CHIP in Health and Disease

Background and Rationale:

Clonal hematopoiesis (CH), including clonal hematopoiesis of indeterminate potential (CHIP), arises from acquired somatic mutations in hematopoietic stem and progenitor cells that accumulate throughout life and drive clonal expansion. The most frequently affected genes include DNMT3A, TET2, and ASXL1. While such mutations are also involved in the pathogenesis of hematologic malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), progression to overt hematologic cancer occurs only in a minority of affected individuals.

In contrast, epidemiological studies have consistently demonstrated a strong association between clonal hematopoiesis and cardiovascular disease. Individuals carrying CHIP-associated mutations have a substantially increased risk of coronary artery disease, myocardial infarction, stroke, and cardiovascular mortality. The magnitude of risk conferred by CHIP has been reported to be comparable to established cardiovascular risk factors such as smoking or hypertension.

Experimental and mechanistic studies suggest that CH-associated mutations remodel immune-cell function through epigenetic reprogramming and activation of inflammatory pathways, including the NLRP3 inflammasome and cGAS-STING signaling. These alterations enhance the proliferation, activation, and inflammatory potential of myeloid immune cells, resulting in chronic sterile inflammation that can promote atherosclerosis, vascular injury, and adverse cardiac remodeling.

Importantly, clonal hematopoiesis is not considered a disease entity itself but rather a common biological state and independent risk factor for a range of age-related disorders. Since clonal hematopoiesis develops in varying degrees in a substantial proportion of the aging population, understanding its biological consequences represents a major opportunity for disease prevention.

Additional support for a causal role of inflammation comes from retrospective analyses of the CANTOS trial, which suggested that individuals carrying somatic TET2 mutations derived particularly strong benefit from anti-inflammatory treatment targeting interleukin-1β. These findings highlight the potential for precision prevention and targeted therapeutic interventions in CH-associated disease. However, a more comprehensive understanding of the cellular and molecular mechanisms linking clonal hematopoiesis to disease development is required for improved risk stratification and the development of novel therapeutic approaches.

Study Objectives:

The primary objective of this study is to define the inflammatory and immunological landscape across the biological continuum of clonal hematopoiesis, from healthy individuals without detectable CH to individuals with high-risk CH and patients with CH-associated hematologic or cardiovascular disease.

Specific objectives include:

• High-resolution characterization of immune-cell composition and activation states across different stages of clonal hematopoiesis.
• Identification of mutation-associated transcriptional, proteomic, and functional alterations, particularly within the myeloid compartment.
• Determination of shared and disease-specific inflammatory pathways associated with progression from CH to clinical hematologic or cardiovascular outcomes.
• Generation of mechanistic hypotheses and therapeutic targets for future anti-clonal and anti-inflammatory interventions.

Study Design:

This is a prospective observational translational research study.

A total of six participant groups will be recruited, with approximately 20 participants per group (total n ≈ 120). The groups will represent different stages along the spectrum of clonal hematopoiesis and disease manifestation, including:

• Healthy individuals without detectable CH or with low-risk molecular profiles.
• Individuals with high-risk clonal hematopoiesis based on established molecular risk characteristics.
• Patients with clonal cytopenia of undetermined significance (CCUS).
• Patients with lower-risk myelodysplastic syndromes (MDS).
• Patients with ST-segment elevation myocardial infarction (STEMI).
• An additional comparator group representing the defined CH-risk spectrum according to study-specific inclusion criteria.

Study Procedures:

Participants will provide peripheral whole-blood samples for comprehensive molecular, cellular, and functional analyses. Planned investigations include:

• UMI-based targeted DNA sequencing for detection and quantification of somatic variants in genes associated with clonal hematopoiesis.
• Single-cell RNA sequencing (scRNA-seq) of freshly collected whole blood combined with single-cell genotyping approaches (Genotyping of Transcriptomes) to link somatic mutations with cellular phenotypes.
• Functional ex vivo immune stimulation assays using freshly collected blood samples.
• Proteomic profiling under baseline and stimulated conditions.
• Flow cytometric characterization of immune-cell composition and activation states.
• Isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) for downstream validation studies.
• Storage of plasma specimens for future analyses, including multiplex cytokine profiling, proteomics, metabolomics, lipidomics, and isolation of extracellular vesicles from platelet-poor plasma.

