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Background: Premature menopause is an independent risk factor for cardiovascular disease in women, but mechanisms underlying this association remain unclear. Clonal hematopoiesis of indeterminate potential (CHIP), the age-related expansion of hematopoietic cells with leukemogenic mutations without detectable malignancy, is associated with accelerated atherosclerosis. Whether premature menopause is associated with CHIP is unknown. Methods: We included postmenopausal women from the UK Biobank (n=11 495) aged 40 to 70 years with whole exome sequences and from the Women’s Health Initiative (n=8111) aged 50 to 79 years with whole genome sequences. Premature menopause was defined as natural or surgical menopause occurring before age 40 years. Co–primary outcomes were the presence of any CHIP and CHIP with variant allele frequency >0.1. Logistic regression tested the association of premature menopause with CHIP, adjusted for age, race, the first 10 principal components of ancestry, smoking, diabetes, and hormone therapy use. Secondary analyses considered natural versus surgical premature menopause and gene-specific CHIP subtypes. Multivariable-adjusted Cox models tested the association between CHIP and incident coronary artery disease. Results: The sample included 19 606 women, including 418 (2.1%) with natural premature menopause and 887 (4.5%) with surgical premature menopause. Across cohorts, CHIP prevalence in postmenopausal women with versus without a history of premature menopause was 8.8% versus 5.5% ( P P =0.004; CHIP with variant allele frequency >0.1: odds ratio, 1.40 [95% CI, 1.10–1.79]; P =0.007). Associations were larger for natural premature menopause (all CHIP: odds ratio, 1.73 [95% CI, 1.23–2.44]; P =0.001; CHIP with variant allele frequency >0.1: odds ratio, 1.91 [95% CI, 1.30–2.80]; P DNMT3A CHIP was significantly associated with premature menopause. Among postmenopausal middle-aged women, CHIP was independently associated with incident coronary artery disease (hazard ratio associated with all CHIP: 1.36 [95% CI, 1.07–1.73]; P =0.012; hazard ratio associated with CHIP with variant allele frequency >0.1: 1.48 [95% CI, 1.13–1.94]; P =0.005). Conclusions: Premature menopause, especially natural premature menopause, is independently associated with CHIP among postmenopausal women. Natural premature menopause may serve as a risk signal for predilection to develop CHIP and CHIP-associated cardiovascular disease.

Background: Clonal hematopoiesis of indeterminate potential (CHIP) refers to clonal expansion of hematopoietic stem cells attributable to acquired leukemic mutations in genes such as DNMT3A or TET2 . In humans, CHIP associates with prevalent myocardial infarction. In mice, CHIP accelerates atherosclerosis and increases IL-6/IL-1β expression, raising the hypothesis that IL-6 pathway antagonism in CHIP carriers would decrease cardiovascular disease (CVD) risk. Methods: We analyzed exome sequences from 35 416 individuals in the UK Biobank without prevalent CVD, to identify participants with DNMT3A or TET2 CHIP. We used the IL6R p.Asp358Ala coding mutation as a genetic proxy for IL-6 inhibition. We tested the association of CHIP status with incident CVD events (myocardial infarction, coronary revascularization, stroke, or death), and whether it was modified by IL6R p.Asp358Ala. Results: We identified 1079 (3.0%) individuals with CHIP, including 432 (1.2%) with large clones (allele fraction >10%). During 6.9-year median follow-up, CHIP associated with increased incident CVD event risk (hazard ratio, 1.27 [95% CI, 1.04–1.56], P =0.019), with greater risk from large CHIP clones (hazard ratio, 1.59 [95% CI, 1.21–2.09], P IL6R p.Asp358Ala attenuated CVD event risk among participants with large CHIP clones (hazard ratio, 0.46 [95% CI, 0.29–0.73], P P =0.08; P interaction =0.003). In 9951 independent participants, the association of CHIP status with myocardial infarction similarly varied by IL6R p.Asp358Ala ( P interaction =0.036). Conclusions: CHIP is associated with increased risk of incident CVD. Among carriers of large CHIP clones, genetically reduced IL-6 signaling abrogated this risk.

