Asif Butt

Circulating Nucleic Acids in Plasma and Serum : Recent Developments

Abstract:  Nucleic acids (DNA and RNA) have been detected in plasma, serum, urine, and other body fluids from healthy subjects as well as in patients. The ability to detect and quantitate specific DNA and RNA sequences has opened up the possibility of diagnosis and monitoring of diseases. With the recent developments in the field of circulating nucleic acids the application in the diagnostic field has increased. The recent discovery of epigenetic changes in placental/fetal DNA and the detection of fetal/placental‐specific RNAs have made it possible to use this technology in all pregnancies irrespective of the gender of the fetus. With the application of mass spectrometry and other techniques to this field, it is now possible to detect very small amounts of specific DNA in the presence of excess of other nonspecific nucleic acids (e.g., detection of mutations in fetal DNA in the presence of excess of maternal DNA). Circulating nucleic acids have now been shown to be useful in other conditions, such as diabetes mellitus, trauma, stroke, and myocardial infarction. In oncology, detection and monitoring of tumors is now possible by the detection of tumor‐derived nucleic acids. In spite of these advances questions regarding the origin and biologic significance of circulating nucleic acids remain to be answered. Furthermore preanalytical and analytical aspects of this field remain to be standardized.

Overview of Circulating Nucleic Acids in Plasma/Serum

The existence of circulating nucleic acids in plasma and serum (CNAPS) was first described almost six decades ago. However, the prognostic and diagnostic utility of this circulating DNA/RNA has only really begun to be appreciated in the last decade. Earlier studies concentrated mainly on investigations concerned with fetal medicine and oncology, and significant progress was made in both specialities. More recently the field of enquiry has extended further, and attention has turned to other pathologic states, including trauma, sepsis, myocardial infarction, stroke, transplantation, diabetes mellitus, and hematologic disorders. In some of these studies, mitochondrial as well as genomic DNA and tissue‐specific mRNA have been analyzed, either quantitatively or qualitatively or both, and have been shown to be modified in the presence of disease. While there is tremendous potential for CNAPS as a clinical modality, many of the emerging studies seem to be confined to a few dedicated labs. Therefore, additional independent studies are necessary in some cases in order to show reproducibility, which will further consolidate the field. Despite this shortcoming, and with the evidently increasing number of applications for CNAPS, it is highly likely that routine testing, as described, will become reality within the next 5 to 10 years.

Role of Cell-Free Plasma DNA as a Diagnostic Marker for Prostate Cancer

Abstract: Recent evidence has shown elevated levels of cell‐free plasma DNA in cancer patients. The aim of the present study was to quantify and compare the levels of cell‐free plasma DNA in patients with prostate cancer, prostatic intraepithelial neoplasia (PIN), and benign prostatic hypertrophy (BPH) to examine if it offered a useful diagnostic test. Blood samples were obtained from 37 patients attending a clinic for prostate biopsies. Samples were taken prior to biopsy, within 1 hour of the biopsy, and then 2 weeks later. DNA was extracted using a QIAamp blood kit (Qiagen) and plasma DNA measured, in genome equivalents/milliliter plasma (GE/mL), using real‐time quantitative PCR for the β‐globin gene. Prior to biopsy, plasma DNA concentration in BPH patients was 936 GE/mL (median; range: 633–2074 GE/mL), while cancer and PIN patients had significantly higher levels of DNA at 1734 GE/mL (median; range: 351–3131 GE/mL; P= 0.01) and 1780 GE/mL (median; range: 1514‐2732 GE/mL; P= 0.04), respectively. Comparison of plasma DNA concentration before and after biopsy showed that 60 minutes after biopsy values were significantly higher in both BPH (1494 GE/mL; range: 613‐2522 GE/mL; P= 0.029) and cancer (2758; range: 1498‐5226 GE/mL; P= 0.007) patients. ROC analysis of the data indicated a sensitivity of 85% and a specificity of 73% when DNA concentration of 1000 GE/mL was taken as an indicator of malignancy or PIN. The data suggest that quantification of cell‐free plasma DNA may have an important diagnostic role in distinguishing benign and malignant prostate disease.

