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. 2020 Jul 8;5(10):1–6. doi: 10.1001/jamacardio.2020.2468

Association of Clonal Hematopoiesis of Indeterminate Potential With Inflammatory Gene Expression in Patients With Severe Degenerative Aortic Valve Stenosis or Chronic Postischemic Heart Failure

Wesley Tyler Abplanalp 1,3, Silvia Mas-Peiro 2,3, Sebastian Cremer 2,3, David John 1, Stefanie Dimmeler 1,3,4, Andreas M Zeiher 2,3,4,5,✉
PMCID: PMC7344831  PMID: 32639511

Key Points

Question

What is the association of clonal hematopoiesis of indeterminate potential (CHIP) with the transcriptome of human circulating monocytes in patients with cardiovascular disease?

Findings

This observational, single-cell RNA–sequencing diagnostic study found that peripheral blood monocytes of patients with severe degenerative aortic valve stenosis or chronic postischemic heart failure who carry DNMT3A or TET2 CHIP-driver sequence variations displayed increased expression of proinflammatory cytokines, interleukin 6 receptor, and cellular receptor CD163, as well as the NLRP3 inflammasome complex and other genes involved in cytokine release syndrome.

Meaning

Monocytes of individuals who carry CHIP-driver sequence variations and have cardiovascular disease may be primed for excessive inflammatory responses and sensitized for cytokine release syndrome.

This diagnostic study aims to (1) determine if clonal hematopoiesis of indeterminate potential is associated with an inflammatory gene expression that sensitizes monocytes to aggravated immune responses and (2) generate hypotheses on the relevance of this to coronavirus disease 2019 infection and cytokine release syndrome.

Abstract

Importance

Cytokine release syndrome is a complication of coronavirus disease 2019. Clinically, advanced age and cardiovascular comorbidities are the most important risk factors.

Objective

To determine whether clonal hematopoiesis of indeterminate potential (CHIP), an age-associated condition with excess cardiovascular risk defined as the presence of an expanded, mutated somatic blood cell clone in persons without other hematological abnormalities, may be associated with an inflammatory gene expression sensitizing monocytes to aggravated immune responses.

Design, Setting, and Participants

This hypothesis-generating diagnostic study examined a cohort of patients with severe degenerative aortic valve stenosis or chronic postinfarction heart failure, as well as age-matched healthy control participants. Single-cell RNA sequencing and analyses of circulating peripheral monocytes was done between 2017 and 2019 to assess the transcriptome of circulating monocytes.

Exposures

Severe degenerative aortic valve stenosis or chronic postinfarction heart failure.

Main Outcomes and Measures

CHIP-driver sequence variations in monocytes with a proinflammatory signature of genes involved in cytokine release syndrome.

Results

The study included 8 patients with severe degenerative aortic valve stenosis, 6 with chronic postinfarction heart failure, and 3 healthy control participants. Their mean age was 75.7 (range, 54-89) years, and 6 were women. Mean CHIP-driver gene variant allele frequency was 4.2% (range, 2.5%-6.9%) for DNMT3A and 14.3% (range, 2.6%-37.4%) for TET2. Participants with DNMT3A or TET2 CHIP-driver sequence variations displayed increased expression of interleukin 1β (no CHIP-driver sequence variations, 1.6217 normalized Unique Molecular Identifiers [nUMI]; DNMT3A, 5.3956 nUMI; P < .001; TET2, 10.8216 nUMI; P < .001), the interleukin 6 receptor (no CHIP-driver sequence variations, 0.5386 nUMI; DNMT3A, 0.9162 nUMI; P < .001;TET2, 0.5738 nUMI; P < .001), as well as the NLRP3 inflammasome complex (no CHIP-driver sequence variations, 0.4797 nUMI; DNMT3A, 0.9961 nUMI; P < .001; TET2, 1.2189 nUMI; P < .001), plus upregulation of CD163 (no CHIP-driver sequence variations, 0.5239 nUMI; DNMT3A, 1.4722 nUMI; P < .001; TET2, 1.0684 nUMI; P < .001), a cellular receptor capable of mediating infection, macrophage activation syndrome, and other genes involved in cytokine response syndrome. Gene ontology term analyses of regulated genes revealed that the most significantly upregulated genes encode for leukocyte-activation and interleukin-signaling pathways in monocytes of individuals with DNMT3A (myeloid leukocyte activation: log[Q value], −50.1986; log P value, −54.5177; regulation of cytokine production: log[Q value], −21.0264; log P value, −24.1993; signaling by interleukins: log[Q value], −18.0710: log P value, −21.1597) or TET2 CHIP-driver sequence variations (immune response: log[Q value], −36.3673; log P value, −40.6864; regulation of cytokine production: log[Q value], −13.1733; log P value, −16.3463; signaling by interleukins: log[Q value], −12.6547: log P value, −15.7977).