Introduction: Premature menopause is an independent risk factor for cardiovascular disease in women, but mechanisms underlying this association remain unclear. Clonal hematopoiesis of indeterminate potential (CHIP), the age-related expansion of hematopoietic cells with leukemogenic mutations, is associated with accelerated atherosclerosis. Whether premature menopause is associated with CHIP is unknown. Methods: We included postmenopausal women from the UK Biobank (N=11,509) aged 40-70 years with whole exome sequences and from the Women’s Health Initiative (WHI, N=8,111) aged 50-79 years with whole genome sequences. Premature menopause was defined as natural or surgical menopause occurring before age 40 years. Co-primary outcomes were the presence of (1) any CHIP and (2) CHIP with variant allele fraction (VAF) >0.1. Logistic regression tested the association of premature menopause with CHIP, adjusted for age, race, the first 10 principal components, smoking, diabetes mellitus, and hormone therapy use. Results: Across cohorts, prevalence of CHIP in women with vs. without a history of premature menopause was 8.8% vs. 5.5% (P0.1: 1.91, 95% CI 1.30-2.80; Figure ). In gene-specific analyses, DNMT3A CHIP had a strong association with natural premature menopause but no association with surgical premature menopause. Among postmenopausal middle-aged women in the UK Biobank and WHI, CHIP was independently associated with incident coronary artery disease (meta-analyzed HR 1.52, 95% CI 1.17-1.99). Conclusions: Premature menopause, especially natural premature menopause, is independently associated with CHIP. CHIP may contribute to the excess cardiovascular risk associated with premature menopause.

Background: Therapy-related clonal hematopoiesis in cancer patients is typically associated with somatic mutations in hematopoietic cell genes that encode regulators of the DNA-damage response (DDR) pathway. The Protein Phosphatase Mg2+/Mn2+ Dependent 1D ( PPM1D ) gene is the most frequently mutated DDR gene associated with therapy-related clonal hematopoiesis. While epidemiological evidence suggests an association between therapy-related clonal hematopoiesis and cardiovascular disease in cancer patients, causal and mechanistic relationships have never been evaluated in an experimental system. Methods: To test whether hematopoietic cell mutations in PPM1D can increase the susceptibility to cardiac stress, we evaluated cardiac dysfunction in response to angiotensin II infusion in a mouse model where clonal-hematopoiesis-associated mutations in Ppm1d were produced by CRISPR-Cas9 technology. Results: Mice transplanted with hematopoietic stem cells containing clinically relevant mutations in exon 6 of Ppm1d exhibited augmented cardiac remodeling following the continuous infusion of angiotensin II. Ppm1d -mutated macrophages showed impairments in the DDR pathway and had an augmented proinflammatory profile. Mice transplanted with Ppm1d mutated cells exhibited elevated IL-1β in the stressed myocardium, and bone marrow derived macrophages produced more IL-1β in response to LPS stimulation. The administration of an NLRP3 inflammasome inhibitor to mice reversed the cardiac phenotype induced by the Ppm1d -mutated hematopoietic stem cells under conditions of Angiotensin II-induced stress. Conclusions: A mouse model of Ppm1d -mediated clonal hematopoiesis was more susceptible to cardiac stress following of angiotensin II infusion. Mechanistically, disruption of the DDR pathway led to elevations in inflammatory cytokine production, and the NLRP3 inflammasome was shown to be essential for this augmented cardiac stress response. These data indicate that therapy-related clonal hematopoiesis involving mutations in PPM1D could contribute to the cardiac dysfunction observed in cancer survivors.

Background: It becomes increasingly clear that age-related clonal hematopoiesis (CH) is associated with cardiovascular diseases. However, CH with a driver mutation that causes myeloproliferative neoplasm (MPN) is not fully understood. Objective: To determine the clinical relevance of CH with MPN-driving mutations on cardiovascular diseases. Methods: This study prospectively enrolled 832 patients with cardiovascular diseases who did not show any hematological disorders. We examined the presence of the known MPN-driving mutations including JAK2V617F, CALRdel52, CALRins5, MPLW515L, and MPLW515K by an allele-specific polymerase chain reaction. Results: Out of 832 patients, 16 patients (1.9%) exhibited MPN-driving mutations including 15 patients with JAK2V617F (1.8%) and 1 patient with CALRins5 (0.1%). We divided the patients into two groups: vascular diseases including coronary artery disease, stroke, aortic aneurysm, aortic dissection, peripheral artery disease, deep vein thrombosis, and pulmonary thromboembolism (n=462, 55%) and non-vascular diseases (n=370, 45%). The prevalence of JAK2V617F-positive CH was significantly higher in patients with vascular diseases than in those with non-vascular diseases (2.8% vs. 0.5%, P = 0.014). In a multivariable analysis adjusted for known classic-atherosclerotic risk factors (age, male, obesity, smoking, hypertension, diabetes mellitus, dyslipidemia), the presence of JAK2V617F-positive CH was independently associated with vascular diseases (odds ratio, 5.448; P = 0.043). Additionally, peripheral leukocyte IL1B mRNA expressions as well as plasma interleukin-1β concentrations were significantly increased in patients with JAK2V617F-positive CH compared to those without JAK2V617F (P = 0.040 and P = 0.030, respectively). Conclusions: CH with JAK2V617F, but not CALR or MPL mutations, was significantly associated with vascular diseases independently on classic-atherosclerotic risk factors, in relation to interleukin-1β-related inflammatory mechanisms. JAK2V617F is a potential diagnostic and therapeutic target for vascular diseases.