Cell-free plasma DNA as a prognostic marker in intensive treatment unit patients

Abstract: Recent evidence suggests that cell‐free plasma DNA has potential use as a prognostic marker in many clinical settings. The aim of the present study was to evaluate the prognostic role of cell‐free plasma DNA in the prediction of clinical outcome in intensive treatment unit (ITU) patients. Cell‐free plasma DNA was measured by real‐time polymerase chain reaction assay for the β‐globin gene and SOFA score, APACHE II score, CRP concentrations, and clinical outcome (duration of stay, ventilation time, and mortality) were noted in 94 patients on admission to the ITU. The median plasma DNA concentration in ITU patients was 5493 GE/mL and this was significantly (P <0.001) higher than the DNA concentration in healthy subjects (1970 GE/mL). DNA concentration demonstrated a significant correlation with serum C‐reactive protein (CRP) (r= 0.363) concentration and S epsis‐related O rgan F ailure A ssessment (SOFA) (r= 0.360) score (P <0.001 for both by Pearson correlation) but not with A cute Physiology And Chronic Health Evaluation (APACHE II) score. Patients on ventilation had significantly higher DNA concentrations compared to nonventilated patients (7362 GE/mL versus 4479 GE/mL; P= 0.004). The median DNA concentration in nonsurvivors was 9148 GE/mL, and this was 2.3‐fold greater than that in survivors (3921 GE/ml, P <0.001). ROC analysis of the data indicated a sensitivity of 85% and a specificity of 80% when DNA concentration of 6109 GE/mL was taken as a predictor of death. The data suggest that cell‐free plasma DNA concentration is potentially useful as a prognostic marker in ITU patients.

Real-Time Quantitative PCR Measurement of Thyroglobulin mRNA in Peripheral Blood of Thyroid Cancer Patients and Healthy Subjects

Abstract: Follow‐up of recurrent differentiated thyroid carcinoma involves the measurement of serum thyroglobulin (Tg). However, Tg autoantibodies are present in a high proportion of thyroid carcinoma patients (up to 25%) and these can interfere with the Tg immunoassays. To overcome this obstacle, investigators have used real‐time quantitative reverse transcriptase polymerase chain reaction (RT‐PCR) to measure Tg mRNA in the blood of patients with differentiated thyroid cancer, with varying degrees of success. In the present study, we demonstrate the first reported use of the PAXgene Blood RNA collection tube and extraction kit method for the preparation of RT‐PCR‐quality RNA with subsequent deployment of the latter in the development of a specific, sensitive, and reproducible Taqman assay for the detection and quantification of thyroglobulin mRNA. Beta‐actin mRNA was also assayed and results are expressed as a ratio of Tg to β‐actin mRNA. The intra‐assay coefficient of variations (CVs) for Tg and β‐actin mRNA assay were 27.7% and 25.4%, respectively. Inter‐assay CVs were 20.8% and 28.8%, respectively, for the two assays. Tg mRNA was detected in all cancer subjects (n= 42) and healthy individuals (n= 20). Tg mRNA was significantly higher in cancer patients than in the healthy subjects (0.00169 ± 0.00013 vs. 0.00051 ± 0.00015; P<0.0001). Fourteen cancer patients had detectable levels of serum Tg, and Tg mRNA levels tended to be higher in these than in cancer subjects with undetectable serum Tg (0.00188 ± 0.00021 vs. 0.00157 ± 0.000178; P= 0.08). Circulatory Tg mRNA measurement may serve a useful role in the assessment of thyroid cancer.

Measurement of circulating neuron-specific enolase mRNA in diabetes mellitus.

In this study we measured the levels of neuron‐specific enolase mRNA as a possible marker of diabetic neuropathy. Blood samples were collected from healthy controls (n= 26), diabetic controls (no known neuropathy or retinopathy) (n= 22), and diabetics with clinically diagnosed neuropathy (n= 24) into PAXgene blood RNA tubes. mRNA was extracted, reverse‐transcribed to cDNA, and measured by real‐time quantitative PCR. Enolase mRNA levels were normalized by the simultaneous measurement of β‐actin mRNA. The results showed that the enolase mRNA was significantly (P= 0.002) higher in the diabetic control (median = 0.018; range = 0.006–0.037) group compared to healthy subjects (median = 0.0086; range = 0.0016–0.039). However, the diabetic neuropathy group showed lower enolase levels (median = 0.0067; range = 0.0025–0.017) compared to both healthy subjects (P= 0.06) and diabetic controls (P < 0.001). In the diabetic neuropathy group patients with preproliferative (median = 0.01; range = 0.008–0.017) or proliferative retinopathy (median = 0.011; range = 0.007–0.015) had significantly (P= 0.001) higher enolase mRNA compared to patients with background retinopathy (media = 0.004; range = 0.0025–0.0092). It is concluded that levels of enolase mRNA are decreased in diabetic neuropathy and this molecular marker may also be useful in differentiating early from advanced eye disease in those diabetics diagnosed with neuropathy.

