Transrenal DNA Research Articles

Investigation of transrenal KRAS mutation in late stage NSCLC patients correlates to disease progression

Purpose: Using transrenal DNA to detect KRAS mutations in non-small cell lung cancer (NSCLC), the study addressed the clinical impact for longitudinal monitoring and prognostic value for disease outcome.Methods: Digital droplet PCR was used to detect the mutant DNA. A total of 200 NSCLC patients were recruited with varying molecular profiles. To ascertain the specificity of transrenal DNA to accurately profile the disease, primary tissues were compared. Subsequently, serial samplings were performed at different treatment cycles to gauge the predictive value.Results: Transrenal DNA was successfully detected in all 200 patients. Overall concordance rate for mutant KRAS DNA within urine specimens and primary tissue biopsies was 95% (k = 0.87; 95% CI: 0.82–0.95). Patients with positive results at baseline had lower median overall survival (OS) than the wildtype group. More importantly, longitudinal monitoring of urine specimens showed an increase in the quantity of transrenal DNA, which were highly associated with disease progression and outcome.Conclusions: Our study showed a highly associative link to the patient’s tumor KRAS profile. Monitoring its variations aided in stratifying patients with worse outcome. Urinary specimens that can be extracted non-invasively presents new opportunities to track patients with KRAS mutation undergoing therapy.

Detection of transrenal DNA for the diagnosis of pulmonary tuberculosis and treatment monitoring.

Molecular diagnostics of patients with MTB tuberculosis from urine samples. We developed a new molecular assay based on the detection of M. tuberculosis-specific transrenal DNA (trDNA) and tested it for the diagnosis of active tuberculosis at the initiation of anti-tuberculosis therapy and during treatment follow-up. The overall sensitivity of trDNA was 96 and 100% when smear-microscopy and trDNA was combined. In a subset of TB treatment naïve patients (n=11) sensitivity and specificity of trDNA was 64 and 100%, respectively. For this subset of patients the sensitivity was 91% when smear-microscopy and trDNA diagnosis were combined. After treatment initiation, trDNA showed a significant reduction in concentration over time reaching undetectable trDNA values at week 12 in 9 of 11 accessible patients (82%). Kinetics in treatment-naïve patients showed low base-line trDNA levels, which increased to maximal trDNA levels within one week indicating bactericidal activity of anti-tuberculosis drugs after the initiation of effective therapy. Maximal trDNA levels correlated positively with a radiological score, suggesting that the process of DNA excretion may reflect the extent of pulmonary disease. Matched samples showed an inverse correlation between the time to positivity of solid culture with maximum trDNA levels as well as the expected positive correlation between smear grade and maximum trDNA values. The detection of M. tuberculosis trDNA from urine specimen is a promising method for the diagnosis tuberculosis. The assay may be a candidate diagnostic tool for patients with paucibacillary and extrapulmonary disease, as method to assess treatment responses and could be helpful to diagnose tuberculosis in children.

Diagnosis of Strongyloides stercoralis: Detection of parasite-derived DNA in urine

Detecting infections of Strongyloides stercoralis is arduous and has low sensitivity. Clinically this is a major problem because chronic infections may disseminate in the host and lead to a life threatening condition. Epidemiologically, S. stercoralis is often missed in surveys as it is difficult to identify by standard stool examination procedures. We present, for the first time, evidence that the infection can be detected in filtered urine samples collected and processed in the field and subsequently assayed for the presence of parasite DNA. Urine specimens (∼40mL) were collected from 125 test and control individuals living in rural and peri-urban regions of Northern Argentina. From the same individuals, fresh stool specimens were processed using three different copropological methods. Urine specimens were filtered in the field through a 12.5cm Whatman No. 3 filter. The filters were dried and packed individually in sealable plastic bags with desiccant and shipped to a laboratory where DNA was recovered from the filter and PCR-amplified with primers specific to a dispersed repetitive sequence. Prevalence of S. stercoralis infection by stool culture and direct examination was 35/125 (28%), In contrast, PCR-based detection of parasite-specific trans-renal DNA in urine indicated that 56/125 (44.8%) carried the parasite. Of the patients that tested positive for urine-based parasite DNA, approximately half also tested positive in their stool specimens. There were 6.4% of cases where parasite larvae were seen in the stool but no DNA was amplified from the urine. As proof of principle, DNA amplification from urine residue reveals significantly more cases of S. stercoralis infection than the current standard stool examination techniques. Additional work is required to establish the relative utility, sensitivity and specificity of urine-based analysis compared to parasitological and nucleic acid detection from stool for clinical and epidemiological detection for S. stercoralis infection.

