Association between CHIP and Coronary Heart Disease

In the samples obtained from the BioImage and MDC studies, we found that the somatic mutations most commonly occurred in the genes DNMT3A, TET2, and ASXL1 and that 72 of 77 participants (94%) with CHIP had a mutation in only a single driver gene1,2 (Table S4 and Fig. S1 in the Supplementary Appendix).

Figure 1. Figure 1. Association between Clonal Hematopoiesis of Indeterminate Potential (CHIP) and Coronary Heart Disease and Early-Onset Myocardial Infarction. Panel A shows a forest plot of hazard ratios for the association between coronary heart disease and CHIP in the BioImage and Malmö Diet and Cancer (MDC) studies. Hazard ratios for coronary heart disease among study participants with CHIP mutations were obtained with the use of a Cox proportional-hazards model after adjustment for age, sex, type 2 diabetes status, total cholesterol, high-density lipoprotein (HDL) cholesterol, smoking status, and hypertension. Panel B shows a forest plot of odds ratios for the association between myocardial infarction and CHIP in the Atherosclerosis, Thrombosis, and Vascular Biology Italian Study Group (ATVB) and the Pakistan Risk of Myocardial Infarction Study (PROMIS). The odds ratios were obtained with the use of a logistic-regression model after adjustment for age, sex, type 2 diabetes status, and smoking status.

The median age of the participants in BioImage at the time of DNA sample collection was 70 years, and the median follow-up time was 2.6 years. We found that 19 of 113 participants with coronary heart disease (17%) were CHIP carriers versus 25 of 257 controls (10%) (hazard ratio, 1.8; 95% confidence interval [CI], 1.1 to 2.9; P=0.03). The median age of the participants in MDC was 60 years at the time of DNA sample collection, and the median follow-up was 17.7 years. CHIP was identified in 21 of 320 participants with coronary heart disease (7%) versus in 12 of 320 controls (4%) (hazard ratio, 2.0; 95% CI, 1.2 to 3.1; P=0.003) (Figure 1A, and Table S5 and Fig. S2 in the Supplementary Appendix). The combined analysis of the two cohorts in a fixed-effects meta-analysis showed that the CHIP carriers had a risk of incident coronary heart disease that was 1.9 times as great as in noncarriers (95% CI, 1.4 to 2.7; P<0.001).

Association between CHIP and Early-Onset Myocardial Infarction

In both ATVB and PROMIS, participants with early-onset myocardial infarction had marked enrichment of CHIP, as compared with controls. In ATVB, 37 of 1753 participants with myocardial infarction (2%) were CHIP carriers versus 6 of 1583 controls (<1%) (odds ratio, 5.4; 95% CI, 2.3 to 13.0; P<0.001). In PROMIS, 52 of 2540 participants with myocardial infarction (2%) were CHIP carriers versus 13 of 1369 controls (1%) (odds ratio, 3.4; 95% CI, 1.8 to 6.5; P<0.001). A combined fixed-effects meta-analysis of these two cohorts showed that CHIP was associated with an odds ratio of 4.0 (95% CI, 2.4 to 6.7; P<0.001) for early-onset myocardial infarction (Figure 1B).

Association between Risk Mutations and Coronary Events

Figure 2. Figure 2. Association between Coronary Heart Disease and Early-Onset Myocardial Infarction among CHIP Carriers, According to Genetic Mutation. Panel A shows a forest plot of hazard ratios for the risk of coronary heart disease in BioImage, MDC, and three prospective cohorts that were unselected for coronary events: the Jackson Heart Study (JHS), Finland–United States Investigation of NIDDM [Non–Insulin-Dependent Diabetes Mellitus] Genetics (FUSION), and the Framingham Heart Study (FHS), according to mutated gene. Hazard ratios for the listed mutations were obtained by a fixed-effects meta-analysis of Cox proportional-hazards models after adjustment for age, sex, type 2 diabetes status, total cholesterol, HDL cholesterol, triglycerides, smoking status, and hypertension. Panel B shows the risk of early-onset myocardial infarction among the participants in ATVB and PROMIS (combined analysis), according to the mutated gene. The odds ratios for myocardial infarction were obtained with the use of Fisher’s exact test; P values were not adjusted for multiple hypothesis testing. Panel C shows the proportion of total mutations (according to gene) among participants with myocardial infarction in the ATVB and PROMIS studies, as compared with those with coronary heart disease in the BioImage and MDC studies.

