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

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.

Circulating cell-free DNA is predominantly composed of retrotransposable elements and non-telomeric satellite DNA.

Circulating cell-free DNAs (cfDNAs) are DNA fragments which can be isolated from mammalian blood serum or plasma. In order to gain deeper insight into their origin(s), we have characterized the composition of human and cattle cfDNA via large-scale analyses of high-throughput sequencing data. We observed significant differences between the composition of cfDNA in serum/plasma and the corresponding DNA sequence composition of the human genome. Retrotransposable elements and non-telomeric satellite DNA were particularly overrepresented in the cfDNA population, while telomeric satellite DNA was underrepresented. This was consistently observed for human plasma, bovine serum and for the supernatant of human cancer cell cultures. Our results suggest that reverse transcription of retrotransposable elements and secondary-structure formation during the replication of satellite DNA are contributing to the composition of the cfDNA molecules in the mammalian blood stream. We believe that our work is an important step towards the understanding of the biogenesis of cfDNAs and thus may also facilitate the future exploitation of their diagnostic potential.

Evaluation of host-based molecular markers for the early detection of human sepsis.

We have identified 24 molecular markers, based on circulating nucleic acids (CNA) originating from the human genome, which in combination can be used in a quantitative real-time PCR (qPCR) assay to identify the presence of human sepsis, starting two to three days before the first clinical signs develop and including patients who meet the SEPSIS-3 criteria. The accuracy was more than 87 % inside of the same patient cohort for which the markers were developed and up to 81 % in blind studies of patient cohorts which were not included in the marker development. As our markers are host-based, they can be used to capture bacterial as well as fungal sepsis, unlike the current PCR-based tests, which require species-specific primer sets for each organism causing human sepsis. Our assay directly uses an aliquot of cell-free blood as the substrate for the PCR reaction, thus allowing to obtain the diagnostic results in three to four hours after the collection of the blood samples.