Abstract LB-133: Triage of high risk HPV positive women before colposcopy with reflex tests in pap smears and screening of high risk HPV in Transrenal DNA isolated from urine, using novel workflows to identify panels of methylated viral and human DNA

Abstract We set out to develp novel workflows to identify panels of methylated human papilloma virus (HPV) and human genes that can discriminate between CIN2+ and normal/CIN1 patients in liquid prep samples and in Transrenal DNA (TrDNA) isolated from urine. Using Human DNA methylation arrays and massively parallel Next Generation Sequencing (NGS) for Discovery and quantitative Methylation Specific PCR (qMSP) for validation, we found that promoter methylation of ZNF516, FKBP6, and INST1 discriminates samples with CIN2+ lesions from no intraepithelial lesions or malignancy (NILM) in cervical brush samples: 88.3% sensitivity, 88.9% specificity, 93.2 Area Under the Curve (AUC), 86.9% positive predictive value (PPV) and 90.2% negative predictive value (NPV). Using custom sequence capture pools of baits, we pulled down genomic and bisulfite converted high-risk HPV DNA before library prep for massively parallel Next Generation Sequencing (NGS) in 454 and MiSeq instruments, respectively to identify high risk HPV and the HPV methylome. Using our NGS results, we designed Syber Greeen qPCR assays to detect high risk HPV DNA in tissue and in Trans Renal DNA (TrDNA). We also designed qMSP primers-probe sets to detect methylated DNA in the HPV16-L1 gene region and ZNF516, FKBP6, and INST1 in TrDNA. TrDNA is circulating DNA that gets filtered by the kidneys and we isolate from urine as fragmented DNA ranging is size from 50 to 250 bp. We replicated the cervical brush epithelium results in liquid prep samples from another cohort, by adding HPV16-L1 methylation to the panel of methylated host genes (ZNF516, FKBP6, and INST1): 90.9% sensitivity, 60.9% specificity, AUC = 90.1, 52.6% positive predictive value (PPV) and 93.3% NPV. These results were verified in plasma DNA (AUC = 80.7) and TrDNA (AUC = 86.1) isolated from a subset of patients who provided liquid prep samples. Our results suggest that a biomarker panel that quantifies DNA methylation in the HPV-LI gene, together with promoter methylation of three host genes, ZNF516, FKBP6, and INST1, may be used as a reflex test in liquid prep to triage high-risk HPV positive women into colposcopy in countries with established cervical cancer screening programs. Our results also suggest that a panel of human and viral DNA methylation markers can be combined with a high-risk HPV DNA assay, to develop urine screening and triage tests in countries where cervical cancer screening programs are not effective due to cultural or infrastructure factors. Country specific DNA methylomes of normal and premalignant cervical epithelium for different age groups can be created as reference methylomes, to better understand the cultural-, socio-economic- and age-specific variations in the cervical epithelium and their correlation with HPV DNA methylomes. Citation Format: Rafael E. Guerrero-Preston, Blanca L. Valle, Anne Jedlicka, Nitesh Turaga, Liliana Florea, Oluwasina Folawiyo, Francesca Pirini, Angelo Vergura, Maartje Noordhuis, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Díaz, Edgardo De Jesus-Rodríguez, Keimari Méndez-Martínez, José Rodríguez-Orengo, Teresa Díaz-Montes, David Sidransky, Josefina Romaguera. Triage of high risk HPV positive women before colposcopy with reflex tests in pap smears and screening of high risk HPV in Transrenal DNA isolated from urine, using novel workflows to identify panels of methylated viral and human DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-133.