Expected Impact:

The study is designed to establish a comprehensive map of inflammatory and immune mechanisms associated with clonal hematopoiesis across its natural history. By defining the biological pathways that connect somatic mutations to hematologic and cardiovascular disease, the project aims to support the development of improved risk stratification tools and enable future preventive, anti-clonal, and anti-inflammatory therapeutic strategies. Ultimately, these efforts may contribute to preventing or delaying progression from healthy aging through clonal hematopoiesis to clinically manifest hematologic and cardiovascular disease.

Inclusion criteria

Participants must be ≥18 years of age and meet at least one of the following criteria:

• Written informed consent has been obtained for participation in the study "Deconvolution of Clonal Hematopoiesis-Associated Inflammation in Health and Disease"; OR
• Participant is enrolled in the Inn.Health study and has provided written informed consent; OR
• Participant is enrolled in the study "Clonal Hematopoiesis of Indeterminate Potential and Infarct Severity in ST-Elevation Myocardial Infarction" and has provided written informed consent.

Group A: Control Group (n=20)

• Age ≥60 years
• No detectable somatic variant identified by peripheral blood next-generation sequencing (NGS)
• No history of stroke or myocardial infarction
• No surgery within the previous 3 months
• No diagnosis of a WHO-defined neoplasm
• No cytopenia at study enrollment Group B: Low-Risk Clonal Hematopoiesis Risk Score (CHRS) Group (n=20)
• Age ≥60 years
• Somatic variant in DNMT3A, TET2, ASXL1, or JAK2 with variant allele frequency (VAF) ≥2% in peripheral blood
• No history of stroke or myocardial infarction
• No surgery within the previous 3 months
• No diagnosis of a WHO-defined neoplasm
• No cytopenia at study enrollment
• Low-risk CHRS (<9.5 points) Group C: Intermediate-/High-Risk CHRS Group (n=20)
• Age ≥60 years
• Somatic variant in DNMT3A, TET2, ASXL1, or JAK2 with VAF ≥2% in peripheral blood
• No history of stroke or myocardial infarction
• No surgery within the previous 3 months
• No diagnosis of a WHO-defined neoplasm
• No cytopenia at study enrollment
• Intermediate- or high-risk CHRS (>9.5 points) Group D: Clonal Cytopenia of Undetermined Significance (CCUS) Group (n=20)
• Age ≥60 years
• Somatic variant in DNMT3A, TET2, ASXL1, or JAK2 with VAF ≥2% in peripheral blood
• No history of stroke or myocardial infarction
• No surgery within the previous 3 months
• No diagnosis of a WHO-defined neoplasm
• Untreated, unexplained cytopenia present for ≥4 months
• Hemoglobin <13 g/dL (men) or <12 g/dL (women), and/or absolute neutrophil count <1.8 × 10⁹/L, and/or platelet count <150 × 10⁹/L
• No diagnostic criteria for a defined myeloid neoplasm based on bone marrow examination Group E: Cardiovascular Disease Group (n=20)
• Age ≥60 years
• Somatic variant in DNMT3A, TET2, ASXL1, or JAK2 with VAF ≥2% in peripheral blood
• ST-segment elevation myocardial infarction (STEMI) treated with percutaneous coronary intervention (PCI) within the previous 4 months
• No history of coronary artery bypass grafting (CABG)
• No history of stroke
• No cytopenia at study enrollment
• No diagnosis of a WHO-defined neoplasm Group F: Low-Risk Myelodysplastic Syndrome (MDS) Group (n=20)
• Age ≥60 years
• Untreated, newly diagnosed (<3 months from diagnosis) low-risk myelodysplastic syndrome according to the Revised International Prognostic Scoring System (IPSS-R score >1.5 to 3.0)
• No history of stroke or myocardial infarction

Exclusion criteria

• Treatment with immunosuppressive medication within the previous 4 weeks, including but not limited to systemic corticosteroids, methotrexate, other disease-modifying antirheumatic drugs (DMARDs), colchicine, TNF-α inhibitors, mTOR inhibitors, calcineurin inhibitors, or immunomodulatory antibodies
• History of rheumatologic, autoinflammatory, or autoimmune disease