In samples obtained from BioImage, MDC, and the three prospective cohorts unselected for coronary events, we specifically tested for associations between coronary heart disease and mutations in DNMT3A, TET2, ASXL1, and JAK2. Participants with mutations in DNMT3A, TET2, and ASXL1 had 1.7 to 2.0 times the risk of incident coronary heart disease as did those with no mutations, whereas the JAK2 V617F mutation was associated with 12.1 times the risk (Figure 2A).

Mutations in TET2, JAK2, and ASXL1 also showed significant enrichment among participants with early-onset myocardial infarction in samples obtained from ATVB and PROMIS, with myocardial infarction identified in 12 of 13 participants with TET2 mutations, in 8 of 8 participants with ASXL1 mutations, and in 16 of 16 participants with JAK2 mutations (Figure 2B). JAK2 V617F accounted for 19% of the total mutations among patients with myocardial infarction in ATVB and PROMIS but for only 4% in BioImage and MDC (Figure 2C).

Association between CHIP and Coronary-Artery Calcification

Figure 3. Figure 3. Association between CHIP and Increased Coronary-Artery Calcification. Panel A shows coronary-artery calcification (CAC) scores in Agatston units among participants with CHIP and those without CHIP in the BioImage study, stratified according to the presence or absence of incident coronary heart disease. The horizontal line in each box indicates the median score, and the top and bottom of the boxes indicate the 75th and 25th percentiles, respectively. Among the participants with no coronary heart disease, the median scores were 92 for no CHIP and 306 for CHIP; among those with coronary heart disease, the median scores were 355 and 650, respectively. P=0.03 for the comparison between CHIP and no CHIP; the P value was obtained with the use of a linear-regression model of logarithm-transformed CAC plus 1, after adjustment for incident coronary heart disease status. Panel B shows a forest plot of odds ratios for the association between a CAC score of 615 or more and mutation status, stratified according to the variant allele fraction (VAF) among participants with incident coronary heart disease in BioImage. The odds ratios were obtained with the use of a logistic-regression model after adjustment for age, sex, type 2 diabetes status, total cholesterol, HDL cholesterol, smoking status, and hypertension. P=0.02 for heterogeneity between a VAF of less than 0.10 and a VAF of 0.10 or more.

We hypothesized that an increased atherosclerosis burden drives the association between CHIP and coronary heart disease, rather than other factors that might cause myocardial infarction, such as increased thrombosis or vasospasm. Among the participants in the BioImage study, we assessed data on coronary-artery calcification, a noninvasive measure of atherosclerosis as detected on cardiac computed tomography. Among those without incident coronary heart disease, CHIP carriers had a median score for coronary-artery calcification that was 3.3 times as high as that in noncarriers (306 versus 92 Agatston units); in those with incident coronary heart disease, the score was 1.8 times as high in CHIP carriers as in noncarriers (650 versus 355 Agatston units) (P=0.03 by linear regression after adjustment for incident coronary heart disease) (Figure 3A).

A coronary-artery calcification score of 615 Agatston units or more has served as an empirical cutoff for identifying older persons at high risk for coronary events.18 Among the participants without incident coronary heart disease, CHIP carriers were 3.0 times as likely to have a coronary-artery calcification score of 615 or more than were noncarriers (P=0.05 by logistic regression after adjustment for age, sex, type 2 diabetes status, total cholesterol, HDL cholesterol, hypertension, and smoking status) (Figure 3B).

We had previously found that the presence of a CHIP clone with a variant allele fraction of at least 10% (which corresponds to ≥20% of nucleated blood cells harboring the mutation) was associated with a greater risk of a hematologic cancer than a CHIP clone below this size.1 Therefore, we tested whether CHIP with a larger clone size was also associated with a greater burden of atherosclerosis. CHIP carriers without incident coronary heart disease but with a variant allele fraction of a least 10% had 12 times the risk of having a coronary-artery calcification score of 615 or more as did noncarriers (P=0.002 by logistic regression after adjustment), whereas participants with a variant allele fraction of less than 10% had no increased risk (P=0.02 for heterogeneity) (Figure 3B, and Fig. S3 in the Supplementary Appendix).