Abstract B11: Viral and host gene methylation in liquid prep: novel molecular screening and triage tools to reduce cervical cancer disparities

Abstract Latino women in the United States have the highest cervical cancer incidence rate, yet the highest death rate from cervical cancer is among Black women. The disproportionate burden of cervical cancer in the United States is primarily due to lack of screening, among the medically underserved, regardless of race or ethnicity. Rural and inner city women living in poverty have lower rates of screening and higher rates of cervical cancer in the US. The advent of molecular diagnostics provides an opportunity to develop novel cervical cancer screening and triage tools that can be packaged as point of care and self-testing solutions. We set out to identify a panel of methylated HPV DNA and human genes that can discriminate between CIN2+ and normal/CIN1 patients in liquid prep samples and in Transrenal DNA (TrDNA) isolated from urine. We used three independent cohorts, from Chile, Baltimore and Puerto Rico, to develop a method that can be used to triage into colposcopy, HPV+ women with abnormal cytology. Participants were women with no cervical intraepithelial lesions or malignancy and women with abnormal cervical biopsies– Cervical Intraepithelial Neoplasia (CIN), carcinoma in-situ and cervical cancer. Using DNA methylation arrays for Discovery and quantitative Methylation Specific PCR (qMSP) for Validation, we found that promoter methylation of ZNF516, FKBP6, and INST1 discriminates samples with CIN2+ lesions from samples with no intraepithelial lesions or malignancy (NILM): 88.3% sensitivity, 88.9% specificity, 93.2 Area Under the Curve (AUC), 86.9% positive predictive value (PPV) and 90.2% negative predictive value (NPV). Using custom sequence capture pools of baits, we pulled down genomic and bisulfite converted high-risk HPV DNA before library prep for NGS in 454 and MiSeq instruments, respectively. Using our NGS results, we optimized a Syber Greeen qPCR assay to detect high risk HPV DNA and a qMSP primer-probe set to detect methylated HPV. We replicated the results in liquid prep samples (n=67), adding HPV16 methylation to the panel: 90.9% sensitivity, 60.9% specificity, 90.1 (AUC), 52.6% positive predictive value (PPV) and 93.3% NPV. These results were verified in plasma DNA (AUC=80.7) and TrDNA (AUC=86.1) isolated from a subset of patients who provided the liquid prep samples. Our results suggest that our panel of viral and host gene methylation markers may be used as a reflex test in liquid prep to triage high risk HPV positive women into colposcopy, or as a screening and triage test in TrDNA, in combination with our high risk HPV test. There are several commercial co-testing options for identification of oncogenic high-risk HPV types (HPV+) in patients with abnormal cytology. However, there are currently no tests that can reliably identify the HPV+ patients with abnormal cytology that need to be referred for colposcopy. Consequently, more than half of the women that are referred to colposcopy either have a negative biopsy, or a grade 1 Cervical Intraepithelial Neoplasia (CIN1) diagnosis, which usually reverts to normal in 12-24 months. A triage test from cytology to colposcopy that can identify in the liquid prep sample those patients with a CIN grade more likely to progress to cervical cancer (CIN2+), would decrease the number of unnecessary cervical mucosa biopsies performed today, decreasing health care cost and improving the quality of health care delivery. These molecular tools can also be packaged as point of care and self-testing solutions to eliminate cervical cancer disparities in medically underserved women. Note: This abstract was not presented at the conference. Citation Format: Rafael Guerrero-Preston, Anne Jedlicka, Blanca L. Valle, Nitesh Turaga, Liliana Florea, Oluwasina Folawiyo, Francesca Pirini, Fahcina Lawson, Angelo Vergura, Maartje Noordhuis, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Díaz, Edgardo De Jesus, Teresa Diaz-Montes, Bruce Trock, Keimari Mendez, Josefina Romaguera, David Sidransky. Viral and host gene methylation in liquid prep: novel molecular screening and triage tools to reduce cervical cancer disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B11.

