Circulating Nucleic Acids in Plasma & Serum

A non-invasive approach

Although DNA was first demonstrated in human blood from healthy donors, pregnant women and clinical patients in 1948, the structure of DNA was still to be determined as was the elucidation of its role as the basis of the gene [Table 1]. Consequently, no interest was shown in the presence of DNA in the circulatory system until high DNA levels were demonstrated in the blood of patients with systemic lupus erythematosus. Similar observations were also made in acute medicine, diabetes, oncology and fetal medicine [Table 2, 4, 5].

Nucleic acid and nuclease content

Both DNA (1.8 - 35 ng mL-1) and RNA (2.5ng mL-1) are found in and plasma and serum from healthy donors. These levels rise in patients with various cancers, trauma, myocardial infarction and stroke with values of over 3,000ng DNA being recorded on occasions. The amount of DNA and RNA present in the plasma and serum will depend upon the health status of the individual and the level of nucleases present in blood. The average blood plasma concentration of DNAase I is 3.2 - 18.4ng mL-1 whilst the average serum RNAase value is 104 units mL -1. Hence the relatively low levels of circulating DNA in healthy individuals may indeed be partially due to peripheral blood DNAase activity, although DNA from cancer patients may be resistant to DNAase and the DNAase levels may have just been low. Similarly, high RNA levels may also be due to RNA resistance to RNAase digestion especially when high RNAase and RNA levels are present together. The RNA may be protected with a glycolipid due to its apoptotic origin. Furthermore, an RNA fraction is associated with the released DNA-complex from healthy cells which appears to be protected from digestion by RNAase (see below).

Nucleic acid sources

There are six possible sources of blood DNA, namely (i) breakdown of bacteria and blood cells; (ii) viruses, (iii) leucocyte surface DNA, (iv) necrosis, (v) apoptosis and (vi) spontaneous release of a newly synthesised DNA / RNA-lipoprotein complex from healthy cells.
DNA

Only small amounts of DNA are yielded by the first three possibilities with just nasopharyngeal carcinoma Barr virus and human papilloma virus carcinoma DNAs having been identified and the breakdown of bacteria and bloods cells yielding only low levels of DNA.

Necrosis is clearly an option for the origin of Circulating Nucleic Acids in Plasma & Serum (CNAPS). However, when the double stranded CNAPS DNA is separated by gel electrophoresis, the fragments tend to form a ladder rather than a smear. The ladder fragments are mainly 180 - 1,000 bp in size and so are likely to be formed by apoptosis. DNA released by necrosis is incompletely and non-specifically digested and so smears on electrophoretic separation due to its fragment sizes of about 10,000bp; this is not a major source of CNAPS.

Apoptosis is confirmed as a major DNA source especially since nucleosomes are present in the blood e.g. of cancer patients. Naked DNA fragments are also found in serum, possibly due to apoptosis.

Therefore, the two major sources of CNAPS are apoptosis and the spontaneously released DNA/RNA-lipoprotein complex. The DNA is newly synthesised and is released from the cell in the form of a complex together with newly-synthesised lipoprotein and RNA. This complex is released homeostatically from the healthy, but not dead or dying, cells whether human or other mammalian cells or avian or amphibian. The DNA is double-stranded and about 2,000 bp in size. Importantly, after leaving the cell, the complex readily enters other cells where it expresses a biological activity that appears to be cell-type specific (Table 3).

RNA

RNA is only recently of importance through its exploitation in clinical diagnosis and prognosis. The stability of RNA in the bloodstream is due to the availability and type of the RNAs and RNAses present (see above).

However, a newly synthesised RNA is released spontaneously from cells together with the DNA-lipoprotein complex. In consequence, RNA is primarily released by apoptosis and through the DNA/RNA-lipoprotein complex. Some RNA may also be derived by necrosis e.g. some m-RNAs.

Applications of CNAPS in diagnosis, prognosis and the monitoring of treatments

CNAPS in diagnosis, prognosis and the monitoring of treatments has been applied in a wide variety of clinical disorders and situations from the emergency and accident ward to foetal medicine. The general approach to clinical application involves the taking of blood samples from which are separated plasma, serum and leucocytes. DNA / RNA are removed from plasma / serum and the surface DNA from leucocytes. The DNA / RNA are then subjected to quantitative real-time PCR and RT-PCR prior to analysis by gel electrophoresis and mass spectrometry. Relating the markers so derived to the clinical condition permits the possibility of early diagnosis, and prognosis as well as the possibility of monitoring the treatment prescribed.

Acute Medicine

• Trauma: Circulating DNA levels increased in patients presenting with injury, the concentration relating to the severity of the injury with up to a 100-fold increase occurring in patients developing organ failure, multiple organ disfunction syndrome, acute lung injury and those who will die when compared to patients with uncomplicated injury. Since the DNA normally has a short half-life in circulation and given the elevated DNA levels in the first few hours after patient admission with potential organ failure, the maintenance of the high DNA levels could be used to anticipate that organ failure.
• Stroke: Circulating DNA levels are elevated after a stroke, the amount being related to the extent of brain damage and it may be possible to use these DNA levels as indicators of short and long-term changes as well as post-stroke mortality.
• Acute Myocardial Infarction (AMI): AMI patients have elevated circulating DNA levels when compared to controls including both AT-rich and GC-rich fragments of DNA. This has yet to be transformed into an early diagnostic approach.
• Organ Transplants: Preliminary studies on rejection monitoring with CNAPS by exploiting donor-DNA fractions showed a good correlation in the case of pancreas-kidney rejection and elevated donor-DNA levels.