We hypothesized that the participants with an increased proportion of mutated cells might also have a greater risk of incident coronary heart disease. In a meta-analysis of data from BioImage and the three prospective cohorts unselected for coronary events, we found that the risk of incident coronary heart disease was 2.2 times as great among the participants with a variant allele fraction of at least 10% as among those without mutations (P<0.001 by Cox proportional hazards after adjustment), whereas those with a variant allele fraction of less than 10% had a risk that was 1.4 times as great (P=0.16; P=0.24 for heterogeneity) (Fig. S4 in the Supplementary Appendix). In this analysis, data from MDC were not included because DNA in this cohort was obtained from granulocytes rather than whole blood, which would probably inflate the variant allele fraction.

Tet2 Knockout Mice and Accelerated Atherosclerosis

Having established a correlation between CHIP and coronary heart disease, we next sought to assess causality experimentally. We selected Tet2 for further study because it is the second most commonly mutated gene in CHIP and has been associated with the risk of coronary heart disease regardless of age. In previous studies, hematopoietic stem cells from mice that are homozygous for loss of function of Tet2 in all hematopoietic cells recapitulate the clonal advantage of mutated TET2 hematopoietic cells seen in humans.6

Figure 4. Figure 4. Loss of Tet2 in Hematopoietic Cells and Atherosclerosis in a Murine Model. Shown are the effects of the transplantation of bone marrow into female, atherosclerosis-prone Ldlr knockout mice, according to whether the donor mice had wild-type (WT) Tet2, heterozygous (HET) Tet2, or knockout (KO) Tet2. Deletions in Tet2 were obtained by using Cre recombinase expressed from the Vav1 promoter. Panel A shows aortic-root sections obtained from mice that had received transplants from WT or KO Tet2 mice after the mice had received a high-cholesterol diet for 5 weeks and 9 weeks (oil red O staining at 5 weeks and Masson’s trichrome staining at 9 weeks). The dashed lines indicate the lesion areas. Panel B shows the quantification of aortic-root lesions in mice that had received transplants from WT or KO Tet2 mice at 5 weeks and 9 weeks. P values were obtained with the use of the Wilcoxon rank-sum test. Panel C shows lesions in the descending aorta that were stained with oil red O at 17 weeks in mice that had received transplants from WT, HET, or KO Tet2 mice. The amount of red dye that is visible indicates the degree of atherosclerosis, according to Tet2 status. Panel D shows the quantification of lesions in the descending aorta at 17 weeks, according to Tet2 status. P values were obtained with the use of Dunn’s Kruskal–Wallis test for multiple comparisons and the Benjamini–Hochberg correction. In Panels B and D, the black horizontal lines represent the median values.

We transplanted bone marrow from these mice and from control mice into irradiated, atherosclerosis-prone Ldlr knockout mice19 and initiated a high-cholesterol diet after allowing time for hematopoietic reconstitution. As compared with mice that had received control bone marrow, mice that had received bone marrow from the Tet2 knockout mice had a median lesion size in the aortic root that was 2.0 times as large after 5 weeks (P=0.02 by the Wilcoxon rank-sum test), that was 1.7 times as large after 9 weeks (P=0.01 by the Wilcoxon rank-sum test), and that was 1.4 times as large after 13 weeks (P=0.03 by Dunn’s test) (Figure 4A and 4B, and Fig. S5A and S5B in the Supplementary Appendix). By 17 weeks, the median lesion size in the descending aorta was 2.7 times as large in the mice that had received bone marrow from Tet2 knockout mice as in those that had received control bone marrow (P=0.02 by Dunn’s test) (Figure 4C and 4D).

Most humans with TET2-associated CHIP have a mutation in only a single allele of the gene.1,2 Therefore, we also tested the phenotype of Tet2 haploinsufficiency. Ldlr knockout mice receiving bone marrow from mice that were heterozygous for the Tet2 deletion had a median aortic-root lesion size that was 1.4 times as large as that in control mice after 13 weeks on the high-cholesterol diet (P=0.05 by Dunn’s test) (Fig. S5A and S5B in the Supplementary Appendix); after 17 weeks, the median lesion size in the descending aorta was 2.7 times as large (P=0.03 by Dunn’s test) (Figure 4C and 4D).