Telomere length and transrenal DNA isolated from transplanted kidney recipients' urine

Transplantation is the preferred method of end stage renal insufficiency treatment due to better quality of life and extended life of transplanted patients. Currently a non-invasive test, which evaluates the risk of acute or chronic rejection or deterioration of the transplanted organ's function, is being sought. An increase of the transrenal DNA concentration in the urine of urinary tract infection patients and in renal graft recipients during an episode of acute rejection was observed. There were also reports on shortening of telomeres in transplanted organ chromosomes, as the result of accelerated aging of cells, and its connection with the onset of chronic allograft nephropathy and the degree of its completion, and thus the deterioration of kidney function. The aim of this paper is to describe the urine genetic analysis through determining the length of the telomeres and the content of transrenal DNA to monitor kidney function and to evaluate the prevalence of acute and chronic rejection in patients after kidney transplantation. The genetic analysis of the biological material collected from patients relies on the determination of transrenal DNA content and length of DNA telomeres isolated from the urine of kidney recipients. The presented methods assume that the genetic profile of the transplanted organ recipient as well as kidney donor can be determined, so the source of the genetic material in the urine of the patient can be identified. A measurable effect of these methods' use would be to complement the evaluation of the prevalence of acute and chronic rejection and transplanted kidney function with a modern, non-invasive method, which is the analysis of telomere length from sediment of urine and the content of transrenal DNA in the urine.

Abstract LB-68: Identification of circulating HPV DNA in urine with a dual sequence capture approach.

Abstract HPV testing is increasingly used for cervical cancer screening in conjunction with cervical cytology. Privacy, cultural, and infrastructure issues challenge the effective implementation of HPV testing in cervical cancer screening. The detection of circulating HPV DNA from urine can be useful for cervical cancer screening. We captured and sequenced the HPV genome after isolating HPV Transrenal DNA (HPV-TrDNA) and then developed a qPCR-based HPV-TrDNA assay to quantify high risk HPV TrDNA. HPV TrDNA molecules are relatively short, fragmented, pieces of circulating DNA (less than 250 nucleotides) that get filtered by the kidneys and can be isolated from urine. Methods: We adsorbed cell-free nucelic acids from urine samples (10mL) of 41 women with 23 LGL and 18 HGL cervical lesions and 31 women with normal cytology on a Q-Sepharose resin followed by a silica-based method. A custom-designed pool of HPV-specific dual sequence capture probes was used for library amplification and target selection (Roche/NimbleGen SeqCap EZ Choice Library). A pre-capture amplification of the library was performed with ligation-mediated PCR (LM-PCR) using primers complementary to the adaptors, followed by two rounds of hybridization to the HPV-specific SeqCap EZ Choice Library. Amplified captured DNA from HPV infected cell lines, HeLa (HPV 18), CSCC7 (HPV16), and HPV TrDNA samples from premalignant cervical lesions were processed for multiplexed sequencing on the GS Junior system (Roche). Following completion of the pyrosequencing run, signal processing was performed, followed by detailed analyses. For the SYBR green qPCR assay we amplified an HPV E1 region common to thirteen high-risk HPV types for cervical cancer. Results: We isolated TrDNA from 10mL of urine samples. The mean concentration, total amount, and 260/280 ratio of isolated TrDNA was 34.2 ng/ul;1ug and 1.7 for premalignant samples and 17.2 ng/ul; 0.5ug; and 1.4 for normal samples. The total number of Pass Filter sequence reads was 100,428, the total number of bases over 41 million and the read length average was 130.3 bp. Most reads (91.63%) mapped to the reference HPV sequences, with the total number of mapped base pairs over 36 million (88%). The number of fully mapped reads was 14,899 (14.84%), and the number of partially mapped reads was 70,686 (88.17%). The average coverage of reference sequences was 78% for HPV 18 in the CSCC7 DNA, 86% for HPV 16 in HeLa DNA, as well as, 100% for HPV 18 and 96% for HPV 16 in TrDNA samples. The average Ct value for Pre-Capture LM-PCR DNAs was 35.74. The average Ct value for Post-Capture LM-PCR DNAs was 19.73; therefore the Delta Ct value was 16.01. Based on an estimated efficiency for the assay, the approximate fold enrichment was greater than 12,000. Conclusions: We demonstrated that the HPV genome can be assembled from HPV TrDNA. We are also the first to show that circulating high risk HPV TrDNA can be quantified using SYBR green qPCR assay after dual sequence capture of the HPV genome. Citation Format: Rafael E. Guerrero-Preston, Anne Jedlicka, Teresa Diaz-Montes, Josefina Romaguera, Juan C. Roa, David Sidransky. Identification of circulating HPV DNA in urine with a dual sequence capture approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-68. doi:10.1158/1538-7445.AM2013-LB-68

