Megakaryocyte- and erythroblast-specific cell-free DNA patterns in plasma and platelets reflect thrombopoiesis and erythropoiesis levels

Abstract

Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determine that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (~26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Surprisingly, we discover that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets.

Fig. 1. Comparative analysis of erythroblast and megakaryocyte methylomes, and their representation in plasma.

A Genomic loci unmethylated specifically in megakaryocytes (n = 96 loci) were identified by comparison to other cell types of hematopoietic origin (left) and subsequently evaluated by WGBS of white blood cells (WBC) and cell-free DNA (cfDNA) of 23 individuals (right). These sites were, on average, 5% unmethylated in WBC and 31% unmethylated in cfDNA. For each individual, the sites were significantly hypomethylated in cfDNA (p < 7.3e-17, paired two-tailed Mann-Whitney test). Average percent of unmethylated sites is marked by a dotted line for cfDNA (red) and WBC (blue). Error bars represent standard deviation. B Genomic loci unmethylated specifically in erythroblasts (n = 1884 loci) were identified by comparison to other cell types of hematopoietic origin (left) and subsequently evaluated by WGBS of white blood cells (WBC) and cell-free DNA (cfDNA) of 23 individuals (right). These sites were, on average, 5% unmethylated in WBC and 9% unmethylated in cfDNA. For each individual, the sites were significantly hypomethylated in cfDNA (p < 3.7e-67, two-tailed Mann-Whitney test). Average percent of unmethylated sites is marked by a dotted line for cfDNA (red) and WBC (blue). Error bars represent standard deviation. C Targeted bisulfite-sequencing of megakaryocyte-specific unmethylated regions in whole blood (n = 26) and cfDNA (n = 62) of healthy individuals, demonstrating a higher percentage of megakaryocyte-derived DNA in cfDNA compared to WBC (p = 1.7e-13, two-tailed Mann-Whitney test). Error bars represent standard deviation. D Targeted bisulfite-sequencing of erythroblast-specific unmethylated regions in whole blood (n = 15) and cfDNA (n = 71) of healthy individuals, demonstrating a higher percentage of erythroblast-derived DNA in cfDNA compared to WBC (p = 1.4e-8, two-tailed Mann-Whitney test). Error bars represent standard deviation. Source data are provided as a Source Data file.

Fig. 2. Platelets contain genomic DNA derived from megakaryocytes.

A DNA extracted from platelet concentrates contains DNA derived from megakaryocytes, leukocytes and hepatocytes, as measured by targeted bisulfite sequencing of cell-type specific unmethylated genomic regions. B Uncentrifuged platelet concentrates (platelets in plasma) were analyzed with or without DNase treatment, demonstrating that DNase treatment reduces leukocyte and liver, but not MK markers in platelets. Source data are provided as a Source Data file.

Fig. 3. Genome-wide analysis of platelet DNA supports megakaryocyte origin.

A Platelet DNA is derived from all human chromosomes; however, the majority is mitochondrial DNA. The mean of three platelet samples is plotted with error bars representing standard deviations. B, C Regions uniquely unmethylated in cell types of hematopoietic origin, hepatocytes and endothelial cells were identified as described (Methods). Megakaryocyte-unique unmethylated regions are unmethylated in platelets as well. HSC Hematopoietic stem cell, MPP Multipotent progenitor, CMP Common myeloid progenitor, CLP Common lymphoid progenitor, MLP Multi-lymphoid progenitor, GMP Granulocyte macrophage progenitor, MEP Megakaryocyte erythroid progenitor. D Deconvolution of platelet DNA methylation demonstrated megakaryocyte DNA as the main component of platelet DNA. Error bars represent 90% confidence intervals calculated by bootstrapping over 10000 iterations of pooled platelet samples. The bar height represents the average across the bootstrapped samples. Source data are provided as a Source Data file.

Fig. 4. Sex-mismatched platelet transfusions suggest that platelets are not the source of megakaryocyte DNA.

The ratio of concentration of DNA from the SRY gene, located on the Y-chromosome, to DNA from the Beta-actin gene, located on chromosome 7, was evaluated in the plasma of female recipients of platelets from male donors, by massive parallel sequencing. At 24–72 h after transfusion there was no remaining male DNA detectable. Each line represents a different individual. Source data are provided as a Source Data file.

Fig. 5. Targeted analysis of megakaryocyte (MK) and erythroblast methylation markers in plasma samples.

A Genome equivalents (GE) per milliliter plasma of megakaryocyte DNA in samples from healthy donors and patients with Idiopathic Thrombocytopenic Purpura (ITP), ET Essential Thrombocythemia (ET) and hypoplastic bone marrow (after chemotherapy). B MK DNA is present in significantly different concentrations between healthy (n = 61), ITP (n = 5), ET (n = 5) and hypoplastic bone marrow (n = 5) (p < 0.05). MK DNA is not significantly different in thalassemia (n = 3) as compared to healthy individuals. All samples are biologically independent samples. The center, bounds of box and whiskers represent the median, upper and lower quartiles, and 1.5x the interquartile range, respectively. C Erythroblast cfDNA is significantly elevated in thalassemia (n = 3) compared to healthy individuals (n = 70), as well as compared to ET (n = 5) (p < 0.05). Data is plotted for hypoplastic bone marrow (n = 5) and ITP (n = 5) as well. All samples are biologically independent samples. The center, bounds of box and whiskers represent the median, upper and lower quartiles, and 1.5x the interquartile range, respectively. *p < 0.05. Source data are provided as a Source Data file.