Mice receiving bone marrow from Tet2 knockout mice had normal levels of peripheral-blood white cells and a normal differential count during the study period. There also was no significant between-group difference in fasting serum lipid levels after 17 weeks on the high-cholesterol diet (Table S6 in the Supplementary Appendix).

Loss of Tet2 Function in Myeloid Cells

The earliest stages of atherosclerosis involve monocyte infiltration into vessel walls and differentiation into macrophages.20 We hypothesized that Tet2 loss alters the function of macrophages (and their precursor monocytes) in plaques to enhance atherosclerosis. We tested this hypothesis by generating mice that lacked Tet2 in the majority of myeloid cells but not in other lineages; to create this murine line, we bred mice with loxP sites flanking exon 3 of Tet2 with mice that express Cre recombinase from the Lyz2 promoter.21 In Ldlr knockout mice that had received bone marrow from these mice, the mean size of the aortic-root lesion was 1.7 times as large as in mice that had received control bone marrow after 10 weeks on the high-cholesterol diet (P=0.003 by the Wilcoxon rank-sum test) (Fig. S5C in the Supplementary Appendix).

Tet2 catalyzes DNA hydroxymethylation,22 an epigenetic modification that can influence gene transcription. Therefore, we hypothesized that Tet2 modulated gene expression in macrophages exposed to excess cholesterol. We cultured bone marrow–derived macrophages from Tet2 knockout or control mice, incubated them with either vehicle or a pathophysiologically relevant dose of native LDL (200 mg per deciliter),23,24 and analyzed the transcriptome using messenger RNA sequencing. Gene set enrichment analysis revealed that the most significantly up-regulated functional class of genes in Tet2 knockout macrophages contained cytokines or chemokines and receptors, whereas the most significantly suppressed class contained genes involved in lysosomal function. Gene class annotations were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Tables S7 and S8 and Fig. S6 in the Supplementary Appendix).

We focused on the set of 217 genes that showed differential regulation by both loss of Tet2 and by LDL treatment. Transcript levels in Cxcl1, Cxcl2, Cxcl3, Pf4, Il1b, and Il6 were among the most highly induced in Tet2 knockout macrophages in this set (Fig. S7A and S7B in the Supplementary Appendix). Cxcl1, Cxcl2, Cxcl3, and Pf4 belong to a single C-X-C motif (CXC) chemokine gene cluster, whereas Il1b and Il6 are classic proinflammatory cytokine genes. Tet2 knockout macrophages also secreted more of these proteins in vitro in response to LDL loading or endotoxin exposure than did control macrophages. Although either LDL or endotoxin strongly induced the CXC chemokines, endotoxin but not LDL caused robust secretion of interleukin-1b and interleukin-6 (Fig. S7C in the Supplementary Appendix).

To assess the in vivo importance of these observations, we measured CXC chemokine levels in the transplanted mice after they had been on an atherogenic diet for 13 to 17 weeks. We found that the levels of Cxcl1, Cxcl2, Cxcl3, Pf4, and Ppbp were 2 to 4 times as high in the serum of mice that had received bone marrow from Tet2 knockout mice as in mice that had received control bone marrow, whereas those that had received bone marrow from mice that were heterozygous for Tet2 deletion showed intermediate levels (Fig. S7D in the Supplementary Appendix).

We also sought evidence of increased inflammation in other tissues. In mice that had received Tet2 knockout bone marrow, there was development of prominent xanthomas in the spleen and middle ear, marked foam-cell accumulation and glomerulosclerosis in the kidney, and large inflammatory infiltrates in the liver and lung (Fig. S8A and S8B in the Supplementary Appendix).

Because we found increased levels of CXC chemokines in the serum of mice that had received bone marrow from Tet2 knockout mice, we tested for an analogous increase in humans with TET2 clonal hematopoiesis. The prototypical CXC chemokine in humans is interleukin-8, which mice lack. Plasma levels of interleukin-8 were available from 2689 controls (age range, 40 to 82 years) in the PROMIS study. The 12 participants with TET2 mutations in this cohort had significantly higher circulating interleukin-8 levels than did those without the mutations (median level, 50 vs. 21 ng per milliliter; P=0.02 by the Wilcoxon rank-sum test on log-transformed values) (Fig. S8C in the Supplementary Appendix).