Putative Origins of Cell-Free DNA in Humans: A Review of Active and Passive Nucleic Acid Release Mechanisms

Abstract

Through various pathways of cell death, degradation, and regulated extrusion, partial or complete genomes of various origins (e.g., host cells, fetal cells, and infiltrating viruses and microbes) are continuously shed into human body fluids in the form of segmented cell-free DNA (cfDNA) molecules. While the genetic complexity of total cfDNA is vast, the development of progressively efficient extraction, high-throughput sequencing, characterization via bioinformatics procedures, and detection have resulted in increasingly accurate partitioning and profiling of cfDNA subtypes. Not surprisingly, cfDNA analysis is emerging as a powerful clinical tool in many branches of medicine. In addition, the low invasiveness of longitudinal cfDNA sampling provides unprecedented access to study temporal genomic changes in a variety of contexts. However, the genetic diversity of cfDNA is also a great source of ambiguity and poses significant experimental and analytical challenges. For example, the cfDNA population in the bloodstream is heterogeneous and also fluctuates dynamically, differs between individuals, and exhibits numerous overlapping features despite often originating from different sources and processes. Therefore, a deeper understanding of the determining variables that impact the properties of cfDNA is crucial, however, thus far, is largely lacking. In this work we review recent and historical research on active vs. passive release mechanisms and estimate the significance and extent of their contribution to the composition of cfDNA.

Keywords: active release of cfDNA; cell-free DNA; circulating DNA; circulating tumor DNA; liquid biopsy; origins of cfDNA; passive release of cfDNA.

Figure 1

Different forms of cell-free DNA (cfDNA) in the human circulatory system. The biological and structural characteristics of the aggregate cfDNA population in a typical biospecimen is highly heterogeneous. While some overlap is common (see the main text), different cellular sources and mechanisms of origin often result in the production of uniquely distinct forms of cfDNA. Exhaustive stratification of the different cfDNA subtypes and an improved understanding of the factors which might modulate these characteristics of cfDNA are vital steps toward understanding the biological role(s) of cfDNA, as well as an expansion of their clinical utility.

Figure 2

Different extracellular vesicle (EV) types in the human body. The three main types of EVs that occur in human body fluids include microvesicles, apoptotic bodies, and exosomes. These vesicles can be differentiated based on the mode of their production, cellular extrusion pathways, and their overall size. In addition, EVs can be stratified according to their content and function.

Figure 3

Breakage-fusion-bridge cycles as a potential source of cell-free DNA (cfDNA). A chromosome that has lost its telomere (1) is replicated (2) and the sister chromatids fuse at the broken ends, forming a dicentric chromosome (3). The two centromeres are, then, pulled to opposite spindle poles during the anaphase, stretching out the chromatin to form an anaphase bridge (4). In many cases an anaphase bridge breaks (5) and the broken telomere-deficient chromosomes are again replicated (6) and once again fuse with their sister chromatids (7). Several repetitions of this process result in the amplification of DNA sequences that are adjacent to the break or fusion point. An anaphase bridge may break in various regions, resulting in the amplification of different genomic regions (e.g., genes, oncogenes, and repetitive DNA). Recombination between homotypic sequences within the amplified DNA generates extrachromosomal circular DNA (8), which can eventually become looped out of the chromosome (9). These mini circles of DNA can subsequently be extruded from the nucleus through budding (10) and eventually become extruded from cells into the extracellular space (11).