findings, mutations in Tet2, Dnmt3a, Jak2, Ppm1d, Trp53, and Asxl1 exacerbated HF in experimental murine models of ischemic and nonischemic HFrEF.^3,19 Additionally, in some of these models, the cardiac pathology was a consequence of a deficiency of the CH driver gene within the myeloid populations and was able to be ameliorated with Nlrp3 inflammasome inhibition.^7,19 Recently, Cochran et al performed ultradeep error-corrected sequencing on 2 cohorts of patients with HFpEF and uncovered that CH was associated with worse diastolic heart function and increased risk of CV-related hospitalization in patients with HFpEF.²⁰ Notably, a variant allele fraction of 0.5% was the most predictive of adverse outcomes, suggesting that these small clones, which are typically neglected by traditional sequencing methodologies, may harbor important prognostic significance. Additionally, TET2-mediated CH was found to be enriched in the HFpEF population compared with a control population without HFpEF. Translating these findings, adoptive transfer of Tet2-deficient bone marrow exacerbated diastolic heart function and cardiac hypertrophy in a murine model of HFpEF.²⁰

Despite the rigorous and reproducible association of CH with worse CVD prognosis and the relatively conserved pathogenesis of CH-exacerbated disease, there exists a paucity of work exploring how CH can aid in clinical management. To date, CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) remains the only trial to examine CH as a potential biomarker for treatment response.²¹ In CANTOS, 10 061 patients with atherosclerotic disease and signs of systemic inflammation were randomized to either placebo or the IL-1β neutralizing antibody canakinumab.²² Despite a 15% reduction in MACE rate, canakinumab failed Food and Drug Administration approval, citing a modest efficacy relative to the side effect profile. Since the initial trial, several retrospective analyses have been published examining the utility of different biomarkers in predicting treatment response to canakinumab. In 2 of these studies, on-treatment levels of the systemic inflammatory biomarkers CRP (C-reactive protein) and IL-6 improved treatment response stratification.^23,24 Specifically, patients who achieved lower levels of CRP on treatment exhibited 25% reduction in MACE rate, whereas patients who achieved lower levels of IL-6 on treatment exhibited a 32% reduction in MACE rate. Recently, Svensson et al²¹ characterized CH via targeted DNA sequencing of peripheral blood samples sourced from baseline visits for CANTOS participants. Notably, patients with mutations in TET2 observed a 62% reduction in MACE rate with canakinumab treatment (P=0.04), whereas individuals without detectable CH exhibited a nonsignificant reduction in MACE rate with canakinumab treatment (hazard ratio, 0.93; P=0.38). Presently, TET2-mediated CH is the only candidate prospective biomarker and displays the greatest power in predicting treatment response. Given the large number of studies ongoing or completed using anti-inflammatory therapies for CVD,²⁵ it will be interesting to determine whether CH can enhance prediction of treatment response in other disease states.

In closing, an exhaustive amount of work has accumulated on the association between CH and increased CVD incidence, burden, and prognosis, and mechanistic studies have demonstrated this relationship to be causal and frequently mediated through inflammatory pathways (Figure). In particular, TET2-mediated CH has emerged as a potential, prospective biomarker for response to IL-1β antagonism in the context of atherosclerotic CVD. However, as patients with non–TET2-driven CH did not observe a benefit from treatment, it will be important to more thoroughly characterize the distinct pathogenesis of other CH driver genes in CVD and exploit this understanding to better tailor CVD treatment for patients. In future studies, it will be interesting to determine how the more definitive analysis of CH through ultradeep error-corrected sequencing can be used to enhance risk stratification and inform treatment responses for various CVDs.

Figure. Summary of hallmark clinical and experimental findings connecting clonal hematopoiesis with cardiovascular disease. More details can be found in an extensive review.³ CHD indicates coronary heart disease; CMD, coronary microvascular disease; DCM, dilated cardiomyopathy; HFD, high-fat diet; HFpEF, heart failure with preserved ejection fraction; HFrEF, heart failure with reduced ejection fraction; L-NAME, N[w]-nitro-l-arginine methyl ester; LAD, left anterior descending artery; MACE, major adverse cardiovascular event; and TAC, transverse aortic constriction.

