Genome-wide DNA methylation profiling of peripheral blood reveals an epigenetic signature associated with severe COVID-19

Abstract

The global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic RNA virus causing coronavirus disease 2019 (COVID-19) in humans. Although most patients with COVID-19 have mild illness and may be asymptomatic, some will develop severe pneumonia, acute respiratory distress syndrome, multi-organ failure, and death. RNA viruses such as SARS-CoV-2 are capable of hijacking the epigenetic landscape of host immune cells to evade antiviral defense. Yet, there remain considerable gaps in our understanding of immune cell epigenetic changes associated with severe SARS-CoV-2 infection pathology. Here, we examined genome-wide DNA methylation (DNAm) profiles of peripheral blood mononuclear cells from 9 terminally-ill, critical COVID-19 patients with confirmed SARS-CoV-2 plasma viremia compared with uninfected, hospitalized influenza, untreated primary HIV infection, and mild/moderate COVID-19 HIV coinfected individuals. Cell-type deconvolution analyses confirmed lymphopenia in severe COVID-19 and revealed a high percentage of estimated neutrophils suggesting perturbations to DNAm associated with granulopoiesis. We observed a distinct DNAm signature of severe COVID-19 characterized by hypermethylation of IFN-related genes and hypomethylation of inflammatory genes, reinforcing observations in infection models and single-cell transcriptional studies of severe COVID-19. Epigenetic clock analyses revealed severe COVID-19 was associated with an increased DNAm age and elevated mortality risk according to GrimAge, further validating the epigenetic clock as a predictor of disease and mortality risk. Our epigenetic results reveal a discovery DNAm signature of severe COVID-19 in blood potentially useful for corroborating clinical assessments, informing pathogenic mechanisms, and revealing new therapeutic targets against SARS-CoV-2.

Keywords: COVID-19; DNA methylation; IFN; SARS-CoV-2; epigenetics.

Graphical Abstract

FIGURE 1

DNA methylation signature associated with severe COVID-19. (A) Overview of experimental design for comparative DNA methylation profiling. (B) Box and whisker plots of DNA methylation cell type deconvolution by the CIBERSORT method showing estimated cell type proportions of B cells, NK cells, CD4 T cells, CD8 T cells, monocytes, and neutrophils for uninfected control (orange), hospitalized influenza (purple), primary HIV+ ART naïve (red), coinfection HIV+/COVID-19 (gray), and severe COVID-19 (blue) PBMCs. (C) DNA methylation (DNAm)-based neutrophil–lymphocyte ratio (NLR). (D) Box and whisker plots of DNA methylation cell-type deconvolution showing estimated cell-type proportions of B cells, NK cells, CD4 T cells, CD8 T cells, monocytes, and neutrophils in postmortem lung tissues from COVID-19 and uninfected controls. (E) Heatmap of hypermethylated CpGs in uninfected control (orange), hospitalized influenza (purple), primary HIV+ ART naïve (red), coinfection HIV+/COVID-19 (gray), and severe COVID-19 (blue) PBMCs; each participant indicated at top of row. GeneID associated with each CpG displayed for each row. Unsupervised hierarchic clustering above columns identified 2 main clusters. Methylation values displayed as ranging from low methylation (0; blue) to high methylation (1, red). (F) Heatmap of hypomethylated CpGs in uninfected control (orange), hospitalized influenza (purple), primary HIV+ ART naïve (red), coinfection HIV+/COVID-19 (gray), and severe COVID-19 (blue) PBMCs. GeneID associated with each CpG displayed for each row. Unsupervised hierarchic clustering above columns identified 2 main clusters. Methylation values displayed as ranging from low methylation (0; blue) to high methylation (1, red). (G–J) Dot plots of DNA methylation levels at CpGs related to IFITM1, ISG20, NLRP3, and MX1 genes. (K and L) Correlation plots of SARS-CoV-2 plasma viral copies/ml and platelets count with DNA methylation levels of CpG at the MX1 genes

FIGURE 2

Comparative epigenetic clock analyses of uninfected controls, influenza infection, primary HIV infection, HIV+/COVID-19 coinfection, and severe COVID-19. (A) Bar graph displaying significant elevation in estimated epigenetic age acceleration in severe COVID-19 participants compared to uninfected controls and influenza. (B) Bar graph displaying significant elevation in estimated mortality risk according to DNAm GrimAge in severe COVID-19 participants compared with uninfected controls and primary HIV. (C) Bar graph displaying significant decrease in DNAm-based telomere length in primary HIV compared with uninfected control. (D and E) Scatter plot showing severe COVID-19 participants significant elevations in DNAm-based biomarker estimates of cystatin C and TIMP1 compared with uninfected control, influenza, HIV+, and coinfection HIV+/COVID-19. One-way ANOVA was utilized to identify significant group differences. Post hoc multiple comparisons testing utilized Tukey test. Significance was set at P = 0.05