Introduction: In the current era of intense LDL lowering, superficial erosion may be on the rise as a cause of ACS (25-30%). Experimental data on human atheromata support a role for neutrophils and the formation of neutrophil extracellular traps (NETs) in the pathogenesis of superficial erosion and subsequent thrombosis. The common mutation of Janus kinase 2 ( Jak2 V617F ) borne by clones of leukocytes derived from bone marrow stem cells sensitizes neutrophils to undergo NETosis and linked NET formation to increased thrombosis. Jak2 V617F associates with accelerated atherosclerosis and venous thrombosis in patients with myeloproliferative neoplasms (MPN) and in individuals harboring this mutation but without a demonstrable MPN. Hypothesis We hypothesized that i) the Jak2 V617F associated with clonal hematopoiesis and increased atherosclerotic and thrombotic risk in humans, predisposes to NETosis and subsequent endothelial injury and thrombosis at sites of flow disturbance in arteries with erosion-like intimas; and ii) the clinically approved Jak-1,2 inhibitor, ruxolitinib (Rux), can preserve endothelial integrity and reduce thrombosis in mice with myeloid Jak2 V617F . Methods: We generated cohorts of mice harboring Jak2 V617F or wild-type Jak2 . We used a surgical preparation recapitulating features of superficial erosion and assessed endothelial integrity, thrombosis, and NET formation in relation to genotype and in vivo Rux treatment. Results and Conclusions: Evans blue extravasation following introduction of flow disturbance at sites of erosion-prone intimas showed significant impairment of endothelial barrier in the group of mice bearing the Jak2 V617F mutation compared to control (WT) mice ( ** pJak2 V617F mutation. Immunohistochemical evaluation of the regions of experimental superficial erosion revealed reduced endothelial continuity and increased NET accumulation in Jak2 V617F vs. WT mice. Rux treatment mitigated the adverse effects of Jak2 V617F on both NET accumulation and endothelial integrity, supporting the translational potential of these observations in individuals with clonal hematopoiesis due to Jak2 V617F and acute coronary syndromes.

Rationale: Pulmonary hypertension (PH) is associated with hematological disorders by unclear multifactorial mechanisms. JAK2 V617F is the most frequent driver mutation among the myeloproliferative neoplasms and is recently identified in clonal hematopoiesis. However, the impact of clonal hematopoiesis on PH remains unknown. Objectives: To elucidate the mechanistic relevance of hematopoietic JAK2V617F in the development of PH. Methods: We applied experimental mouse models, mimicking hematological clinical scenarios using Jak2 V617F-transgenic (JAK2 V617F ) mice and recipient mice transplanted with JAK2 V617F bone marrow cells (JAK2 V617F recipients), exposed to chronic hypoxia to induce PH. The presence of JAK2 V617F was also examined prospectively in PH patients. Measurements and Main Results: Increases in right ventricular systolic pressure and right ventricular hypertrophy accompanied by pulmonary arterial structural remodeling after exposure to chronic hypoxia were significantly exacerbated in both JAK2 V617F mice which showed a myeloproliferative neoplasm-phenotype and JAK2 V617F recipients which modeled clonal hematopoiesis compared to corresponding controls. JAK2V617F-expressing neutrophils were specifically accumulated in pulmonary arterial regions, accompanied by increases in neutrophil-derived elastase activity and chemokines. RNA sequencing identified progressive upregulation of Acvrl1 (encoding ALK1) during the differentiation from bone marrow stem/progenitor cells peripherally into mature neutrophils of pulmonary arterial regions in the enrichment of JAK-STAT-related molecules by JAK2V617F. JAK2V617F-mediated STAT3 phosphorylation upregulated ALK1-Smad1/5/8 signaling. Strikingly, ALK1 inhibition completely rescued the development of PH in JAK2 V617F mice. Furthermore, clonal hematopoiesis with JAK2 V617F was significantly more common in PH patients than in healthy subjects. Conclusions: Our study reveals clonal hematopoiesis with JAK2V617F as a crucial factor that causally leads to PH through ALK1.

