Background: With increasing age, an accumulation of sub-clinical clones of hematopoietic cells with somatic mutations in genes associated with myeloid neoplasia is observed. This condition is known as clonal hematopoiesis of indeterminate potential (CHIP). CHIP mutations have been reported to increase the risk of coronary artery disease and CHIP mutant macrophages are thought to be mediators of atherosclerotic pathomechanisms. In fact, macrophages with mutations in one of the two most common CHIP genes DNMT3A and TET2 have been shown to express increased levels of chemokines and inflammatory cytokines, to increase their interaction with endothelium, and to accelerate atherosclerosis in a murine disease model.

Aim: We analyzed blood and atherosclerotic arterial tissue of patients with peripheral artery disease (PAD) for CHIP mutations.

Methods: Patients (n=32) with PAD who underwent open vascular surgery to treat femoral artery stenosis and occlusion were informed about the study and gave written consent to participate. Peripheral blood, plaque tissue from the atherosclerotic femoral lesion, subcutaneous and perivascular tissue and collateral vessel samples were collected. At follow-up examination peripheral blood was withdrawn and used for magnetic antibody cell sorting of monocytes (CD14 positive) and progenitor cells (CD34 positive). DNA was isolated and CHIP was assessed using a next-generation sequencing approach targeting the most commonly mutated loci (DNMT3A, TET2, ASXL1, JAK2) covering more than 80% of the genomic loci currently implicated in CHIP.

Results: CHIP mutations were detected in 18 (56%) patients, seven of whom carried more than one mutation. The most commonly affected target genes were TET2 (12 mutations) and DNMT3A (8 mutations). In 14 of the 18 CHIP carriers, CHIP mutations were detected in tissue from the femoral lesion. Unexpectedly, seven patients carried CHIP mutations in DNA prepared from fat deposits. In blood samples, mutation frequencies were often increased in enriched cell populations but the pattern was heterogeneous, with mutations being detected in monocytes (7 cases), progenitor cells (7 cases) or in the remaining population (7 cases). In three patients, mutations were detectable in enriched blood populations or vessel material, but not in DNA from whole blood.

Conclusion: CHIP mutations are common in PAD patients, with TET2 mutations in this small PAD cohort apparently being more common than in CAD or hematological cohorts. Since these mutations can be detected in atherosclerotic lesions, an involvement of inflammatory cells affected by CHIP mutations in disease progression seems plausible. Detection of CHIP in subcutaneous depots of PAD patients indicates inflammatory invasion, which raises important questions concerning a potential association of subcutaneous fat tissue derived adipokines, CHIP and PAD pathomechanisms.