Conclusions and Relevance

Monocytes of individuals who carry CHIP-driver sequence variations and have cardiovascular disease appear to be primed for excessive inflammatory responses. Further studies are warranted to address potential adverse outcomes of coronavirus disease 2019 in patients with CHIP-driver sequence variations.

Introduction

The novel coronavirus designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which initially manifests by fever and pneumonia and leads to acute respiratory distress syndrome in the most severe cases. Previous epidemics with members of the coronavirus family demonstrated cytokine release syndrome (CRS) to be the major cause of mortality and morbidity in patients infected with SARS-CoV and Middle Eastern respiratory syndrome virus.1 Elevated serum concentrations of the cytokine interleukin 6 (IL-6) and other proinflammatory cytokines are hallmarks of CRS.2 Indeed, CRS is also common in patients with COVID-19, and most importantly, serum levels of the prototypical marker IL-6 correlate with acute respiratory distress syndrome and are associated with adverse clinical outcomes in patients infected with SARS-CoV-2.3,4 Clinically, by far the most important risk indicators for adverse outcomes in COVID-19 are advanced age and the presence of cardiovascular comorbidities.5

Clonal hematopoiesis of indeterminate potential (CHIP), defined as the presence of an expanded somatic blood cell clone because of acquired leukemic sequence variations in persons without other hematological abnormalities,6 increases with age7 and is also termed age-related clonal hematopoiesis. Importantly, CHIP was shown to be associated with cardiovascular diseases, such as coronary artery disease,8 chronic heart failure (HF),9 and severe calcified aortic valve stenosis.10 The CHIP-driver sequence variations DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and Tet methylcytosine dioxygenase 2 (TET2) are the most common age-dependent sequence variations contributing to CHIP and account for almost 70% of all acquired sequence variations associated with clonal expansion of hematological cells.7 Experimental studies provided robust mechanistic evidence that sequence variations in either DNMT3A or TET2 are accompanied by increased inflammation because of activation of the inflammasome complex, which raises IL-6 and IL-1β expression in murine hematopoietic cells.8,11,12 Most recently, genetically reduced IL-6 signaling by a frequently occurring sequence variation of the IL-6 receptor was associated with a reduced risk for cardiovascular events in individuals who carry DNMT3A or TET2 CHIP-driver sequence variations.13

Because CHIP is an important link between aging and inflammation in cardiovascular disease, we hypothesize that patients with cardiovascular disease harboring DNMT3A or TET2 CHIP-driver sequence variations are sensitized for inflammatory activation, which makes them susceptible to the development of CRS. To support such reasoning, we applied single-cell sequencing analyses to find an activation of the inflammatory cascade in circulating monocytes of patients with degenerative aortic valve stenosis and chronic ischemic HF.

Methods

We analyzed single-cell sequencing signatures of gene expression in circulating monocytes obtained from patients with severe degenerative aortic valve stenosis or chronic postischemic HF, as well as age-matched, healthy control participants. Patients with evidence for acute inflammatory or hematological disease were excluded, and all patients were recruited prior to the outbreak of COVID-19 in Europe (December 2017, December 2018, August 2019, and November 2019). All patients provided written informed consent for the study. The ethics review board of the Goethe University of Frankfurt, Germany, approved the protocol, and the study complies with the Declaration of Helsinki.

The CHIP sequence variations were identified by targeted next-generation sequencing of TET2 and DNMT3A in samples sent to MLL Dx GmbH (mean coverage ×2147) for patients with aortic valve stenosis or sequencing of 56 CHIP-driver gene sequence variations for patients with chronic HF (mean coverage ×4282), as previously described.9,10 A variant allele frequency of more than 2% was set as the cut-off.

For single-cell RNA sequencing, blood was obtained from patients and centrifuged on a Ficoll gradient, and mononuclear cells (whole or monocyte enriched) were used for droplet single-cell RNA sequencing (with Chromium Controller with Chromium Next GEM Single Cell 3’ GEM, Library and Gel Bead Kit version 3.1 reagents [10X Genomics]), as previously described.14 All single-cell RNA sequencing libraries were prepared using Chromium Single Cell 3′ version 2 or version 3 Reagent Kits (10X Genomics) per the manufacturer’s protocol, and libraries were sequenced using paired-end sequencing in samples sent to GenomeScan. Single-cell expression data were processed using the Cell Ranger Single Cell Software Suite version 3 (10X Genomics) to perform quality control, sample demultiplexing, barcode processing, and single-cell 3′ gene counting and were then aligned to the human reference genome GRCh38.