Introduction: Recent evidence has demonstrated that Clonal Hematopoiesis of Indeterminate Potential (CHIP) increases the risk of cardiovascular events. However, the exact role played by CHIP in early stages of coronary atherosclerosis, characterized by coronary endothelial dysfunction remains to be elucidated. Hypothesis: The current study was designed to test the hypothesis that the presence of CHIP in peripheral blood cells is associated with coronary endothelial dysfunction and increased levels of inflammatory markers. Methods: Next generation sequencing was used to detect mutations among patients who had coronary endothelial dysfunction and control group (normal coronary endothelial function). Endothelial dysfunction was defined by ≥ 20% decrease in coronary artery diameter or ≤50% increase in blood flow in response to acetylcholine injection compared to baseline. Plasma cytokine levels of Interleukin (IL)-6 and IL-8 were also assessed. Results: Clonal hematopoiesis relevant gene mutations were found in 1 out of 64 patients with normal endothelial function (1.5%) and 11 out of 119 cases in the endothelial dysfunction group (9.2%) (P = 0.04) with ASXL1 (Additional sex combs-like 1) being the most frequent gene (4.2%) mutated. Cytokine analysis demonstrated that mutations in ASXL1, DNMT3A (DNA methyltransferase 3A) and TET2 (Ten-eleven-translocation-2) in the endothelial dysfunction group were associated with increased levels of IL-6 and IL-8 (P = 0.001, P = 0.003; respectively). Conclusions: The current study supports a potential role for CHIP in mechanisms of coronary artery disease starting at early stage of atherosclerosis. Furthermore, enhanced expression levels of IL-6 and IL-8 seem to be related to mutations in DNMT3A, ASXL1 and TET2 genes, more than other gene mutations relevant CHIP.

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related disorder associated with increased risk of atherosclerotic cardiovascular disease (ASCVD). Mechanistic studies of CHIP implicate inflammatory pathways underlying this relationship. Although inflammation is a well-established component of chronic kidney disease (CKD), linked to both CKD progression and ASCVD in CKD patients, CHIP has not been investigated in a CKD setting. Objective: To estimate the prevalence of CHIP in patients with CKD and to examine the cross-sectional associations between CHIP and biomarkers that were previously related to CKD progression and ASCVD among CKD patients. Methods: The current study was conducted among 598 Chronic Renal Insufficiency Cohort (CRIC) study participants with whole-exome sequencing data and baseline measures of fibrinogen, interleukin (IL)-6, serum albumin, and tumor necrosis factor-α. CHIP was detected from sequencing data using MuTect2. CHIP was defined as the presence of a somatic hematologic malignancy-associated mutation with a variant allele frequency (VAF) of at least 2%. CHIP was also categorized ordinally by clone size as no CHIP, small CHIP clone (VAF Results: CHIP was detected in a total of 28 individuals from the WES study (4.7%). Among those 70 years of age and older, we detected a CHIP prevalence of nearly 16%. As expected, participants with CHIP were older than those without CHIP (mean age: 67 and 58 years, respectively; P. Age-adjusted models showed strong associations between CHIP and both fibrinogen and IL-6 with odds ratios of 2.49 (95% CI: 1.09, 5.71) and 2.45 (95% CI: 1.05, 5.72), respectively. After multivariable adjustment, only fibrinogen remained associated with CHIP with an odds ratio of 4.70 (95% CI: 1.69, 13.0). Consistent with these findings, there was a strong dose-dependent association between CHIP clone size and fibrinogen in multivariable adjusted analyses (P for linear trend=0.009). Compared to those without CHIP, odds ratios for higher fibrinogen tertile were 3.0 (95% CI: 0.84, 11.3) and 4.7 (95% CI: 1.29, 16.7) among those with small and large CHIP clone sizes, respectively. Conclusions: CHIP prevalence was higher among CKD patients in the current study compared to previous estimates from the general population. Furthermore, a strong, dose-dependent association between CHIP and fibrinogen was identified.