High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing

BackgroundFetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine.MethodologyWe have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine.Principal FindingsCell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length.ConclusionsWith the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.

Diagnostic Accuracy and Applicability of a PCR System for the Detection of Schistosoma mansoni DNA in Human Urine Samples from an Endemic Area

Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA.

Urine as a Specimen to Diagnose Infections in Twenty-First Century: Focus on Analytical Accuracy

Urine as a clinical specimen to diagnose infections has been used since ancient times. Many rapid technologies to assist diagnosis of infections are currently in use. Alongside traditional enzyme immunoassays (EIA), new technologies have emerged. Molecular analysis of transrenal DNA to diagnose infections is also a rapidly growing field. The majority of EIAs utilize the detection of excreted sugar compounds of the outer microbial cell-wall shed into the bloodstream and excreted into the urine. This mini-review focuses on current knowledge on rapid urinary antigen detection tests to diagnose most common infections, and highlights their diagnostic utility. The past and the future of urinalysis are also briefly discussed. The analysis of the literature shows that some methods are not quantitative, and analytical sensitivity may remain suboptimal. In addition, the performance criteria and technical documentation of some commercial tests are insufficient. Clinical microbiologists and physicians should be alert to assay limitations.

From Transrenal DNA to Stem Cell Differentiation: An Unexpected Twist

In this issue of Clinical Chemistry , Hung et al. describe the results of a study originally devoted to investigation of the transrenal DNA (Tr-DNA)1 phenomenon. These authors report on the presence of donor-derived, cell-free DNA and cells in the urine of sex-mismatched hematopoietic stem cell transplant (HSCT) recipients(1). The term Tr-DNA defines DNA molecules that appear in the urine from sources located outside of the urinary system. The investigators who found such molecules in urine for the first time suggested that DNA fragments from cells dying throughout the body appear in the bloodstream as so-called cell-free circulating DNA (cfcDNA) and then cross the kidney barrier into the urine(2). Otherwise, it is difficult to explain how fetal DNA appears in the urine of pregnant women or how tumor-specific DNA markers appear in the urine of patients with tumors located outside of the urinary system. This original observation was reproduced in many laboratories(2)(3)(4)(5)(6)(7). At the same time, several groups could not detect fetal DNA in the urine of pregnant women(8)(9), raising doubts about the concept of Tr-DNA. The latter results can be explained by the fact that Tr-DNA fragments are shorter than cfcDNA, and shorter amplicons should be used for their detection(4)(7)(10), especially in prenatal models in which concentrations of fetal DNA in maternal urine are low. It is also important to remember that urinary DNA from other sources complicates analysis of Tr-DNA. Other sources of urinary DNA include epithelial cells shed from the organs of the urinary system, often lymphocytes and …