Schematic illustration has been created with BioRender (BioRender.com).

This work was supported by the University of Virginia Medical Scientist Training Program T32GM007267 to J.D. Cochran; the National Institutes of Health grants AG073249, HL142650, and HL152174; and NASA (National Aeronautics and Space Administration) grant 80NSSC21K0549 to K. Walsh.

Disclosures None.

For Sources of Funding and Disclosures, see page 771.

The American Heart Association celebrates its 100^th anniversary in 2024. This article is part of a series across the entire AHA Journal portfolio written by international thought leaders on the past, present, and future of cardiovascular and cerebrovascular research and care. To explore the full Centennial Collection, visit https://www.ahajournals.org/centennial

1948 年，弗雷明汉心脏研究 (FHS) 受委托阐明可能导致心血管疾病 (CVD) 的共性。¹通过这项工作，CVD 的许多核心风险因素在 20 世纪 70 年代末已确定。尽管取得了这些重大进展，人们早已认识到传统的可改变危险因素并不能完全解释心血管疾病的发病率。²有鉴于此，高龄是最大的 CVD 危险因素，但它是一个难以捉摸的治疗目标，传统上被视为不可改变的危险因素。自 FHS 取得开创性发现以来，为了更好地了解 CVD 的分子发病机制，人们开展了大量工作，部分原因是人类基因组计划的完成。尽管取得了这些重要的进展，但直到最近的研究发现年龄相关的克隆性造血（CH）是 CVD 的一个致病危险因素之前，人们对年龄相关 CVD 的新的和普遍的原因仍知之甚少。

每天，身体数十亿个细胞会产生新的细胞，以维持适当的稳态功能。当这些细胞复制时，由于 DNA 聚合酶和修复过程的不完美保真度，它们可能会发生突变。通常，这些突变是同义的、内含子的或可有可无的，因此具有良好的耐受性。然而，随着个体年龄的增长，这种突变负担会累积，特别是在造血细胞等周转率高的组织中。在 CH 过程中，造血干细胞在特定位点表示的驱动基因中发生这些突变，当突变时，这些基因会赋予竞争性生长优势，使突变细胞能够战胜野生型细胞并进行克隆扩张。³与癌症等其他形式的体细胞嵌合体不同，CH 是一种癌前状态，随着年龄的增长，这种现象几乎普遍存在。由于这种情况与明显的血液学变化无关，因此通常被称为潜在不确定性 CH 或 CHIP。

在有关 CH 的开创性报告出现之前，年龄是众所周知的全因死亡率和 CVD 的危险因素。⁴然而，这种附加风险的基础仍然未知。 2014 年，Genovese 等人⁵和 Jaiswal 等人发表了 2 项关于 CH 的开创性研究。 ⁶在这两份报告中，对 10 000 多名患者的外周血样本进行了全外显子组测序，以通过传统的下一代测序方法来识别和量化 CH。两项研究都发现，CH 患病率随着年龄的增长而增加，并且这种情况与全因死亡率相关。预计 CH 的癌前状态与血液癌风险增加相关。然而，Jaiswal 等人⁶也指出，发生冠心病和缺血性中风的风险增加。作为回应，Fuster 等人⁷研究了 CH 驱动基因Tet2（tet 甲基胞嘧啶双加氧酶 2）的缺陷是否会改变动脉粥样硬化的进展。事实上，在动脉粥样硬化的小鼠模型中，Tet2 缺陷细胞的竞争性骨髓移植通过骨髓驱动的 Nlrp3（包含 3 的 NLR 家族吡啶结构域）炎性体/IL-1β（白细胞介素 1 β）导致动脉粥样硬化进展加剧。介导机制。在建立这种因果关系后，Jaiswal等人证实了Fuster的发现，验证了CH与冠心病之间的关联，并发现CH与冠状动脉钙化增加和心肌梗死风险增加有关。^{8此外，在驱动基因分层后，} DNMT3A、TET2、ASXL1和JAK2的突变分别与冠心病风险增加相关。自这些开创性研究以来，更多的研究表明，CH 与动脉粥样硬化 CVD 事件发生率增加和全因死亡率增加有关，该队列由 13 129 名患有动脉粥样硬化 CVD 的个体组成。⁹鉴于 CH 与冠状动脉大血管疾病之间存在明显且明确的关联，Akhiyat 等人¹⁰最近报道，CH 与冠状动脉血流储备较差以及冠状动脉微血管疾病患者主要不良心血管事件 (MACE) 发生率增加相关。