Stability of β‐Actin mRNA in Plasma

It has long been thought that mRNA is labile and easily prone to degradation. However, a recent study demonstrated that GAPDH mRNA in cell‐free plasma may remain stable up to 24 hours after blood collection. As there are no other independent studies, we attempted to reproduce the findings of that study. In our study, blood was collected from a healthy male volunteer into Vacutainer tubes containing EDTA. Blood samples were placed on ice and plasma separated by double‐centrifugation at times 0, 1, 2, and 5 hours after blood collection. mRNA was extracted from four aliquots of the blood sample by means of the QIAamp Viral RNA kit. Extracted mRNA was converted to cDNA by reverse transcription before real time quantitative PCR measurement of the housekeeping β‐actin gene. Plasma β‐actin mRNA at 2 hours (0.012; 0.0031–0.0297, median and range) was significantly lower (P= 0.022) than at 0 hours (0.12; 0.057–0.165) (P= 0.016). The levels decreased further at 5 hours (0.0037; 0.0024–0.011) (P= 0.004). The results show that plasma β‐actin mRNA levels decrease with time after blood collection and that this is likely to be due to degradation in vitro.

Circulating Nucleic Acids and Diabetic Complications

Abstract:  Diabetes mellitus is a major health problem across the world. Diabetic retinopathy (DR) and nephropathy are two of the major complications of diabetes. DR is the leading cause of blindness and diabetic nephropathy is the leading cause of end‐stage renal failure. We have examined the potential value of circulating nucleic acids in the detection and monitoring of these two complications of diabetes. mRNA for nephrin was significantly higher in all diabetics compared to healthy controls and it was significantly higher in normoalbuminuric patients compared to healthy controls. This may indicate progression to microalbuminuric stage. Circulating rhodopsin mRNA was detectable in healthy subjects and in diabetic patients. It was significantly raised in diabetic patients with retinopathy. Higher rhodopsin mRNA in diabetic patients without retinopathy suggests that some of them may go on to develop it or already have it subclinically. Circulating nucleic acids have the potential to be noninvasive molecular tests for diabetic complications.

Effect of hypoxia on circulating levels of retina-specific messenger RNA in type 2 diabetes mellitus.

Previously it was shown that the circulating rhodopsin mRNA level was higher in diabetic retinopathy (DR). Recent evidence suggests that hypoxia may also be associated with DR. The aim of this study was to investigate the effect of oxygen desaturation on circulating retina‐specific mRNA in type 2 diabetic patients. Thirty‐five type 2 diabetic patients underwent overnight oximetry. Two parameters from oximetry were used to measure oxygen desaturation: the number of times per hour the oxygen saturation decreased by 4% or greater (number of dips/hr) and percentage of sleep time with oxygen saturation (SpO2) <90%. Blood samples were collected into PAXgene Blood RNA tubes. Total RNA was extracted from the samples and reverse‐transcribed into cDNA, and retina‐specific markers were measured by quantitative real time PCR. In patients with ≥5 dips/hr, mRNA values for rhodopsin (P= 0.05) and RPE65 (P= 0.044) were significantly higher than in patients with <5 dips/hr. No change was seen in retinoschisin mRNA expression. In patients with preproliferative or proliferative DR, median levels for rhodopsin mRNA and RPE65 mRNA were 30% and 80% higher and retinoschisin mRNA was lower in patients with ≥5 dips/hr when compared to patients with <5 dips/hr. These results indicate that hypoxia may modulate expression of genes in the retina.

Retina-Specific mRNA in the Assessment of Diabetic Retinopathy

In a previous study we demonstrated the presence and diagnostic usefulness of circulating rhodopsin mRNA in the assessment of diabetic retinopathy (DR). In the present study we investigated three further retina‐specific markers in blood to determine their suitability as markers of DR. The markers were RPE65, retinoschisin, and melanopsin. Whole blood was collected from diabetic patients and healthy controls into PAXgene Blood RNA tubes and RNA was extracted using the PAXgene Blood RNA System. Quantitative real‐time PCR was used to quantify mRNA for RPE65, retinoschisin, and melanopsin. β‐actin mRNA was used for normalization. RPE65, retinoschisin, and β‐actin mRNA were detected in 100% of subjects; melanopsin was not detected in either controls or diabetic patients. Circulating RPE65 mRNA concentration was 63% higher in diabetic patients than in healthy individuals (P= 0.019), whereas retinoschisin showed no change between the two groups. Compared with healthy controls, circulating RPE65 mRNA concentration was higher in diabetics with no retinopathy (30%; P= NS), background DR (93%; P= 0.01), preproliferative DR (20%; P= NS), and proliferative DR (107%; P= 0.004). Compared with diabetics with no retinopathy, levels of RPE65 mRNA were also significantly higher (60%) in the presence of proliferative DR (P= 0.029). In contrast, levels of retinoschisin mRNA were lower in background DR (34%; P= 0.033), preproliferative DR (43%; P= 0.026), and proliferative DR (47%; P= 0.038) compared to that in diabetics without retinopathy. We conclude that not all retina‐specific mRNA species are detectable in circulation (e.g., melanopsin). This may be related to differences in expression levels for the individual markers. Both RPE65 and retinoschisin were detectable and demonstrated contrasting trends in diabetics with and without retinopathy. In combination with rhodopsin, RPE65, and retinoschisin, mRNA may offer a useful tool in developing a blood test for DR.