Background: Clonal hematopoiesis of indeterminate potential (CHIP) occurs when there is expansion of leukocyte clones in the blood due to mutations in hematopoietic stem cells. CHIP is associated with a high risk of hematologic malignancy and increased risk of cardiovascular disease (CVD). Although the environmental risk factors for CHIP remain poorly understood, radon is a ubiquitous mutagen and may deliver non-negligible doses of α-radiation to bone marrow that trigger CHIP development. Methods: We conducted a cross-sectional study of 10,495 postmenopausal women without hematologic malignancy from the Women’s Health Initiative clinical trials and observational study (mean age: 69 years; 82% white; 9% African American; 4% Hispanic/Latina; 3% Asian). Using blood from the 1993-1998 screening visit or a subsequent annual visit, and whole genome sequence data from the Trans-Omics for Precision Medicine project, we identified CHIP using the GATK Mu TECT2 somatic variant caller, a pre-specified list of leukemogenic driver mutations in 74 genes, and a threshold variant allele fraction > 0.02. We linked geocoded participant addresses at the time of blood draw to county-level, indoor, screening radon concentrations that were predicted by the Environmental Protection Agency (EPA) in 1993 using data on indoor radon, geology, aerial radioactivity, soil parameters, and foundation types. Radon exposure was classified as follows: Zone 3 (< 2 pCi/L), Zone 2 (2-4 pCi/L), and Zone 1 (> 4 pCi/L), the level at which EPA recommends remediation. We estimated the radon-related risk of CHIP as an odds ratio (OR, 95% CI) using logistic regression with and without adjustment for study design, participant sampling, age, race/ethnicity, smoking, and other sociodemographic / behavioral covariates. Results: We identified CHIP in 887 (8.5% of) participants. 3106 (29.6%), 3982 (37.9%), and 3407 (32.5%) had Zone 3, 2, and 1 radon exposures. The corresponding percentage of participants with CHIP across zones was 7.6%, 8.6%, and 9.1%. Relative to participants in Zone 3, those in Zones 2 and 1 had a higher adjusted risk of CHIP: OR (95% CI) = 1.13 (0.95, 1.35) and 1.24 (1.03, 1.48). Conclusion: Risk of CHIP was positively associated with indoor radon concentrations in this cross-sectional study of post-menopausal women. Longitudinal study of temporally integrated, in-home radon exposures, incident CHIP, and CVD would help confirm this novel radon-CHIP association and place it in context.

Background: Although the etiology of clonal hematopoiesis of indeterminate potential (CHIP) remains unclear, CHIP-defining somatic mutations within hematopoietic stem cells appear to expand peripheral blood leukocyte populations, promote inflammation, and thereby increase cardiovascular disease (CVD) risk. Moreover, high noise exposures have been linked to chromosomal aberrations in bone marrow and DNA repair in peripheral blood leukocytes. We therefore examined the potential of aircraft noise exposure as a novel risk factor for CHIP. Methods: We leveraged cross-sectional data on 10,050 postmenopausal women without prior hematologic malignancy in a Women’s Health Initiative (WHI) ancillary study of 5,309 women with stroke and/or venous thromboembolic disease and 4,741 controls. We ascertained CHIP using Trans-Omics for Precision Medicine (TOPMed) whole-genome sequencing data, the GATK Mu TECT2 somatic variant caller, a pre-specified list of leukemogenic driver mutations in 74 genes, and a threshold variant allele frequency of > 0.02. In cooperation with the Federal Aviation Administration, we generated day-night-level (DNL) noise contours around 90 major U.S. airports using the Aviation Environmental Design Tool noise modelling software. The DNL imposes a penalty for nighttime noise exposure and is the primary means for assessing and regulating noise exposure in the U.S. We estimated geocoded participant address-specific, annual average DNL aircraft noise exposures in decibels (dB) from the contours on the day of blood draw and categorized individuals as exposed or non-exposed (DNL ≥ or < 45dB). We estimated the noise-related risk of CHIP as an odds ratio, 95% confidence interval (OR, 95% CI) using logistic regression before and after adjustment for age, race / ethnicity, case-control status, smoking, alcohol use, body mass index, physical activity, hearing loss, education, and neighborhood socioeconomic status. Results: Among this population of postmenopausal women (Mean age: 68.7 years; White: 82.1%; African American: 12.3%), 19.4% were exposed to aircraft noise, and 8.4% had CHIP. The age-adjusted CHIP proportion was higher among whites (8.1%) than African Americans (7.3%) and other racial/ethnic groups (6.4%). CHIP was also more common among women aged ≥ 70 years (11.5%) than those aged 60-69 years (7.5%) or 50-59 years (3.8%). Compared to non-exposed women, those exposed to aircraft noise were not at increased risk of CHIP: OR unadjusted (95% CI) = 0.95 (0.80, 1.14) and OR adjusted (95% CI) = 0.97 (0.81, 1.16). Results were insensitive to dichotomization of noise exposure at 55 dB and further exclusion of women with hearing loss. Conclusion: Our study found no evidence of an association between aircraft noise and CHIP suggesting that aircraft noise may not be a factor contributing to CHIP-defining somatic mutations linked to CVD.