鉴于 CH 与缺血性中风和动脉粥样硬化性疾病的最初关联，Bhattacharya 等人¹¹分析了 8 项 CH 前瞻性研究中的 86 178 名患者。在本报告中，CH 与总卒中风险增加相关。此外，DNMT3A和TET2介导的CH与出血性中风发病率增加相关，而TET2介导的CH也与缺血性中风发病率增加相关。 Arends 等人¹²扩展了这些发现，通过纠错、靶向 DNA 测序分析了 581 名首次缺血性中风患者的外周血样本，并进行了 3 年随访，这使得 CH 克隆检测具有更高的灵敏度。在该队列中，CH 与大动脉粥样硬化和白质病变负荷增加有关。此外，CH 患者发生 MACE 的风险更大。在不同的潜水员基因突变中，TET2和PPM1D介导的 CH 表现出血管事件或死亡的最大风险。尽管小鼠缺血性和出血性中风模型具有广泛的可及性和良好的特征，但尚未发表CH与中风之间的因果证据，并且CH在改变中风后结果中的机制作用仍然突出。

CH 在心力衰竭 (HF) 中的病理作用已得到充分记录。在来自 5 项前瞻性研究的 56 597 名个体队列中，CH 与 HF 发病风险增加 25% 相关。¹³ ASXL1、TET2和JAK2的克隆突变分别与心力衰竭发病率增加相关。 Shi 等人进一步证实了这些发现，¹⁴他们发现 CH 与较高的心力衰竭发生率相关，特别是射血分数保留的心力衰竭 (HFpEF) 发生率较高。在临床和实验上，CH 还与射血分数降低的心力衰竭 (HFrEF) 的较差预后密切相关。在 HFrEF 患者中，无论病因如何，CH，特别是DNMT3A和TET2驱动的 CH，与 HF 相关死亡或 HF 相关住院风险增加相关。¹⁵此外，Assmus 等人¹⁶对 419 名慢性缺血性心力衰竭患者的骨髓源性单核细胞或外周血单核细胞进行了靶向纠错 DNA 测序，足以识别变异等位基因分数≥0.5% 的驱动基因中的突变（即 1% 的细胞带有突变）。在优化的变异等位基因分数截止值为 0.73% 和 1.15% 时，TET2或DNMT3A介导的 CH 患者的死亡风险增加了 77%。 Kiefer 等人¹⁷扩展了这些发现，证明其他 CH 驱动基因（包括CBL、CEBPA、EZH2、GNB1、PHF6、SMC1A和SRSF2）的突变与慢性缺血性心力衰竭患者死亡率增加相关。最后，在急性心肌梗塞后发生心源性休克的患者中，TET2和ASXL1介导的 CH 与 30 天生存率降低相关。¹⁸根据这些发现，在缺血性和非缺血性 HFrEF 实验鼠模型中， Tet2、Dnmt3a、Jak2、Ppm1d、Trp53和Asxl1的突变会加剧心力衰竭。 ^3,19此外，在其中一些模型中，心脏病理学是骨髓细胞群内 CH 驱动基因缺陷的结果，并且可以通过 Nlrp3 炎性体抑制得到改善。^7,19最近，Cochran 等人对 2 组 HFpEF 患者进行了超深纠错测序，发现 CH 与 HFpEF 患者舒张心功能较差以及心血管相关住院风险增加有关。²⁰值得注意的是，0.5% 的变异等位基因分数最能预测不良结果，这表明这些通常被传统测序方法忽视的小克隆可能具有重要的预后意义。此外，与没有HFpEF的对照群体相比， TET2介导的CH被发现在HFpEF群体中富集。根据这些发现，在 HFpEF 小鼠模型中，Tet2 缺陷型骨髓的过继转移会加剧心脏舒张功能和心脏肥大。²⁰