Presence of Donor-Derived DNA and Cells in the Urine of Sex-Mismatched Hematopoietic Stem Cell Transplant Recipients: Implication for the Transrenal Hypothesis

The term "transrenal DNA" was coined in 2000 to signify that DNA in urine may come from the passage of plasma DNA through the kidney barrier. Although DNA in the urine has the potential to provide a completely noninvasive source of nucleic acids for molecular diagnosis, its existence remains controversial. We obtained blood and urine samples from 22 hematopoietic stem cell transplant (HSCT) recipients and used fluorescence in situ hybridization, PCR for short tandem repeats, mass spectrometry, quantitative PCR, and immunofluorescence detection to study donor-derived DNA in the urine. All HSCT recipients exhibited high amounts of donor-derived DNA in buffy coat and plasma samples. Male donor-derived DNA was detected in supernatants of urine samples from all 5 female sex-mismatched HSCT recipients. Surprisingly, the amount of DNA in urine supernatants was not correlated with the plasma value. Moreover, cell-free urine supernatants contained DNA fragments >350 bp that were absent in plasma. Donor-derived polymorphs were detected in urine by fluorescence in situ hybridization. Coincidentally, donor-derived cytokeratin-producing epithelial cells were discovered in urine samples from 3 of 10 sex-mismatched HSCT recipients as long as 14.2 years after transplantation. This report is the first to demonstrate the presence of donor-derived DNA in the urine of HSCT recipients; however, we show that much of this DNA originates from donor-derived cells, rather than from the transrenal passage of cell-free plasma DNA. Our discovery of donor-derived cytokeratin-producing epithelial cells raises interesting biological and therapeutic implications, e.g., the capacity of marrow stem cells to serve as an extrarenal source for renal tubule regeneration.

Optimization of Transrenal DNA Analysis: Detection of Fetal DNA in Maternal Urine

Fragments of DNA from cells dying throughout the body are detectable in urine (transrenal DNA, or Tr-DNA). Our goal was the optimization of Tr-DNA isolation and detection techniques, using as a model the analysis of fetal DNA in maternal urine. We isolated urinary DNA using a traditional silica-based method and using a new technique based on adsorption of cell-free nucleic acids on Q-Sepharose resin. The presence of Y chromosome-specific SRY (sex-determining region Y) sequences in urine of pregnant women was detected by conventional and real-time PCR using primers/probe sets designed for 25-, 39-, 65-, and 88-bp PCR targets. Method of DNA isolation and PCR target size affected fetal Tr-DNA detection. Assay diagnostic sensitivity increases as the PCR target is shortened. Shorter DNA fragments (50-150 bp) could be isolated by Q-resin-based technique, which also facilitated fetal Tr-DNA analysis. Using DNA isolated by Q-resin-based method and an "ultrashort" DNA target, we successfully detected SRY sequences in 78 of 82 urine samples from women pregnant with male fetuses (positive predictive value 87.6%). Eleven of 91 urine samples from women pregnant with female fetuses produced SRY false-positive results (negative predictive value 95.2%). Single-copy fetal DNA sequences can be successfully detected in the urine of pregnant women when adequate methods for DNA isolation and analysis are applied. Strong precautions against sample contamination with male cells and DNA are necessary to avoid false-positive results.

On Targeting Cell-Free DNA in Urine: A Protocol for Optimized DNA Analysis

This is not the first editorial in Clinical Chemistry dedicated to the testing of urine for cell-free DNA. In 2000 Lo(1) highlighted the findings of Botezatu et al.(2), who were the first to describe a transfer of DNA across the kidney barrier into urine. The origins of detected transrenal DNA sequences were carcinomas, blood transfusions, and, in pregnant women, the fetus. Following this first report, some pioneering work was done in transplant medicine. Urinary microchimerism was found in women after receiving kidney transplants from males(3)(4). In a quantitative analysis it was demonstrated that transplant-derived DNA was increased during graft rejection, indicating a potential marker for transplant control(4). Later, attempts were made to detect cell-free fetal DNA in maternal urine. However, maternal urine analysis was faced with technical difficulties, and it was suggested that the concentration of transrenal DNA might be too low for standard PCR protocols. …

