Epigenetic liquid biopsies reveal elevated vascular endothelial cell turnover and erythropoiesis in asymptomatic COVID-19 patients【bioRxiv 2023年8月1日】


The full spectrum of tissues affected by SARS-CoV-2 infection is crucial for deciphering the heterogenous clinical course of COVID-19. Here, we analyzed DNA methylation and histone modification patterns in circulating chromatin to assess cell type-specific turnover in severe and asymptomatic COVID-19 patients, in relation to clinical outcome. Patients with severe COVID-19 had a massive elevation of circulating cell-free DNA (cfDNA) levels, which originated in lung epithelial cells, cardiomyocytes, vascular endothelial cells and erythroblasts, suggesting increased cell death or turnover in these tissues. The immune response to infection was reflected by elevated B cell and monocyte/macrophage cfDNA levels, and by evidence of an interferon response in cells prior to cfDNA release. Strikingly, monocyte/macrophage cfDNA levels (but not monocyte counts), as well as lung epithelium cfDNA and vascular endothelial cfDNA, predicted clinical deterioration and duration of hospitalization. Asymptomatic patients had elevated levels of immune-derived cfDNA but did not show evidence of pulmonary or cardiac damage. Surprisingly, these patients showed elevated levels of vascular endothelial cell and erythroblast cfDNA, suggesting that sub-clinical vascular and erythrocyte turnover are universal features of COVID-19, independent of disease severity. Epigenetic liquid biopsies provide non-invasive means of monitoring COVID-19 patients, and reveal sub-clinical vascular damage and red blood cell turnover.


SARS-Cov-2 infection directly injures the respiratory system, and inflicts damage to multiple additional tissues including the heart, blood vessels, pancreas, kidneys, and liver. In most of these cases it is not clear if the damage is caused directly by the viral infection, or whether tissues suffer damage due to the massive host’s immune response. This immune response involves both the innate and adaptive arms of the system, and is protective in most cases but can often cause severe inflammatory damage. Despite the extensive information that has accumulated since the emergence of the COVID-19 pandemic, the full spectrum of tissues affected by the infection is still not clear. This may partly explain why we still lack tools to predict the clinical course of disease in infected individuals, which ranges from fatal lung failure to a completely asymptomatic presentation. Clinical heterogeneity during the acute phase can potentially account for variation in late-onset clinical phenotypes such as cardiovascular complications, new-onset diabetes, and the diverse array of long-covid symptoms.

Cell-free chromatin fragments released from dying cells contain extensive information on the identity of cells that released these fragments, and on gene expression programs that operated in the cells prior to their death. In methylation-based liquid biopsies, the tissue origins of cfDNA are inferred based on its tissue-specific methylation patterns. Since the half-life of cfDNA is extremely short (estimated at 15-120 minutes), such analysis can determine the rate of cell death or turnover in specific host tissues close to the time of sampling. Two previous studies characterized the methylome of cfDNA in COVID-19 patients, and deconvoluted it using a partial human cell-type methylome reference atlas. The key finding of these studies was that erythroblast turnover is elevated in severe COVID-19 patients and predicts mortality. However, while methylome deconvolution studies can provide an unbiased overview of cfDNA composition, they depend on the quality and breadth of the reference atlas and are typically limited in sensitivity such that tissue contributions to cfDNA amounting to <1% of the total are not detected. To overcome this limitation, we employed deep whole genome bisulfite sequencing (WGBS) of plasma cfDNA followed by deconvolution using a novel extensive human methylome atlas. In addition, we used a highly sensitive targeted panel of tissue-specific methylation markers to assess tissue turnover in a cohort of COVID-19 patients including severe hospitalized and asymptomatic patients. Finally, we employed a novel method for cell-free chromatin followed by immunoprecipitation (cfChip-seq) to assess gene expression programs in cells of COVID-19 patients prior to cfDNA release.