Diabetes

• Diabetic Retinopathy (DR): Comparison of diabetic patients without DR, with DR and healthy subjects showed diabetic patients as a whole to have about 2.5 times the rhodopsin mRNA than the control subjects. Diabetic control patients levels were about 60 per cent higher than those of healthy individuals whilst the background retinopathy and pre-proliferative retinopathy patients showed increasing rhodopsin mRNA levels with increasing severity of the retinopathy. Diabetic patients without clinical features of retinopathy also showed significantly higher levels of rhodopsin mRNA so indicating that retinal damage could have already occurred. In this case, rhodopsin mRNA levels in peripheral blood could offer an early detection of DR. Additional early predictive markers include increased retinal specific mRNA, RPE65 levels and reduced retinoschisin mRNA levels.
  
• Diabetic Nephropathy (DN): DN patients have a higher mean amount of circulating nephrin mRNA when compared with a control healthy cohort possibly due to a loss of nephrin mRNA from glomerular epithelial cells which correlates well with pathological assays.

Prenatal Medicine

Although fetal DNA accounts only for 3-6 per cent of the total maternal CNAPS, its identification and isolation is facilitated by the majority of the fetal DNA fragments being primarily >300 bp whereas the maternal DNA fragments are >300 bp.

Successful sex determination has been performed on fetal DNA in maternal blood using either paternal derived fragments of the Y chromosome or paternal X-chromosome derived fragments of the amelogenin gene and multicopy DAZ sequence (Table 4).

Working with the maspin gene sequences, it was shown that if regions of the DNA from the father were methylated, but unmethylated from the mother then is was possible to distinguish the parental origin of the DNA. Furthermore, hypermethylated DNA was derived from the maternal blood cells, whilst the hypomethylated form was derived from the placenta and hence of foetal origin. Although the hypomethylated form was normally cleared from the blood in pregnant women, it increased by about six-fold in the case of pre-eclampsia. Higher levels of ß-globulin and SRY genes were also present in pregnant mothers who went on to develop pre-eclampsia and intrauterine growth retardation.

When the foetus-specific circulating mRNA for corticotrophin-releasing hormone increased ten-fold, the levels relate to the severity of pre-eclampsia (Table 4).
Foetal DNA can also be used for foetal blood group genotyping with the Rh status of the foetus being determined successfully. There are strong indications for the successful identification of other blood types including Rhc, RhE.

Other approaches include the possibility to determine Mendelian inherited disorders especially through the paternally-inherited alleles as can be seen through the diagnosis of Huntington’s disease, achondroplasia and mytonic dystrophy. Less easily detected are the aneuploid disorders due to the smaller increases in fetal DNA. However, modest results have been achieved with foetal chromosome 21 status by measuring either the relative concentrations of foetal-specific epigenetic markers on chromosome 21 to those on one or more reference chromosomes or placental-specific mRNA species transcribed from a chromosome involved in an aneuploidy, e.g. the PLAC4 gene on chromosome 21 for Down syndrome using the RNA-SNP (single-inherited nucleotide polymorphisms) allelic ratio method, which has a high sensitivity of 90 per cent and a high specificity of 96.5 per cent.

Pre-natal detection of ß-thalassemia is also feasible using fetal DNA isolated from the maternal blood using an allele-specific based real-time PCR method and using eleven paternally inherited SNPs with a high degree of heterozygosity from the ß-globulin gene for the diagnosis of ß-thalassemia.

Oncology

Initially, circulating DNA was used as (a) an early marker for cancer seen as an increased amount circulating and (b) in monitoring treatment when the DNA levels returned to normal levels upon successful treatment. However, there was no specific correlation between the DNA fragments involved and a particular type of cancer (see Table 5). Nucleosomes form one source of DNA released into the blood stream but are not considered to be suitable for cancer diagnosis due to elevation of nucleosome levels in patients with benign diseases. Nevertheless, circulating nucleosomes can be informative for monitoring cytotoxic therapy with strongly decreasing levels being mainly found in patients with remission of disease whereas constantly high or increasing values are associated with progressive disease during chemo- and radiotherapy.

Lung, Colorectal, Prostate, Liver, Ovary, Breast, Oesophageal Cancers

A range of markers have been proposed for the identification of a particular cancer, though there is frequent conflict in the literature as to the effectiveness of particular probes. However, recently, hypermethylated CpG in the promotor region of tumour suppressor genes has been suggested to trigger local gene silencing. Aberrant methylation of the p26 tumour suppressor gene was the first to be detected in liver, breast and lung cancer. Other frequently methylated tumour suppressor genes (Table 5) have been used with varying success.

CNAPS

Although a relatively recent addition to methodologies available for early diagnosis, prognosis and treatment monitoring, CNAPS offers a non-invasive approach to a wide range of clinical disorders that will allow the basic information necessary not only for use in predictive medicine but also for direct use in acute medicine.

AUTHOR BIO

Peter Gahan is Emeritus Professor of Cell Biology at King’s College London where he continues to teach. He is a director of the European Association for Predictive , Preventive and Personalised Medicine and researches the biology of the DNA/RNA lipo-protein complex found in CNAPS and its possible role in cancer.