Data integration was performed by Seurat versions 2 and 3 (Satija Lab) and statistical analysis of differential expression of genes was done with the FindMarkers function in the Seurat package. Gene ontology terms were generated using the functional annotation tool Metascape (Metascape Team). The significance threshold was set to an adjusted P value of .001 (after Bonferroni correction) for all single-cell RNA-sequencing gene expression comparisons, and a P value of .05 was used as a significance threshold for inclusion of gene ontology and Reactome terms provided by Metascape.

Results

Single-cell RNA sequencing analyses of circulating peripheral monocytes were performed in 8 patients with severe degenerative aortic valve stenosis, 6 patients with chronic postinfarction HF, and 3 age-matched, healthy control participants (Figure 1). The mean age of the patients was 75.7 (range, 54-89) years, and 6 patients were female. Mean CHIP-driver gene variant allele frequency was 4.2% (range, 2.5%-6.9%) for DNMT3A and 14.3% (range, 2.6%-37.4%) for TET2. As illustrated in Figure 1, peripheral blood monocytes of individuals with aortic valve stenosis who carried DNMT3A or TET2 CHIP-driver sequence variations displayed increased expression of IL-1β (IL1B; no CHIP-driver sequence variations, 1.6217 normalized Unique Molecular Identifiers [nUMI]; DNMT3A, 5.3956 nUMI; P < .001; TET2, 10.8216 nUMI; P < .001), the IL-6 receptor (IL6R; no CHIP-driver sequence variations, 0.5386 nUMI; DNMT3A, 0.9162 nUMI; P < .001; TET2, 0.5738 nUMI; P < .001), as well as the NOD-, LRR- and pyrin domain-containing protein 3 inflammasome complex (NLRP3; no CHIP-driver sequence variations, 0.4797 nUMI; DNMT3A, 0.9961 nUMI; P < .001; TET2, 1.2189 nUMI; P < .001).

Figure 1. Single-Cell RNA Sequencing Analysis Showing Relative Expression of Genes Associated With Inflammatory Signaling in TET2 and DNMT3A Mutant Carriers.

Figure 1.

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A, Expression of indicated genes in circulating monocytes of patients with aortic valve stenosis with DNMT3A (n = 3; 2 were women); TET2 (n = 3; 2 were women) or age-matched controls with no DNMT3A or TET2 CHIP sequence variations (n = 2; 1 was a woman) shown in violin plots or with individual points representing individual cells. Violin plots are similar to box plots, except that they also show the probability density of the data at different values. B, Expression of indicated genes in circulating monocytes of patients with chronic postinfarction heart failure (HF; n = 4; 1 was a woman), heart failure with DNMT3A sequence variation (HF+DNMT3A; n = 2; 0 were female) and age-matched healthy controls (n = 3; 0 were female). Monocyte clustering and data analysis were performed as previously described.14 Data are expressed as violin plots. In single-cell RNA sequencing analysis, mean expression values include the many 0 values that occur becuase of low expression or gene dropout. The trending change is still the same; however, the violin plot provides easy visualization of density and range for zero and nonzero values. Normalized Unique Molecular Identifiers are the counts of the transcripts detected in the sequencing. Statistical analysis was performed by using the FindMarkers function in the analysis tool Seurat versions 2 and 3 (Satija Lab) for differential single-cell gene expression.

aAdjusted P value <.001. CCL indicates CC chemokine ligand; IL6R, interleukin-6 receptor; IL6ST, interleukin-6 receptor subunit β; NLRP3, NOD-, LRR- and pyrin domain-containing protein 3 inflammasome complex.

Gene expression was similarly altered in individuals with HF with vs without DNMT3A sequence variations in IL6 expression (individuals with HF and DNMT3A sequence variation, 0.1628 nUMI; individuals with HF: 0.1253 nUMI; P < .001; healthy individuals, 0 nUMI; P < .001), IL1B expression (individuals with HF and DNMT3A sequence variation, 19.6110 nUMI; individuals with HF: 9.7807 nUMI; P < .001; healthy individuals: 0.6146 nUMI; P < .001), IL6R expression (individuals with HF and DNMT3A sequence variation, 0.3633 nUMI; individuals with HF: 0.2754 nUMI; P < .001), NLRP3 expression (individuals with HF and DNMT3A sequence variation: 2.0952 nUMI; individuals with HF: 1.3285 nUMI; P < .001; healthy individuals: 0.6546 nUMI; P < .001).