尽管 CH 与较差的 CVD 预后存在严格且可重复的关联，并且 CH 恶化性疾病的发病机制相对保守，但探索 CH 如何帮助临床管理的工作却很少。迄今为止，CANTOS（卡那奴单抗抗炎性血栓结果研究）仍然是唯一检验 CH 作为治疗反应潜在生物标志物的试验。²¹在 CANTOS 中，10061 名患有动脉粥样硬化疾病和全身炎症体征的患者被随机分配到安慰剂组或 IL-1β 中和抗体卡那奴单抗组。²²尽管 MACE 发生率降低了 15%，但卡那奴单抗未能获得美国食品和药物管理局的批准，理由是相对于副作用而言疗效有限。自初始试验以来，已经发表了几项回顾性分析，研究了不同生物标志物在预测卡那单抗治疗反应方面的效用。在其中 2 项研究中，治疗中全身炎症生物标志物 CRP（C 反应蛋白）和 IL-6 的水平改善了治疗反应分层。^23,24具体来说，治疗中 CRP 水平较低的患者 MACE 发生率降低 25%，而 IL-6 治疗水平较低的患者 MACE 发生率降低 32%。最近，Svensson 等人²¹通过对来自 CANTOS 参与者基线访问的外周血样本进行靶向 DNA 测序来表征 CH。值得注意的是， TET2突变的患者在接受卡那单抗治疗后观察到 MACE 发生率降低了 62%（P = 0.04），而没有检测到 CH 的个体在接受卡那单抗治疗后 MACE 发生率没有显着降低（风险比为 0.93；P = 0.38）。目前，TET2介导的CH是唯一候选的前瞻性生物标志物，并且在预测治疗反应方面表现出最大的能力。鉴于使用抗炎疗法治疗 CVD 的大量研究正在进行或已完成，²⁵确定 CH 是否可以增强对其他疾病状态治疗反应的预测将很有趣。

最后，关于 CH 与 CVD 发病率、负担和预后增加之间的关联，已经积累了大量的工作，并且机制研究已经证明这种关系是因果关系，并且经常通过炎症途径介导（图）。特别是，TET2介导的 CH 已成为动脉粥样硬化 CVD 背景下 IL-1β 拮抗反应的潜在、前瞻性生物标志物。然而，由于非TET2驱动的 CH 患者没有观察到治疗带来的益处，因此更彻底地表征 CVD 中其他 CH 驱动基因的独特发病机制并利用这种理解来更好地为患者制定 CVD 治疗非常重要。在未来的研究中，确定如何通过超深纠错测序对 CH 进行更明确的分析来增强风险分层并为各种 CVD 的治疗反应提供信息将会很有趣。

数字。 将克隆造血与心血管疾病联系起来的标志性临床和实验结果摘要。更多细节可以在广泛的评论中找到。³ CHD表示冠心病； CMD，冠状动脉微血管疾病； DCM，扩张型心肌病； HFD，高脂肪饮食； HFpEF，射血分数保留的心力衰竭； HFrEF，射血分数降低的心力衰竭； L-NAME，N[w]-硝基-L-精氨酸甲酯； LAD，左前降支； MACE，主要不良心血管事件；和TAC，横主动脉缩窄。

示意图是使用 BioRender (BioRender.com) 创建的。

这项工作得到了弗吉尼亚大学医学科学家培训计划 T32GM007267 JD Cochran 的支持；美国国立卫生研究院拨款 AG073249、HL142650 和 HL152174；和 NASA（国家航空航天局）将 80NSSC21K0549 授予 K. Walsh。

披露无。

有关资金来源和披露信息，请参阅第 771 页。

美国心脏协会将于 2024 年庆祝成立 100 周年。^本文是国际思想领袖撰写的整个 AHA 期刊系列文章的一部分，内容涉及心脑血管研究和护理的过去、现在和未来。要探索完整的百年纪念收藏，请访问 https://www.ahajournals.org/centennial