Somatic mutations reveal clonal cell populations in atherosclerotic plaques

Background In recent years, the potential involvement of clonal cell populations in atherosclerosis has gained renewed interest. Evidence from independent research groups unambiguously showed that smooth muscle cells clonally expand in experimental atherosclerosis, and clonal hematopoiesis of indeterminate potential (CHIP) has been proposed as a novel risk factor for atherosclerotic cardiovascular disease in humans. However, direct evidence for the presence of clonal cell populations within human atherosclerotic tissue is lacking. Here, we investigated whether characterizing the mutational landscape of human atherosclerotic plaques could provide insights into the role of clonal cell populations in human atherosclerosis. Methods and Results We conducted in-depth whole-exome sequencing of 65 samples derived from 13 carotid plaques obtained from patients undergoing carotid endarterectomy. This approach unveiled a landscape of somatic mutations exclusively confined to plaque tissue ( i.e. , not detected in patient-matched buffy coats). Leveraging variant allele frequencies, we inferred that distinct locally expanded clones often accounted for over 10% of the cell population within the plaque samples, and certain clones extended across multiple segments within the same plaque, necessitating a physical clone size of at least 1 cm. Furthermore, clones carrying multiple mutations spanned diverse plaque regions, encompassing the media, sub-core intima, and necrotic core. We did not detect any mutational hotspots, however, 21 of the mutated genes appeared in the Integrative Onco Genomics database of 619 mutational cancer driver genes (χ2 test: p = 0.015). Moreover, unbiased pathway enrichment analysis disclosed enrichment of terms linked to cell-cell adhesion and the contractile apparatus, and genes underlying these enrichments were primarily expressed by smooth muscle cells, endothelial cells, and fibroblasts, as corroborated by analysis of single-cell RNA sequencing data. Finally, six patients were CHIP carriers, and in several of these patients, hematopoietic clones constributed significantly to the cell population of plaque segments. Conclusions In summary, our study provides compelling evidence that somatic mutations are an intrinsic aspect of human plaques. Moreover, we substantiate that specific mutation-carrying cells within plaque tissue undergo expansion, giving rise to clones of substantial size. We propose that unraveling the fundamental underpinnings of clonal biology and the phenotypic traits of expanding clones may unveil novel mechanisms to impede lesion development or enhance plaque stability. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The study was supported by a grant from Odense University Hospital to the Center for Individualized medicine in Arterial diseases (CIMA) and a grant from the Novo Nordisk Foundation to LMR (NNF20OC0065744). LBS is supported by Danish Diabetes Academy (supported by the Novo Nordisk Foundation). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Danish National Committee on Health Research Ethics (Project ID: CVK-2006749) and is registered in the Registry of Research Projects in the Region of Southern Denmark (file no. 18/20280). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The datasets generated and analyzed during the current study are not publicly available due to hospital guidelines and legislation regarding personal data. Data will be available from the corresponding author on reasonable request and with permission from Odense University Hospital Legal Department.