Importantly, expression of CD163, a cellular receptor capable of mediating infection and macrophage activation syndrome, and other genes involved in CRS are upregulated in monocytes obtained from individuals who carry DNMT3A or TET2 CHIP-driver sequence variations (no CHIP-driver sequence variations, 0.5239 nUMI; DNMT3A, 1.4721 nUMI; P < .001; TET2, 1.0684 nUMI; P < .001) and individuals with HF with or without DNMT3A (individuals with HF and DNMT3A sequence variation, 1.0469 nUMI; individuals with HF, 0.3966 nUMI; P < .001; healthy individuals, 0.3965 nUMI; P < .001) (Figure 1A and B).

Finally, exploring the top regulated genes by gene ontology term analyses revealed that the most significantly upregulated genes in monocytes derived from individuals who carried DNMT3A or TET2 CHIP-driver sequence variations that encode for leukocyte-activation and interleukin-signaling pathways (Figure 2). Terms upregulated in DNMT3A were for myeloid leukocyte activation (GO Biological Processes, GO:0002274, log[Q value], −50.1986; log P value, −54.5177), regulation of cytokine production (GO Biological Processes, GO:0001817: log[Q value], −21.0264; log P value, −24.1993), and signaling by interleukins (Reactome Gene Sets, R-HSA-449147: log[Q value], −18.0710: log P value, −21.1597) (Figure 2A and B). Terms upregulated in TET2 included cell activation involved in immune response (GO Biological Processes, GO:0002263; log[Q value], −36.3673; log P value, −40.6864), regulation of cytokine production (GO Biological Processes, GO:0001817: log[Q value], −13.1733; log P value, −16.3463), and signaling by interleukins (Reactome Gene Sets, R-HSA-449147: log[Q value], −12.6547: log P value, −15.7977).

Figure 2. Gene Ontology (GO) Term Analysis.

Figure 2.

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A and B, Gene ontology term analysis of top 1000 significantly (adjusted P value <.05) regulated genes in monocytes derived from patients with aortic valve stenosis with DNMT3A (A) or TET2 (B) clonal hematopoiesis of indeterminate potential (CHIP)–driver sequence variations, compared with participants with no CHIP-driver sequence variations. R-HSA indicates Reactome–Homo Sapiens.

Discussion

The results of this study demonstrate that individuals who carried monocytes of CHIP-driver sequence variation and had cardiovascular disease appear to be primed for excessive inflammatory responses. In line with our previous studies,9,10 we did not observe increased plasma levels of IL-6 or high-sensitivity C-reactive protein in this cohort of patients, who were free of acute inflammatory disease. However, we do hypothesize that the degree of evoked cytokine expression that might happen in patients with COVID-19 will be profoundly amplified in individuals who carry DNMT3A or TET2 CHIP-driver sequence variations. Indeed, a recent study reported IL-6–mediated sustained cytokine production and hyperinflammation produced by monocytes, even in the presence of lymphopenia as a unique signature of immune dysregulation in patients with severe COVID-19.15 Taken together, the apparent sensitization of monocytes in individuals who carry DNMT3A and TET2 CHIP-driver sequence variations may provide a putative explanation for the increased morbidity and mortality of COVID-19 in elderly patients and patients with cardiovascular disease.

Limitations

Although single-cell sequencing is an extremely powerful method to decipher the transcriptional signatures of individual cells in an unbiased manner, its very high costs prohibit large-scale studies using this technique. Therefore, the present study is limited by its small sample size and should be regarded as hypothesis generating.

Conclusions

Further implications of a potential mechanistic link between the presence of CHIP and an exaggerated CRS may be 2-fold: first, patients known to harbor DNMT3A or TET2 CHIP-driver sequence variations might be identified as high risk for adverse outcomes of COVID-19, and second, patients infected with SARS-CoV-2 could be tested for the presence of DNMT3A or TET2 CHIP-driver sequence variations to personalize treatment strategies with IL-6 or IL-6R antagonists to mitigate CRS, because preliminary results of clinical trials so far provided inconclusive results for IL-6–blocking therapies in patients with COVID-19. Thus, a proposed hypothesis linking CHIP with adverse outcomes of COVID-19 could be immediately tested by interrogating existing, large-scale biobanks for the prevalence of CHIP sequence variations in patients with COVID-19, as well as by establishing prospective biorepositories to address a potential adverse outcome of COVID-19 in patients with CHIP-driver sequence variations.

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