Elevated transrenal DNA (cell-free urine DNA) in patients with urinary tract infection compared to healthy controls

Objective To study whether levels of transrenal DNA (Tr-DNA) are influenced by the presence of urinary tract infections (UTI). Methods Tr-DNA concentrations were measured in 124 donors and 55 patients with UTI. Two methods for DNA extraction were compared. Results UTI patients showed higher concentrations than the donors (normal range: 0–147 GE/mL). QIAamp MiniElute Virus Spin Kit was more efficient than QIAamp DNA Blood Mini Kit. Conclusion Increased concentrations of Tr-DNA urine DNA are shown in presence of UTI.

Removal of High‐Molecular‐Weight DNA by Carboxylated Magnetic Beads Enhances the Detection of Mutated K‐ras DNA in Urine

We have previously demonstrated that mutated DNA derived from the circulation can be detected in urine and predominantly exists as DNA fragments <1 kb. To preferentially isolate the trans-renal DNA from urine, we developed a method using carboxylated beads to separate high-MW (1 kb or larger) from low-MW DNA in urine. A primer set for 18s rRNA (generating a PCR product of 872 bp) was designed and optimized for real-time PCR quantification of high-MW DNA templates. To evaluate the method, urine samples from 5 volunteers with no known diseases and 36 patients with various colorectal diseases were collected and tested. It was found that the average removal efficiency of high-MW DNA from total urine DNA using carboxylated beads is 92.72%+/- 1.42%. Furthermore, compared with using total urine DNA, our method provides a greater ability to detect mutated K-ras in the urine of colorectal cancer patients. The concurrence of K-ras mutations detected in disease tissue and the corresponding urine specimen is significantly higher (P= 0.0015) when the samples were enriched in low-MW DNA.

Transrenal DNA testing: progress and perspectives

Transrenal DNA (Tr-DNA) is a recently discovered class of extracellular urinary DNA that originates from cells dying throughout the body. Postapoptotic DNA is known to appear in the circulating plasma, but it is now recognized that a portion of these fragments cross the kidney barrier and appear in urine in the form of 150–200-bp fragments. Tr-DNA containing fetal sequences has been isolated from the urine of pregnant women, tumor-specific mutations have been detected in Tr-DNA from patients with colon and pancreatic tumors, and donor DNA has been found in Tr-DNA isolated from recipient urine. Furthermore, proviral HIV DNA, bacterial and parasite DNA sequences have been detected in Tr-DNA from infected patients. Potential applications of Tr-DNA-based tests cover a very broad area of molecular diagnostics and genetic testing, including prenatal detection of inherited diseases, tumor diagnostics and therapeutic monitoring and detection of infectious agents. The Tr-DNA test is expected to have utility in treatment monitoring, transplantation monitoring, drug development and broad public health screening, where a noninvasive, common-platform diagnostic technology has particular value. This review describes some of the highlights of Tr-DNA technology applications, advantages over existing technologies and potential problems anticipated in test development.

Circulating nucleic acids and apoptosis.

It is well documented that plasma contains DNA from tissues throughout the body, including developing fetuses, and tumors. A portion of this DNA crosses the kidney barrier and appears in urine (i.e., transrenal DNA). However, molecular, cellular, and physiological mechanisms of the circulating DNA phenomenon and renal clearance are in an early phase of investigation. Here, we discuss possible forms of circulating DNA, factors affecting representation of different tissues and genomic sequences in plasma DNA, possible mechanisms of renal DNA clearance, and technical problems encountered in DNA isolation from urine. We suggest that apoptotic cells are an important source of DNA in both plasma and urine. Further analysis of the data has led us to propose that a significant portion of circulating DNA can be represented in apoptotic bodies.